# **METAL ECONOMY IN HOST-MICROBE INTERACTIONS**

**Topic Editors Frédéric Veyrier and Mathieu Cellier**

CELLULAR AND INFECTION MICROBIOLOGY

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**ISSN** 1664-8714 **ISBN** 978-2-88919-497-1 **DOI** 10.3389/978-2-88919-497-1

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## **METAL ECONOMY IN HOST-MICROBE INTERACTIONS**

Topic Editors: **Frédéric Veyrier,** Institut Pasteur, France **Mathieu Cellier,** Institut national de la recherche scientifique, Canada

This Research Topic presents knowledge on transition metal metabolism in various infections from the dual perspective of offender and defender.

1) Host Nutritional Immunity: depriving or poisoning.

To date, the implication of divalent metals have been described in two different immune strategies that aim to fight microbial invaders. One consists in depriving microbes of essential divalent metals whereas the other aims at overloading invaders with toxic concentrations of metal. The contributions in this section present, in different situations, various aspects of this metal economy at the host-microbe interface.

Two papers deal with metal homeostasis as hosts interact with bacteria. Diaz-Ochoa et al. (2014) review immunological mechanisms to sequester Fe, Mn and Zn in the inflamed gut and strategies of commensals and pathogens to evade mucosal defenses and obtain such nutrients. Lisher & Giedroc (2013) detail chemical and structural mechanisms to capture Mn, an antioxidant used by pathogens to adapt to human hosts, and the impact of Fe and Zn on Mn bioavailability during infections.

The most coveted metal, iron is key to nutritional immunity and microbial virulence. Using amoeba as model phagocyte, Bozzaro et al. (2013) present the tug of war between a bacterial predator, sequestering intracellular iron to resist invasion, and pathogens which elude such defense mechanisms. On mammalian defense against intracellular bacteria and protozoan parasites, Silva-Gomes et al. (2013) outline divergent approaches: iron-withholding to prevent microbial replication or iron-based oxidative injury to kill invaders.

Host may also target invaders with toxic doses of Cu and Zn, normally kept at low concentrations. Neyrolles et al. (2013) present an opinion article on bacterial Zn and Cu poisoning in the context of Mycobacterium tuberculosis infection. Chaturvedi & Henderson (2014) summarize the specific properties of copper and its toxic effect on bacteria cells. Argüello et al. (2013) review how bacteria integrate homeostatic mechanisms to avoid Cu toxicity by sensing and regulating ion chelation, chaperoning and membrane transport.

2) Microbial adaptation to host defenses: metallo-transporters or exporters. To overcome host resistance to infection, numerous mechanisms have been selected through the course of microbial evolution, in particular transporters that can feed the bacteria even at low metal concentration or, on the contrary, metallo-exporters that can expel metals outside the cell to avoid toxic accumulation. The articles in this section describe the microbial

transport arsenal, and its regulation, which play major roles to influence metal economy at the host-microbe interface.

Bacterial and fungal strategies to acquire Fe is the subject of four contributions. Liu & Biville (2013) discuss erythrocyte parasitism by Bartonella, transmitted by arthropod vectors and relying principally on heme capture and oxidative stress defense to cause persistent infections. Runyen-Janecky (2013) highlights some of the recent findings on heme iron acquisition system and the regulation of their expression in Gram-negative pathogens. Cornelis & Dingemans (2013) recap how Pseudomonas adapts means of iron capture to the type of infection it establishes, acute or chronic. Caza & Kronstad (2013) contrast strategies of virulent bacteria and fungi to subvert host immunity and steal iron from hemoglobin, heme, transferrin and lactoferrin or elemental iron using specialized uptake systems and siderophores.

Five papers deal with microbial homeostasis of other metals Mn, Ni and Zn. Honsa et al. (2013) review the roles of importers and exporters of Mn, Fe, Zn and Cu in Streptococcus pneumoniae gene regulation and tissue-specific pathogenesis. Guilhen et al (2013) focus on families of exporters and the role of metal efflux in the evolution of Neisseria meningitidis virulence and naso-pharyngeal colonization. de Reuse et al. (2013) present the specific nickel needs of the gastric pathogen Helicobacter pylori and the homeostasis of nickel in this bacterium. Finally, Zn homeostasis is the subject of two articles. Staats et al. (2013) present the role of Zn in bacteria-host relationship and how this metal represents a very promising target for the development of novel antimicrobial strategies. Cerasi et al. (2013) emphasize the role of Zn in the host-fungi relationships and the impact of Zn bioavailability on the expression of virulence genes.

Lastly, metallo-regulation of bacterial gene expression is discussed in relation to virulence. Porcheron et al. (2013) review the enterobacterial metallo-transporters and their regulation, discussing strain-specific differences. Troxell & Hassan (2013) review Fe-dependent regulations of transcription by the Ferric Uptake Regulator to control iron metabolism, oxidative stress defense and virulence. Finally, Troxell & Yang (2013) present the metalloregulation in the causative agent of Lyme disease (Borrelia burgdorferi) a bacterium that does not require iron for its metabolism.

# Table of Contents



## Metal economy in host-microbe interactions

#### *Frédéric J. Veyrier 1,2\* and Mathieu F. Cellier <sup>2</sup> \**

*<sup>1</sup> Institut Pasteur, Paris, France*

*<sup>2</sup> Institut National de la Recherche Scientifique-Institut Armand Frappier, Laval, Canada \*Correspondence: frederic.veyrier@iaf.inrs.ca; mathieu.cellier@iaf.inrs.ca*

#### *Edited and reviewed by:*

*Yousef Abu Kwaik, University of Louisville School of Medicine, USA*

**Keywords: metal, virulence, host, pathogen, transporter, exporter, regulation**

This Research Topic presents knowledge on transition metal metabolism in various infections from the dual perspective of offender and defender.

## **HOST NUTRITIONAL IMMUNITY: DEPRIVING OR POISONING**

To date, the implication of divalent metals have been described in two different immune strategies that aim to fight microbial invaders. One consists in depriving microbes of essential divalent metals whereas the other aims at overloading invaders with toxic concentrations of metal. The contributions in this section present, in different situations, various aspects of this metal economy at the host-microbe interface.

Two papers deal with **metal homeostasis as hosts interact with bacteria**. Diaz-Ochoa et al. (2014) review immunological mechanisms to sequester Fe, Mn, and Zn in the inflamed gut and strategies of commensals and pathogens to evade mucosal defenses and obtain such nutrients. Lisher and Giedroc (2013) detail chemical and structural mechanisms to capture Mn, an antioxidant used by pathogens to adapt to human hosts, and the impact of Fe and Zn on Mn bioavailability during infections.

The most coveted metal, **iron is key to nutritional immunity and microbial virulence**. Using amoeba as model phagocyte, Bozzaro et al. (2013) present the tug of war between a bacterial predator, sequestering intracellular iron to resist invasion, and pathogens which elude such defense mechanisms. On mammalian defense against intracellular bacteria and protozoan parasites, Silva-Gomes et al. (2013) outline divergent approaches: iron-withholding to prevent microbial replication or iron-based oxidative injury to kill invaders.

Host may also target invaders with **toxic doses of Cu and Zn**, normally kept at low concentrations. Neyrolles et al. (2013) present an opinion article on bacterial Zn and Cu poisoning in the context of *Mycobacterium tuberculosis* infection. Chaturvedi and Henderson (2014) summarize the specific properties of copper and its toxic effect on bacteria cells. Arguello et al. (2013) review how bacteria integrate homeostatic mechanisms to avoid Cu toxicity by sensing and regulating ion chelation, chaperoning and membrane transport.

### **MICROBIAL ADAPTATION TO HOST DEFENSES: METALLO-TRANSPORTERS OR EXPORTERS**

To overcome host resistance to infection, numerous mechanisms have been selected through the course of microbial evolution, in particular transporters that can feed the bacteria even at low metal concentration or, on the contrary, metallo-exporters that can expel metals outside the cell to avoid toxic accumulation. The articles in this section describe the microbial transport arsenal, and its regulation, which play major roles to influence metal economy at the host-microbe interface.

**Bacterial and fungal strategies to acquire Fe** is the subject of four contributions. Liu and Biville (2013) discuss erythrocyte parasitism by *Bartonella*, transmitted by arthropod vectors and relying principally on heme capture and oxidative stress defense to cause persistent infections. Runyen-Janecky (2013) highlights some of the recent findings on heme iron acquisition system and the regulation of their expression in Gram-negative pathogens. Cornelis and Dingemans (2013) recap how Pseudomonas adapts means of iron capture to the type of infection it establishes, acute or chronic. Caza and Kronstad (2013) contrast strategies of virulent bacteria and fungi to subvert host immunity and steal iron from hemoglobin, heme, transferrin and lactoferrin or elemental iron using specialized uptake systems and siderophores.

Five papers deal with **microbial homeostasis of other metals Mn, Ni, and Zn**. Honsa et al. (2013) review the roles of importers and exporters of Mn, Fe, Zn, and Cu in *Streptococcus pneumoniae* gene regulation and tissue-specific pathogenesis. Guilhen et al. (2013) focus on families of exporters and the role of metal efflux in the evolution of *Neisseria meningitidis* virulence and naso-pharyngeal colonization. De Reuse et al. (2013) present the specific nickel needs of the gastric pathogen *Helicobacter pylori* and the homeostasis of nickel in this bacterium. Finally, Zn homeostasis is the subject of two articles. Staats et al. (2013) present the role of Zn in bacteria-host relationship and how this metal represents a very promising target for the development of novel antimicrobial strategies. Cerasi et al. (2013) emphasize the role of Zn in the host-fungi relationships and the impact of Zn bioavailability on the expression of virulence genes.

Lastly, **metallo-regulation of bacterial gene expression** is discussed in relation to virulence. Porcheron et al. (2013) review the enterobacterial metallo-transporters and their regulation, discussing strain-specific differences. Troxell and Hassan (2013) review Fe-dependent regulations of transcription by the Ferric Uptake Regulator to control iron metabolism, oxidative stress defense and virulence. Finally, Troxell and Yang (2013) present the metallo-regulation in the causative agent of Lyme disease (*Borrelia burgdorferi*) a bacterium that does not require iron for its metabolism.

## **REFERENCES**


an emerging paradigm. *Front. Cell. Infect. Microbiol.* 3:89. doi: 10.3389/fcimb.2013.00089


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 15 December 2014; accepted: 19 December 2014; published online: 13 January 2015.*

*Citation: Veyrier FJ and Cellier MF (2015) Metal economy in host-microbe interactions. Front. Cell. Infect. Microbiol. 4:190. doi: 10.3389/fcimb.2014.00190*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2015 Veyrier and Cellier. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Transition metal ions at the crossroads of mucosal immunity and microbial pathogenesis

## *Vladimir E. Diaz-Ochoa1,2, Stefan Jellbauer 1,2, Suzi Klaus 1,2 and Manuela Raffatellu1,2\**

*<sup>1</sup> Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA, USA <sup>2</sup> Institute for Immunology, University of California, Irvine, Irvine, CA, USA*

#### *Edited by:*

*Mathieu F. Cellier, Institut National de la Recherche Scientifique, Canada*

#### *Reviewed by:*

*Simon C. Andrews, University of Reading, UK Guenter Weiss, Medical University of Innsbruck, Austria François Canonne-Hergaux, Institut National de la Santé et de la Recherche Médicale, France*

#### *\*Correspondence:*

*Manuela Raffatellu, Department of Microbiology and Molecular Genetics, University of California, Irvine, B251 Medical Sciences, Irvine, CA 92697-4025, USA e-mail: manuelar@uci.edu*

Transition metal ions are essential micronutrients for all living organisms. In mammals, these ions are often protein-bound and sequestered within cells, limiting their availability to microbes. Moreover, in response to infection, mammalian hosts further reduce the availability of metal nutrients by activating epithelial cells and recruiting neutrophils, both of which release metal-binding proteins with antimicrobial function. Microorganisms, in turn, have evolved sophisticated systems to overcome these limitations and acquire the metal ions essential for their growth. Here we review some of the mechanisms employed by the host and by pathogenic microorganisms to compete for transition metal ions, with a discussion of how evading "nutritional immunity" benefits pathogens. Furthermore, we provide new insights on the mechanisms of host-microbe competition for metal ions in the mucosa, particularly in the inflamed gut.

**Keywords: infection, nutritional immunity, iron, zinc, manganese, lipocalin-2, calprotectin, S100 proteins**

## **INTRODUCTION**

Transition metal ions are involved in many biological processes crucial for sustaining life. These metals can serve as cofactors in proteins, enabling their biological function, regulating their activity, and/or stabilizing their structure (Aisen et al., 2001; Andreini et al., 2008; Waldron et al., 2009). This mini-review focuses on three metal ions targeted by host sequestration strategies and the means by which microbes acquire them; namely, iron, zinc, and manganese.

Among transition metal ions, iron is the most abundant in the human body. Of the 3–5 g of iron in adults, 65–75% is located within erythrocytes bound to heme, the tetrapyrrole cofactor of hemoglobin, and utilized for oxygen transport (Andrews, 2000). Iron is also critical for other cellular processes in mammals, such as nucleic acid and protein synthesis, electron transport, and cellular respiration (Griffiths, 1999; Lieu et al., 2001). Most of the iron in the body is intracellular, and extracellular iron is associated with high-affinity iron binding proteins, namely transferrin and lactoferrin, so iron that microorganisms need for survival is severely restricted. Much like eukaryotic cells, microorganisms also utilize iron in DNA synthesis, electron transport, oxygen binding, and superoxide metabolism (Griffiths, 1999). Outside the body, the bioavailability of iron is generally limited due to the low solubility of ferric iron (Fe3+) at physiological pH (7.4), likely facilitating microbial adaptation to low iron conditions (Raymond et al., 2005). In light of this, microbes that possess multiple iron uptake mechanisms, or those that can utilize alternative metal ions like zinc and manganese, are able to thrive when usable iron is scarce.

Zinc is an essential metal nutrient with an estimated dietary requirement in humans of 15 mg per day (Tapiero and Tew, 2003; King, 2011). Approximately 95% of zinc in humans is intracellular, where it serves structural and functional roles for a large number of macromolecules and enzymes (Tapiero and Tew, 2003; King, 2011). For prokaryotes, it is estimated that 5–6% of their proteome may consist of zinc binding proteins, which emphasizes the need for mechanisms of zinc acquisition in these cells (Andreini et al., 2006). In contrast to iron and zinc, only trace concentrations of manganese are found in human serum (*<*10 nM) and tissue (*<*4μM) (Keen et al., 2000), which is likely to pose significant challenges for microorganisms that have adapted to thrive on earth's biosphere where manganese is widely available (Morgan, 2000). Only a handful of strictly manganese-dependent enzymes are known in both eukaryotes and prokaryotes because manganese in metalloenzymes appears to be readily interchangeable with other divalent cations (Andreini et al., 2008). Manganese in microbes is largely known for its role as a cofactor for some free radical detoxifying enzymes, but it also plays a key role in central carbon metabolism (Kehres and Maguire, 2003).

Transition metal ions are important biological catalysts because they can undergo changes in oxidation states involving one electron. To limit the unspecific reactive potential of transition metals, their availability in vertebrate hosts needs to be tightly regulated at all times and especially limited during infection, a process termed nutritional immunity (the sequestration of nutrients from pathogens). Host mechanisms of nutritional immunity are varied and include: the induction of hepcidin, a master hormone regulator that controls the levels of iron in the body (Drakesmith and Prentice, 2012); the expression of the Natural Resistance-Associated Macrophage Protein 1 (NRAMP1), an ion transporter that pumps iron and manganese out of pathogencontaining phagosomes (Jabado et al., 2000; Forbes, 2003; Cellier et al., 2007); and the expression of antimicrobial proteins that sequester metal ions at sites of infection (Aujla et al., 2008; Corbin et al., 2008; Raffatellu et al., 2009; Hood et al., 2012; Liu et al., 2012). Whereas all of these strategies aid the host in limiting the replication of infecting microbes, some microorganisms have evolved or acquired mechanisms of metal uptake that circumvent the nutritional immune response. Here we review some of the mechanisms that the mammalian host utilizes to sequester metal ions in response to infection, we describe how microbes can evade this nutritional immunity (with a focus on mucosal sites), and we discuss how circumventing this host defense benefits pathogens. Although transition metal toxicity and active intoxication are also established strategies in antimicrobial host responses, we will set our focus on the starvation of essential metal nutrients.

#### **MICROBIAL MECHANISMS OF ACQUIRING IRON**

Iron is required by numerous microbial species because it serves as a cofactor for important cellular processes including DNA replication, central metabolism and respiration. Microbes have thus evolved or acquired a variety of specialized iron uptake systems to overcome iron limitation. These systems are generally categorized as unbound iron, siderophore, or heme acquisition systems. Bacteria can uptake unbound iron using ferrous iron (Fe2+) transport systems like Feo proteins, mechanisms that appear to be important mainly during low oxygen conditions, when ferrous iron remains more stable and predominate over ferric iron (Andrews et al., 2003). Such systems likely play a negligible role in bacterial iron acquisition under inflammatory conditions, where unbound iron is rarely found.

Under iron-limiting conditions, many pathogenic bacteria and some fungi synthesize and secrete siderophores; small, highaffinity iron-chelating compounds (Neilands, 1995). Siderophore effectiveness resides in their ability to bind ferric iron (Fe3+) with an affinity that can exceed that of host Fe3+-binding proteins like transferrin or lactoferrin (Griffiths, 1999), enabling siderophores to "steal" iron from these host proteins. Microbial uptake of Fe3<sup>+</sup> from siderophore-Fe3<sup>+</sup> complexes is achieved by either the reduction of iron from the siderophore at the extracellular surface or by the internalization of the complex (Miethke and Marahiel, 2007). Filamentous fungi are capable of iron uptake by both routes (Philpott, 2006). Though the mechanisms of extracellular reduction by bacteria are not well-understood, the internalization of siderophore-Fe3<sup>+</sup> complexes is well-studied (Crosa and Walsh, 2002; Krewulak and Vogel, 2008; Braun and Hantke, 2011). In Gram-negative bacteria, several outer-membrane receptors that transport siderophore-Fe3<sup>+</sup> complexes have been identified; examples include the FepA receptor for enterobactin, and the FhuA receptor for ferrichrome (Chakraborty et al., 2007; Braun, 2009). The energy required by these receptors for the transport of the substrate originates from the proton motive force of the inner membrane and is transduced through the TonB protein complex (Braun and Braun, 2002; Moeck and Coulton, 2002; Postle and Kadner, 2003). Once in the periplasm, substrate-binding proteins (SBPs) shuttle the siderophore-Fe3<sup>+</sup> complex to the corresponding ATP-binding cassette (ABC) transporter, which then translocates the complex into the cytoplasm (Biemans-Oldehinkel et al., 2006). ABC transporters in the cytoplasmic membrane of Gram-positive bacteria are also involved in the uptake of siderophore-Fe3<sup>+</sup> complexes. Unlike Gram-negative bacteria, their cognate SBPs are responsible for initial binding of the complex and are tethered to the cytoplasmic membrane (Sutcliffe and Russell, 1995; Biemans-Oldehinkel et al., 2006). Once in the cytoplasm, iron can be liberated from siderophores through reduction of Fe3<sup>+</sup> to Fe2<sup>+</sup> or by enzymatic degradation of the siderophore (Miethke and Marahiel, 2007). Among bacteria, siderophore-based iron acquisition systems are widespread.

One of the most studied siderophores is enterobactin, also called enterochelin, which is synthesized by commensal and pathogenic Enterobacteriaceae including *Escherichia coli*, *Klebsiella pneumoniae*, and *Salmonella* spp (O'Brien and Gibson, 1970; Pollack and Neilands, 1970; Rogers et al., 1977; Perry and San Clemente, 1979; Lawlor and Payne, 1984). Enterobactin has high affinity for iron *(Ka* <sup>=</sup> <sup>10</sup><sup>51</sup> <sup>M</sup>−1), which is higher than the affinity of host proteins like transferrin (*Ka* <sup>=</sup> <sup>10</sup><sup>20</sup> <sup>M</sup>−1) (Aisen et al., 1978; Carrano and Raymond, 1979). Therefore, bacteria that synthesize enterobactin can efficiently scavenge iron from the host; however, the host innate immune response has evolved a mechanism to counteract enterobactin-mediated iron acquisition (discussed in detail below) (Fischbach et al., 2006). Although siderophores are generally secreted into the host extracellular environment, some siderophores aid iron acquisition by pathogens with a predominantly intracellular lifestyle. *Mycobacterium tuberculosis* (Mtb), for example, expresses siderophores known as mycobactins that diffuse out of Mtb-containing phagosomes, chelate iron from cytoplasmic stores, and re-enter the phagosome via lipid droplets (Luo et al., 2005).

In addition to siderophores, microbial pathogens can utilize different uptake systems to obtain iron from a variety of sources, which allow them to inhabit diverse niches and to respond to host mechanisms of iron sequestration. In the case of *Candida albicans*, uptake of unbound iron via the high-affinity iron permease *FTR1* is critical for establishing systemic infection in mice (Ramanan and Wang, 2000). In contrast, uptake of iron-bound siderophores is necessary for *C. albicans* colonization of epithelial layers but not for the development of a bloodborne infection (Heymann et al., 2002). Because *C*. *albicans* lacks the genes for the biosynthesis of siderophores (Haas, 2003), it depends on other microorganisms for the production of siderophores. Therefore, *C. albicans* uptake of iron via siderophores is likely restricted to sites where siderophore-producing microorganisms are found (e.g., mucosal surfaces in the gut). Another source of iron for microbes is the biggest pool of iron in the human body: iron from heme and heme-binding proteins.

Similar to the uptake of siderophore-bound iron, the first step in bacterial heme transport involves the binding of heme or hemoglobin to a surface receptor. In Gram-negative bacteria, TonB-dependent receptors are involved in the transport of heme into the periplasm, where heme-specific SBPs bind the molecule (Braun and Hantke, 2011). For hemoglobin, both Gram-negative and Gram-positive bacteria extract the heme group prior to transfer to an SBP. Heme-specific ABC transporters then translocate heme into the cytoplasm, where iron is released by heme-degrading enzymes (Braun and Hantke, 2011; Nobles and Maresso, 2011). Heme oxygenases catalyze the oxidative cleavage of heme with an electron donor to liberate iron (Nobles and Maresso, 2011). Subsequent catabolism of the heme is required to reduce the toxicity associated with the heme porphyrin (Nobles and Maresso, 2011).

#### **HOST MECHANISMS OF SEQUESTERING IRON**

Iron is essential for the replication of many pathogenic organisms, so it is not surprising that the host has evolved sophisticated strategies to limit the availability of iron to pathogens. Conversely, both iron supplementation and diseases characterized by iron overload, such as hemochromatosis, increase the host's susceptibility to infection (reviewed in Griffiths, 1999). In humans, the levels of unbound iron are low; most iron is bound by heme in the context of hemoglobin. Moreover, free heme can be captured by hemopexin and free hemoglobin by haptoglobin. Other proteins, like transferrin in serum, or lactoferrin in neutrophils and human secretions, bind strongly to ferric iron. In most cells, ferritin is responsible for storing iron for normal cellular use, but in specialized cells, i.e., hepatocytes and macrophages, ferritin is used for long-term iron storage and sequestration during iron overload, respectively (Andrews, 2000). Additionally, macrophages increase iron uptake and ferritin synthesis when converting to their inflammatory phenotype, suggesting that ferritin-based sequestration may be a key mechanism for intracellular iron withholding during infection (Birgegård and Caro, 2009).

An additional mechanism of regulating iron metabolism is mediated by the hormone hepcidin, which controls hostprotective responses by integrating signals from iron status and threat of infection. Initially identified as an antimicrobial peptide (Krause et al., 2000), hepcidin is considered to be the master hormonal regulator of iron metabolism, controlling both the overall level of iron and its localization (Nicolas et al., 2001; Park et al., 2001; Nicolas, 2002; Nemeth et al., 2003). Upon microbial infection, the upregulation of hepcidin, concomitant with a reduction of serum transferrin saturation, causes an overall decrease in iron levels (Nemeth et al., 2003; Armitage et al., 2011). Hepcidin upregulation is partially mediated through expression of pro-inflammatory cytokines like interleukin (IL-) 6, which stimulates the production of hepcidin in the liver (Nemeth et al., 2003, 2004a; Rodriguez et al., 2013). Hepcidin then inhibits both cellular iron efflux and duodenal iron absorption by binding to and inducing the degradation of the cellular iron transporter ferroportin 1, which exports iron into the plasma from cells that store or transport iron, including hepatocytes, macrophages, and absorptive enterocytes (Nemeth et al., 2004b; Ross et al., 2012). Subcutaneous infection with either Gram-negative or Gram-positive bacteria has been shown to induce hepcidin synthesis by neutrophils and macrophages, suggesting that local production of hepcidin may limit iron availability at sites of infection (Peyssonnaux et al., 2006). Overall, the induction of hepcidin upon infection results in hypoferremia and anemia of inflammation, which represent important host defense strategies to limit the availability of iron to pathogens.

One of the most studied host transporters in the context of bacterial pathogenesis is NRAMP1,—a proton-dependent transporter of divalent metal ions expressed by professional phagocytes, such as macrophages and neutrophils (Cellier et al., 2007). This transporter is localized in the phagosomal membrane and exports Fe2<sup>+</sup> and Mn2<sup>+</sup> out of the phagosomal compartment, presumably to reduce access to these metals of pathogens residing within the phagosome (Cellier et al., 2007). While this export function occurs during infection, NRAMP1 is also known to contribute to hemoglobin iron recycling by reticuloendothelial macrophages that phagocytose senescent erythrocytes (Cellier et al., 2007; Soe-Lin et al., 2009). In addition to its phagosome metal-withholding function, expression of a functional NRAMP1 also restricts microbial growth by enhancing macrophage production of the antimicrobial effector molecule nitric oxide (NO) through sustained transcription of inducible nitric oxide synthase (iNOS) (Fritsche et al., 2003). Although the mechanism for iNOS induction is not fully understood, both STAT-1-mediated expression of the transcription factor IRF-1, as well as suppressed production of the inhibitory cytokine IL-10, contribute to NRAMP1-dependent prolonged activation of iNOS transcription (Fritsche et al., 2003, 2008). Similarly, another recent study using macrophage cell lines suggests that NRAMP1-mediated stimulation of the expression of lipocalin-2, an antimicrobial peptide that binds iron-loaded bacterial siderophores including enterobactin, is a novel mechanism by which NRAMP1 confers resistance to infection with the intracellular pathogen *Salmonella enterica* serovar Typhimurium (*S. Typhimurium*) (Fritsche et al., 2012). The importance of NRAMP1 in the host response to infection is further underlined by many studies showing that mice with a functional *Nramp1* (*Slc11a1*) allele are more resistant to infection with a variety of intracellular pathogens including *Mycobacterium bovis* BCG, *Leishmania donovanii*, and *S. Typhimurium* (Forbes and Gros, 2001; Cellier et al., 2007).

Host mechanisms discussed thus far effectively reduce available iron, but they are not sufficient to completely prohibit bacterial iron acquisition during an infection. As discussed above, pathogenic bacteria can deploy an efficient weapon in the battle for iron: siderophores. However, as mammals have evolved for millions of years together with siderophore-producing bacteria, it is not surprising that we have evolved an anti-siderophore mechanism: secretion of lipocalin-2 (also known as siderocalin, neutrophil gelatinase-associated lipocalin, uterocalin, or 24p3) (Goetz et al., 2002; Flo et al., 2004; Correnti and Strong, 2012). Lipocalin-2 is one of the most abundant antimicrobial proteins released by epithelial cells and neutrophils during infections in the gut and respiratory mucosa with pathogens like *S. Typhimurium* and *K. pneumoniae*, respectively (Aujla et al., 2008; Bachman et al., 2009; Raffatellu et al., 2009). Lipocalin-2 sequesters a subset of catecholate siderophores, including enterobactin, thereby limiting bacterial access to iron (Goetz et al., 2002; Flo et al., 2004; Berger et al., 2006). In a sepsis model, lipocalin-2 induction is dependent on Toll-like receptor 4 signaling (Flo et al., 2004; Srinivasan et al., 2012). It is also known that lung and intestinal epithelial cells express and secrete lipocalin-2 in response to signaling by pro-inflammatory cytokines released by T helper 17 (Th17) cells, like interleukin IL-17 and IL-22 (Aujla et al., 2008; Raffatellu et al., 2009).

In addition to lipocalin-2, IL-17 and IL-22 also stimulate epithelial secretion of neutrophil chemoattractants, known as CXC chemokines, which mediate the recruitment of neutrophils to the mucosa (Awane et al., 1999; Andoh et al., 2005; Kao et al., 2005; McAllister et al., 2005; Aujla et al., 2008; Raffatellu et al., 2009). Neutrophils play a key role in nutritional immunity because they constitute the largest proportion of circulating white blood cells in humans, quickly mobilize to sites of infection, and express high levels of antimicrobial proteins that sequester metal ions, including lipocalin-2, lactoferrin, and, as detailed below, calprotectin (Masson et al., 1969; Steinbakk et al., 1990; Goetz et al., 2002). Thus, the coordinated expression and release of metalbinding antimicrobial proteins by epithelial cells and neutrophils during infection promotes host sequestration of essential metal nutrients.

In this tug of war for iron, pathogens have evolved mechanisms to counteract the sequestration of siderophores. To circumvent this arm of nutritional immunity, pathogens including *Salmonella* species, *Klebsiella* species and uropathogenic *E. coli* (UPEC) species synthesize salmochelin, a C-glucosylated derivative of enterobactin (Hantke et al., 2003; Bachman et al., 2011), which lipocalin-2 cannot bind, thus enabling iron uptake in these species and enhancing their colonization of host tissues (Fischbach et al., 2006; Crouch et al., 2008; Raffatellu et al., 2009; Bachman et al., 2011). Evasion of lipocalin-2-mediated iron sequestration is thus regarded as a virulence mechanism. However, work in our laboratory has recently shown that a probiotic strain of the Enterobacteriaceae family (*E. coli* Nissle 1917) also evades iron sequestration by lipocalin-2 in the inflamed gut via secretion of siderophores including salmochelin (Deriu et al., 2013). In this case, iron acquisition and evasion of lipocalin-2 is beneficial to the host, because *E. coli* Nissle 1917 reduces *S. Typhimurium* intestinal colonization by outcompeting it for iron acquisition (Deriu et al., 2013). Therefore, evasion of lipocalin-2 by the secretion of modified siderophores can confer a fitness advantage to probiotic strains like *E. coli* Nissle 1917 and enhance the host response against bacterial pathogens by further sequestering iron.

### **MICROBIAL MECHANISMS OF ACQUIRING ZINC AND MANGANESE**

While the role of iron in cellular processes is well-characterized, increasing evidence suggests that other transition metal ions such as zinc and manganese also play a crucial role in microbial physiology (Keen et al., 2000; Hantke, 2005). For example, in order to circumvent host-mediated iron sequestration, *Borrelia burgdorferi* lacks most genes that code for iron-binding proteins, and, for the few metalloproteins it does express, *B. burgdorferi* uses manganese instead of iron (Posey and Gherardini, 2000). In many bacterial species, manganese also serves as a metal cofactor for proteins involved in central carbon metabolism and for the detoxification of reactive oxygen species (ROS) (Kehres and Maguire, 2003). Zinc-dependent enzymes that can detoxify ROS have also been identified (Battistoni, 2003). Furthermore, zinc was found to be associated with up to 5% of all bacterial proteins, of which more than 80% are enzymes (Andreini et al., 2006). In line with their essential role in many bacterial functions, acquisition of zinc and manganese has subsequently been shown to contribute to bacterial pathogenesis (reviewed in Kehl-Fie and Skaar, 2010).

Similar to siderophore and heme transport across the cytoplasmic membrane, ABC-type transporters are involved in bacterial uptake of zinc (Zn2+) and manganese (Mn2+) ions (Claverys, 2001). These transporter systems are composed of a cation binding protein that shuttles its substrate to its cognate transporter, a cytoplasmic ATP-binding protein that facilitates active transport, and the transmembrane protein that mediates transport through the cytoplasmic membrane. In Gram-negative bacteria, the cation binding protein is soluble and localized to the periplasm, while in Gram-positive bacteria it is a lipoprotein anchored to the extracellular membrane (Gilson et al., 1988; Tam and Saier, 1993; Sutcliffe and Russell, 1995). High-affinity ABC-type zinc transporters include ZnuABC of Gram-negative bacteria (Patzer and Hantke, 1998; Hantke, 2005), and AdcBCA of the Grampositive streptococci (Dintilhac et al., 1997; Panina et al., 2003). ABC-type manganese transporters have also been identified in several Gram-positive and Gram-negative bacteria (Claverys, 2001; Papp-Wallace and Maguire, 2006). In *S. Typhimurium*, for example, the ABC-type transporter SitABCD is found within a pathogenicity island and is not present in the closely related organism *E. coli*, indicating this transporter could have been acquired by horizontal gene transfer (Zhou et al., 1999). Of note, studies have shown some manganese transporters to facilitate the uptake of other divalent cations such as Zn2+, Cd2+, and Fe2<sup>+</sup> at lower affinities, with a *Kd* in the μM range (Kolenbrander et al., 1998; Kehres et al., 2002).

In addition to ABC-type transporters, bacteria also express homologs of the eukaryotic NRAMP transporter family (Kehres et al., 2000; Makui et al., 2000; Que and Helmann, 2000; Horsburgh et al., 2002). One example is the MntH protein of *Salmonella* and *Escherichia*, a membrane-bound, proton-coupled symporter with high specificity for manganese (Kehres et al., 2000); similar to the ABC-type manganese transporters, MntH can also transport other divalent cations at higher concentrations (Papp-Wallace and Maguire, 2006). Another discrete transporter of zinc and manganese uptake is ZupT, a permease with broad cation specificity belonging to the ZIP protein family (Grass et al., 2005; Karlinsey et al., 2010). Though metal uptake via ZupT is less specific than via the high-affinity ABC-type or NRAMP transporters, studies in *E*. *coli* have demonstrated this transporter to prefer zinc over manganese, copper, and iron (Grass et al., 2002, 2005; Taudte and Grass, 2010).

In addition to the role of these metals in essential cellular functions, evidence is mounting that specialized mechanisms of zinc and manganese acquisition contribute to bacterial pathogenesis; for instance, zinc and manganese are important cofactors in neutralizing reactive oxygen and nitrogen species, suggesting an important role for these metals in resisting these types of host antimicrobial responses (Lynch and Kuramitsu, 2000; Bowman et al., 2011). Supporting this, mutant strains of the pathogens *Brucella abortus*, *Pasteurella multocida*, and *S. Typhimurium* that lack the ZnuABC transporter are attenuated in systemic models of disease in mice (Campoy et al., 2002; Garrido et al., 2003; Kim et al., 2004; Ammendola et al., 2007). Furthermore, the expression of zinc transporters promotes *Campylobacter jejuni*, *S. Typhimurium*, and *Acinetobacter baumannii* colonization of mucosal tissues (Davis et al., 2009; Hood et al., 2012; Liu et al., 2012). For manganese acquisition, both the ABC-type transporters and the bacterial NRAMP homologs are known to contribute to systemic *S. aureus* and *S. Typhimurium* infection (Karlinsey et al., 2010; Kehl-Fie et al., 2013). Taken together, these studies indicate an important role for zinc and manganese sequestration by the host in controlling microbial infections with different pathogens.

## **HOST MECHANISMS OF SEQUESTERING ZINC AND MANGANESE**

Compared to iron, less is known about the mechanisms the host employs to limit microbial access to metal micronutrients like zinc and manganese. Nevertheless, multiple strategies to limit the availability of these nutrients to pathogens have been identified in the mammalian host.

As described in the section on host iron sequestration, NRAMP1 is a proton-dependent exporter of Fe2<sup>+</sup> and Mn2+across the phagosomal membrane of vertebrates that confers resistance to various intracellular pathogens (Cellier et al., 2007). Another host protein known to sequester metal ions is the antimicrobial protein calprotectin (Corbin et al., 2008), a heterodimer of the two EF-hand calcium-binding proteins S100A8 and S100A9 (Teigelkamp et al., 1991), which exerts antimicrobial activity against several bacterial and fungal organisms by sequestering zinc and manganese (Corbin et al., 2008; Urban et al., 2009; Hood et al., 2012; Liu et al., 2012). Upon dimerizing, S100A8 and S100A9 form two metal binding sites, both of which can bind strongly to Zn2+, though one is also capable of binding manganese (Kehl-Fie et al., 2011; Damo et al., 2013). Like lactoferrin and lipocalin-2, calprotectin is expressed by neutrophils, where it constitutes approximately 50% of their cytosolic content (Hessian et al., 1993). Calprotectin is thought to be secreted by apoptotic neutrophils, where it is associated with their extracellular traps, also called NETs (Urban et al., 2009). Similar to lipocalin-2, the two subunits of calprotectin, S100A8 and S100A9, are also induced by IL-17 and IL-22 in mucosal epithelial cells (Zheng et al., 2008; Liu et al., 2012; Zindl et al., 2013).

To successfully colonize the host, pathogens have evolved mechanisms to resist the effects of calprotectin-dependent zinc and manganese sequestration. Manganese is most notably important as a cofactor for enzymes that detoxify ROS (Aguirre and Culotta, 2012). Consistent with this role, manganese binding by neutrophil-derived calprotectin inhibits the growth of *S. aureus* in tissue abscesses and increases the susceptibility of this pathogen to oxidative stress (Kehl-Fie et al., 2011). To counteract this, the specialized manganese transporters MntABC and MntH contribute to systemic *S. aureus* infection by competing with calprotectin for manganese (Kehl-Fie et al., 2013). In *S. Typhimurium*, expression of the high-affinity zinc transporter ZnuABC aids the pathogen in overcoming calprotectin-mediated zinc sequestration and promotes the growth of *S. Typhimurium* in the inflamed gut as well as *Salmonella* competition with the microbiota (Liu et al., 2012). Genes encoding a similar ABC-type zinc transporter are present in *A. baumannii,* where they also mediate resistance to zinc sequestration by calprotectin and serve to increase pathogenesis (Hood et al., 2012).

S100A12 (calgranulin C) is another calgranulin protein like S100A8 (calgranulin A) and S100A9 (calgranulin B) which binds to zinc and other divalent cations. Similar to S100A8 and S100A9, S100A12 is also predominantly expressed by neutrophils, monocytes and activated macrophages (Robinson and Hogg, 2000). However, unlike S100A8 and S100A9, S100A12 is not found in rodents and its role in metal sequestration is not well-defined. S100A12 seems to be mainly pro-inflammatory through the activation of mast cells but may also play a role in chemotaxis (Hsu et al., 2009; Perera et al., 2010). S100A12 has antiparasitic activity against filarial nematodes (Gottsch et al., 1999), although this activity does not appear to be dependent on metal sequestration. Calcitermin, a 15-residue C-terminal cleavage fragment of S100A12, can be found in the human airways and exhibit antimicrobial activity against *E. coli*, *Pseudomonas aeruginosa*, and *C. albicans*, both at low pH and in media with zinc (Cole et al., 2001).

Another S100 protein with antimicrobial and immunomodulatory activity is S100A7, which is largely expressed in the skin and other epithelia. This protein, also known as psoriasin, was originally discovered as an abundant protein in psoriatic keratinocytes (Gläser et al., 2005). Psoriasin is secreted by keratinocytes and has antimicrobial activity against *E. coli,* possibly by sequestering zinc (Gläser et al., 2005). The molecule is considered an important effector molecule of the cutaneous barrier and, like S100A8 and S100A9, is also induced by IL-17 and IL-22 (Liang et al., 2006).

#### **HOST vs. PATHOGENS: THE BATTLE FOR METALS AT THE INTERSECTION OF HEALTH AND DISEASE IN THE MUCOSA**

Sequestration of metal ions is one of the most important host strategies to limit the growth of bacterial and fungal pathogens. Metal limitation in the host is further enhanced during infection by the secretion of antimicrobial proteins that sequester metal ions, such as lipocalin-2 and calprotectin. Lipocalin-2 appears to be most effective in limiting the growth of commensal bacteria, as a number of pathogens have evolved or acquired additional siderophores to evade this response. In contrast, calprotectin restricts the growth of a variety of bacterial and fungal pathogens, including *S. aureus, C. albicans, B. burgdorferi*, *A. baumannii, and Aspergillus nidulans* (Lusitani et al., 2003; Urban et al., 2009; Moore et al., 2013). While both antimicrobial proteins are constitutively expressed by neutrophils, their expression—as well as the expression of other S100 proteins—may be induced in epithelial cells by pro-inflammatory stimuli like the Th17 cytokines IL-22 and IL-17 (Boniface et al., 2005; Zheng et al., 2008; Kerkhoff et al., 2012; Lee et al., 2012; Liu et al., 2012; Bando et al., 2013; Zindl et al., 2013).

Altogether, the secretion of antimicrobial proteins and the production of reactive oxygen and nitrogen species at the site of infection can reduce the growth of many microorganisms. Susceptibility of commensal bacteria to ROS may be exacerbated as a result of lipocalin-2 and calprotectin expression because these proteins sequester metals that serve as cofactors in bacterial enzymes responsible for neutralizing free radical species. However, in the harsh environment these responses create, microbes with metal scavenging ability can often survive and replicate, sometimes even dominating when commensal competition is reduced. This is the case for *S. Typhimurium*, which overcomes both lipocalin-2- and calprotectin-mediated metal sequestration to colonize the inflamed gut and compete with the microbiota (Raffatellu et al., 2009; Liu et al., 2012), a theme that likely applies to other pathogens (**Figure 1**). Therefore, the secretion of antimicrobial proteins like lipocalin-2 and calprotectin may have a detrimental effect on the host by killing commensal bacteria that are more susceptible to oxidative damage, neutrophil enzymatic activity, and metal nutrient deprivation. Elevated lipocalin-2 and calprotectin levels observed in patients with inflammatory bowel disease (Cayatte et al., 2012; Østvik et al., 2013; Wang et al., 2013) may also be detrimental to the host because antimicrobial activity toward commensal bacteria likely contributes to the microbial imbalance observed in the digestive tract of these patients, known as dysbiosis (Salzman and Bevins, 2008; Manichanh et al., 2012). Sustained intestinal dysbiosis can lead to the overgrowth of potentially harmful bacteria termed pathobionts (Stecher et al., 2013).

The fact that transition metals are essential for proper development and function of the host further complicates the host metal economy during infection. For example, zinc is needed for immune development and function, but it also has to be

**FIGURE 1 | A battle for metals in the intestinal mucosa: mechanisms of host metal sequestration and microbial metal acquisition.** To limit microbial growth, the mammalian host sequesters free iron, zinc, and manganese ions by expressing proteins in the mucosa that directly bind metals or metal-binding agents in a process termed nutritional immunity. Lactoferrin binds to iron (Fe) and calprotectin binds to zinc (Zn) and manganese (Mn). Hemopexin can limit the amount of circulating iron-bound heme (He) and lipocalin-2 sequesters the bacterial iron-scavenging siderophore enterobactin. Upon infection, inflammatory mediators increase the expression of metal-sequestering proteins, which is detrimental to microbes lacking mechanisms to survive metal deprivation. Inflammatory cytokines, IL-17 and IL-22, produced by T cells induce epithelial cells to express antimicrobial proteins including lipocalin-2 and calprotectin. Furthermore, activated epithelial cells secrete CXC chemokines that recruit neutrophils to the site of infection; neutrophils also express high levels of

lactoferrin, lipocalin-2, and calprotectin. Microbial infection and inflammation can stimulate the production of hepcidin in the liver and in macrophages, which further reduces iron availability by inducing the degradation of the cellular iron exporter ferroportin 1. In addition, the divalent metal ion transporter NRAMP1 can export manganese and iron out of the macrophage phagosome to further restrict metal availability to intracellular pathogens. To overcome metal starvation, pathogens (red ovals) employ several strategies to acquire iron, zinc and manganese. Highly specialized ABC-type transporters facilitate the uptake of zinc and manganese as well as iron bound to heme and siderophores. Siderophores, such as enterobactin, are iron-scavenging agents. Although lipocalin-2 can sequester enterobactin to limit microbial access to iron, some pathogens use salmochelin, a C-glucosylated derivative of enterobactin that cannot be bound by lipocalin-2. Some pathogens also express NRAMP family transporters and ZIP family transporters for the uptake of manganese and zinc.

sequestered from staphylococcal abscesses in order to zinc-starve *S*. *aureus* during infection (Kehl-Fie et al., 2013). Furthermore, the amounts of metals vary in different organs (Kehl-Fie et al., 2013), which may be a basis for site-specific differences in the host's metal sequestration strategies. A contributing factor in these differences may include microbial colonization. In the healthy gut, and possibly at other mucosal sites colonized by commensal bacteria, host interactions with the microbiota likely regulate the low-level expression of metal binding proteins as well as the concentration of transition metals. At these mucosal sites and in other tissues and organs, it is plausible that other antimicrobial proteins besides lipocalin-2 and calprotectin may sequester metal ions but have yet to be identified.

In concert with other host defense strategies, nutritional immune responses at the mucosa can lead to beneficial outcomes for the host by reducing the colonization of invading pathogens. However, they can also alter the normal microbial flora, which may enhance the colonization of pathogens like *S.Typhimurium* or result in dysbiosis. Thus, it is important to take into account that metal sequestration strategies can be beneficial to the host, but may also potentially benefit pathogens or pathobionts that evade these responses. Moreover, investigating the mechanisms of host-microbe competition for metal ions may pave the way for developing novel therapeutics that are in critical need given the mounting global threat of antibiotic-resistant pathogens and pathobionts.

#### **ACKNOWLEDGMENTS**

We would like to thank Sean-Paul Nuccio for critical reading of the manuscript and Janet Z. Liu for contributing the artwork for our figure. Work in MR lab is supported by Public Health Service Grants AI083663 and by the Pacific Southwest Regional Center of Excellence for Biodefense and Emerging Infectious Disease Research funds (supported by award number U54AI065359 from the National Institute of Allergy and Infectious Diseases).

#### **REFERENCES**


agent that interferes with siderophore-mediated iron acquisition. *Mol. Cell* 10, 1033–1043. doi: 10.1016/S1097-2765(02)00708-6


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 15 October 2013; paper pending published: 05 November 2013; accepted: 04 January 2014; published online: 24 January 2014.*

*Citation: Diaz-Ochoa VE, Jellbauer S, Klaus S and Raffatellu M (2014) Transition metal ions at the crossroads of mucosal immunity and microbial pathogenesis. Front. Cell. Infect. Microbiol. 4:2. doi: 10.3389/fcimb.2014.00002*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2014 Diaz-Ochoa, Jellbauer, Klaus and Raffatellu. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Manganese acquisition and homeostasis at the host-pathogen interface

## *John P. Lisher <sup>1</sup> and David P. Giedroc 1,2\**

*<sup>1</sup> Graduate Program in Biochemistry, Indiana University, Bloomington, IN, USA*

*<sup>2</sup> Department of Chemistry, Indiana University, Bloomington, IN, USA*

#### *Edited by:*

*Mathieu F. Cellier, Institut National de la Recherche Scientifique, Canada*

#### *Reviewed by:*

*Eric P. Skaar, Vanderbilt University, USA John Helmann, Cornell University, USA Adam Linstedt, Carnegie Mellon University, USA*

#### *\*Correspondence:*

*David P. Giedroc, Department of Chemistry, Indiana University, 800 E. Kirkwood Avenue, Bloomington, IN 47405-7102, USA e-mail: giedroc@indiana.edu*

Pathogenic bacteria acquire transition metals for cell viability and persistence of infection in competition with host nutritional defenses. The human host employs a variety of mechanisms to stress the invading pathogen with both cytotoxic metal ions and oxidative and nitrosative insults while withholding essential transition metals from the bacterium. For example, the S100 family protein calprotectin (CP) found in neutrophils is a calcium-activated chelator of extracellular Mn and Zn and is found in tissue abscesses at sites of infection by *Staphylococcus aureus*. In an adaptive response, bacteria have evolved systems to acquire the metals in the face of this competition while effluxing excess or toxic metals to maintain a bioavailability of transition metals that is consistent with a particular inorganic "fingerprint" under the prevailing conditions. This review highlights recent biological, chemical and structural studies focused on manganese (Mn) acquisition and homeostasis and connects this process to oxidative stress resistance and iron (Fe) availability that operates at the human host-pathogen interface.

**Keywords: manganese, ATP-binding cassette, metal transport, homeostasis, nutritional immunity, iron**

## **INTRODUCTION**

The most abundant transition metals in humans are iron (Fe) and zinc (Zn) (for reviews, see Maret, 2010; Hood and Skaar, 2012) and it is generally accepted that a clinical deficiency in host levels of either metal increases the incidence of infectious disease and mortality (Haider and Bhutta, 2009; Kumar and Choudhry, 2010; Lassi et al., 2010). These deficiencies reduce the ability of the host to utilize these metals to restrict bacterial growth. In the case of Fe, many peroxide- and nitrous oxide-generating enzymes are iron-dependent, and a limitation of iron would compromise this aspect of innate immunity against bacterial pathogens (Kumar and Choudhry, 2010). Zinc deficiency compromises function of the human immune system (Kitamura et al., 2006) and the ability of the host to induce zinc-mediated cellular toxicity as a means to control bacterial infections (for a review, see Stafford et al., 2013); as a result, this condition is associated with an increased incidence of serious infectious disease (Lassi et al., 2010). In contrast, although manganese (Mn) overload is connected to neurological dysfunction (for a review, see Rivera-Mancía et al., 2011) there is not as yet, strong support for the idea that host Mn(II) sufficiency is in any way coupled to the incidence or severity of infectious disease.

There is now, however, emerging evidence that the invading microbe utilizes Mn as a key micronutrient to resist the effects of host-mediated oxidative stress and thus plays a significant role in adaptation of pathogenic bacteria to the human host. This review summarizes recent work in the area of "Mn-centric" nutritional immunity (Weinberg, 1975) placed in the context of the inorganic physiology of the cell and the "fight over metals" implied by recent studies of Mn speciation and chemistries of low molecular weight (LMW) Mn complexes (McNaughton et al., 2010; Barnese et al., 2012; Sharma et al., 2013), and the structures and metal binding affinities of the bacterial high affinity import systems for Mn relative to the extracellular antibacterial protein calprotectin (Corbin et al., 2008; Damo et al., 2013; Hayden et al., 2013). Calprotectin possesses functional properties consistent with that of an extracellular Mn chelator that withholds this metal from the invading pathogen (Kehl-Fie et al., 2013) and is thus formally analogous to siderochalins that capture Fe-siderophores synthesized by the pathogen itself (Flo et al., 2004; Sia et al., 2013). Finally, recent insights into the coordinate regulation and crosstalk that govern intracellular Mn vs. Fe and Mn vs. Zn bioavailability will also be discussed.

#### **INORGANIC CHEMISTRY OF THE CELL: TOTAL METAL AND CELLULAR METAL SPECIATION**

Six first-row 3*d*-block elements extending from manganese to zinc are essential micronutrients that function as inorganic cofactors in up to 25% of all proteins in cells (**Figure 1A**) (Waldron and Robinson, 2009). Mn, Fe, Cu and Zn are ubiquitous in biology (Maret, 2010) while cobalt (Co) and nickel (Ni) play more specialized roles in methyl transfer chemistry (Gherasim et al., 2013) and as a cofactor for a limited number of Ni(II)-containing metalloenzymes (Kaluarachchi et al., 2010), respectively. Zn is unique among these metals in that it is redox-inert and thus stable in the 2+ oxidation state. As such, Zn functions as nature's principal Lewis acid catalyst, where it activates a substrate for catalysis. Zn is a cofactor in a wide range of hydrolytic enzymes, constitutes the largest fraction of metalloproteins in the cell (Maret, 2001) and stabilizes protein structure in the reducing environment of the cytoplasm. Fe readily accesses Fe(II) and Fe(III) oxidation states in 2Fe-2S and 4Fe-4S iron-sulfur proteins involved in electron transfer, with higher valence Fe(IV)-oxo species in both heme and non-heme Fe-containing enzymes responsible

*(Continued)*

#### **FIGURE 1 | Continued**

Modified serum-free media with Exyte (SF-E)] (Posey and Gherardini, 2000), *Deinococcus radiodurans* (Defined Minimal media + 2.5μM Mn) (Daly et al., 2004), *Streptococcus pneumoniae* (Brain Heart Infusion, BHI) (Jacobsen et al., 2011), *Neisseria meningitidis* (Gonococcal Broth, GCB) (Veyrier et al., 2011), *Deinococcus geothermalis* (Tryptone Glucose Yeast extract media, TGY) (Daly et al., 2004), *Deinococcus radiodurans* (TGY) (Daly et al., 2004), *Enterococcus faecium* (TGY) (Daly et al., 2004), *Escherichia coli* (Luria Broth, LB) (Outten and O'Halloran, 2001), *Saccharomyces cerevisiae* (Yeast extract Peptone Dextrose media, YPD) (Rosenfeld and Culotta, 2012), *Neisseria gonorrhoeae* (GCB) (Veyrier et al., 2011), *Caulobacter crescentus* (Peptone Yeast Extract media, PYE) (Hughes et al., 2013), *Escherichia coli* (TGY) (Daly et al., 2004), *Shewanella oneidensis* (TGY; Mn/Fe 0.0005 ± 0.00004) (Daly et al., 2004), and *Pseudomonas putida* (TGY; *<*0.001) (Daly et al., 2004).

for significant oxygen insertion and oxygen activation chemistry (Andrews et al., 2003). Fe is also the obligate cofactor for Fesuperoxide dismutase (Fe-SOD) and nearly all ribonucleotide reductases, although more recently, di-Mn-containing variants have been discovered and characterized (for a review, see Cotruvo and Stubbe, 2012).

Mn, like Zn, is a Lewis acid cofactor in a number of hydrolytic enzymes, e.g., protein phosphatases, and in key enzymes of intermediary metabolism, but is redox-active [to Mn(III), Mn(IV) and Mn(V)] and is most strongly linked to oxidative stress resistance mediated by Mn superoxide dismutase (Mn-SOD) (Culotta et al., 2006) and non-heme di-Mn catalases (Whittaker, 2012). As discussed below, simple small molecule Mn(II)-complexes, unique to Mn(II), may have substantial antioxidant activity inside cells (Barnese et al., 2008, 2012). Finally, the bacterial requirement for intracellular Cu, outside of the photosynthetic bacteria, is generally accepted to be low (Waldron et al., 2009); as a result, there is emerging evidence that the human host harnesses the cytotoxic power of Cu to kill invading bacterial pathogens (White et al., 2009; Rowland and Niederweis, 2012; Samanovic et al., 2012); this need not, however, be the case for all microbial pathogens (Raja et al., 2013).

The inorganic "fingerprint" of unstressed cells is defined as the *total* concentration of all cell-associated metals summed over all cellular fractions (membrane, cytoplasm, periplasm, etc.). This is generally expressed in nmol or ng of each metal per mg total protein and is readily measured by inductively coupled plasma mass spectrometry (ICP-MS) of acid-solubilized cells. A remarkable aspect of the inorganic fingerprint relevant to this discussion is the fact that the total Mn:Fe ratio varies by over *seven orders* of magnitude when various single-celled organisms are compared with one another (**Figure 1B**). If *Escherichia coli* is taken as a typical bacterium, then Zn is as abundant as Fe, with Mn and Cu present at ≈10-fold lower concentration, and Ni and Co about 10–50 fold lower still (Outten and O'Halloran, 2001; Maret, 2010), giving a Mn:Fe ratio of ≈ 0*.*1. This fingerprint tends to characterize "Fe-centric" bacteria, like *E. coli* and the yeast *Saccharomyces cerevisiae* (Outten and O'Halloran, 2001; Rosenfeld and Culotta, 2012) with some Fe-centric species accumulating only vanishingly small amounts of Mn (Mn:Fe ratio ≤0.001) (**Figure 1B**) (Daly et al., 2004). On the other hand, for some Gram-positive bacteria, total cell-associated Mn levels are on par with that of Zn, with Fe levels correspondingly lower, resulting in a Mn:Fe ratio of ≥1 for these Mn-centric organisms (**Figure 1B**) (Daly et al., 2004; Jacobsen et al., 2011; Veyrier et al., 2011). These bacteria include the lactic acid bacteria *Streptococcus pneumoniae* and *Lactobacillus plantarum*, the pathogen *Neisseria meningitidis*, and the UV-resistant *Deinococcus radiodurans*. In *Deinococcus radiodurans,* Mn is known to play a direct role in protecting this organism from the effects of extreme γ-radiation (Daly et al., 2004). As a general rule, lactic acid bacteria tend to have less in the way of an intracellular Fe requirement relative to *E. coli*, and this may be a consequence of the unusual lifestyle of these organisms which lack a respiratory chain and yet generate millimolar hydrogen peroxide (H2O2) when grown in the presence of oxygen (Archibald and Fridovich, 1981b; Ramos-Montañez et al., 2010). This would play havoc with an Fe-centric bacterium and in fact is used by lactobacilli to kill other bacteria in the microbial community (see **Figure 2** below).

It is important to emphasize that the total metal content of an organism does not dictate the relative concentrations of *free* metal, which we define here as the fraction of total metal that is rapidly exchangeable with LMW chelates or cellular metabolites and is thus bioavailable (**Figure 1A**). This bioavailable metal tends to track with the left-to-right arrangement of the *d*-block elements in the periodic table, which is inversely related to metal competitiveness within a cellular environment. Metal competitiveness, in turn, is roughly governed by the intrinsic chelate stability, which provides a measure of the equilibrium ability of one metal to displace another metal from an enzyme active site, for example (Fraústo da Silva and Williams, 2001) (**Figure 1A**). Both Zn(II) and Cu(I) are highly competitive *d*<sup>10</sup> metals and thus will outcompete all other divalent transition metals in the first row, in particular Mn(II) and Fe(II), if left unregulated (for reviews, see Waldron and Robinson, 2009; Reyes-Caballero et al., 2011). In short, there must be a cellular overcapacity to chelate Zn(II) in order to keep this highly competitive metal in check (Outten and O'Halloran, 2001) (**Figure 1A**). In contrast, for Mn(II) (McNaughton et al., 2010; Sharma et al., 2013) and perhaps Fe(II) in *E. coli* (Imlay, 2008) a significant fraction of the total cell-associated metal is found in rapid equilibrium with a chelatable pool of LMW metabolite-metal complexes, e.g., amino acids, nucleosides, nucleotides, orthophosphate, citrate, and carbonate. This has significant implications for Mn(II) and microbial pathogenesis, as discussed below.

### **TRANSITION METAL HOMEOSTASIS AND THE FIGHT OVER METALS**

The cellular bioavailability of transition metals is governed by continuous cycles of adaptation and recovery to changes in extracellular metal availability*,* e.g., that which might occur along an infection axis. This process is termed transition metal homeostasis. Metal homeostasis systems maintain both total and bioavailable metal concentrations to maximize cell viability under the prevailing extracellular milieu. This process is orchestrated by a panel of metal sensor proteins that regulate the transcription of genes encoding metal uptake, metal efflux and metal sequestration proteins. Metal sensor proteins are typically repressors whose DNA operator-promoter binding or transcription activation activity is reversibly modulated by the binding of one or more cognate (-like) metal ions to the exclusion of all others (Giedroc and Arunkumar, 2007; Ma et al., 2009). This regulatory process has a tremendous impact on the survival and pathogenesis of microbial pathogens (Andreini et al., 2008; Botella et al., 2012). For example, Cu(I) and Zn(II) compete with native metals leading to mismetallation of metalloenzymes with more weakly bound metals and loss of function (Aguirre and Culotta, 2012; Botella et al., 2012; Cotruvo and Stubbe, 2012), while ROS and Fe cause deleterious reactions leading to oxidative damage of proteins, DNA and lipids (Imlay, 2013) (see **Figure 2**; discussed more fully below).

For bacterial pathogens, proper metallation of critical proteins also competes against host defenses that have evolved to limit or sequester these required micronutrients to quell a bacterial infection, thus creating a "fight over metals" (for a review, see Hood and Skaar, 2012). Furthermore, this fight is intermingled with ongoing global stress mediated by reactive oxygen species (ROS) (Imlay, 2013), reactive chlorine species (HOCl) (Gray et al., 2013) and/or reactive nitrogen species (RNS) (Stern et al., 2013) to which the pathogen must adapt and ultimately exploit (Hoffmann et al., 2006). The ability of the host immune system to sequester transition metals is an important aspect of nutritional immunity (Kehl-Fie and Skaar, 2010). Although long recognized for Fe limitation (Weinberg, 1974, 1975; Forbes and Gros, 2001; Flo et al., 2004; Skaar, 2010; Hammer and Skaar, 2012), it is now established that both Zn and Mn availability are actively limited by the host as well, through the extracellular action of calprotectin (CP) (Corbin et al., 2008) and perhaps other molecules. Indeed, the host strategy of limiting Mn, in particular, results in sensitization to oxidative stress (Anjem et al., 2009; Ogunniyi et al., 2010; Kehl-Fie et al., 2011) which limits the ability of Mn(II) to function as an antioxidant as discussed below.

### **MANGANESE AS AN ANTIOXIDANT MICRONUTRIENT**

Manganese as a micronutrient is critical to the viability and virulence of many Gram-positive and Gram-negative bacterial pathogens (Tseng et al., 2001; Johnston et al., 2004; Ogunniyi et al., 2010; Kehl-Fie et al., 2011; Wichgers Schreur et al., 2011; Perry et al., 2012). In these bacteria, deletion of either the manganese import system(s) or the associated Mn(II)-specific metal sensor protein compromises virulence and/or viability often through sensitizing the bacteria to various ROS, e.g., superoxide anion radical (denoted here as O2 −) or hydrogen peroxide (H2O2) (Johnston et al., 2006; Abrantes et al., 2013) (**Figure 2**). Major consequences of O2 <sup>−</sup> and H2O2 toxicity in cells is the autocatalytic production of the highly damaging hydroxyl radical, OH•, via the Fenton reaction from these partially reduced forms of O2, and the oxidative attack and dissociation of solventexposed Fe(II) atoms from enzymes harboring mononuclear Fe and 4Fe-4S cluster cofactors (**Figure 2**). Here, a solvent-exposed Fe(II) atom will allow for direct coordination of H2O2 leading to a local generation of OH• and protein oxidation and Fe(III) dissociation, essentially analogous to that which occurs in the peroxide sensor Fe-PerR (Lee and Helmann, 2006) (**Figure 3**). As such, all bacteria encode regulatory strategies to quickly respond to various ROS. In *E. coli*, low (μM) H2O2 induces the OxyR regulon

from upregulated Mn(II) import. (Note: not all processes are known to occur in all cells). The primary molecular targets of ROS in Fe-centric bacteria are Fe (*rust*-filled circles)-release from mononuclear Fe enzymes and from 4Fe-4S clusters (*pink* boxes, lower left), which must be avoided to due to the autocatalytic formation of the freely diffusible hydroxyl radical (OH•). (*pink*, upper left). Four cellular responses involving changes in Mn or Fe speciation as a result of ROS are schematized and highlighted (*bold-face*). (1) Fe sequestration: Fe(II) is scavenged by Dps (*green* triangle) to form Fe-Dps which oxidizes Fe to insoluble Fe oxide using O2 or H2O2 as an oxidant and limiting catalytic

enzymes (*yellow* ellipses; *right*) and perhaps of Dps (*lower right*) with Mn, and (4) enhances the metallation and increased activity of Mn SOD (*middle, right*). Hosts attempt to limit these responses through direct competition of the Mn(II) transporter with host-encoded calprotectin (CP; *purple* hexagon) for Mn (*upper right*) (see text for details). Increased intracellular Mn(II) will ultimately be sensed by the Mn-activated repressor (DtxR, PsaR, etc.) to repress uptake, while excess Mn(II) will be effluxed from the cell by a limited number of organisms (Rosch et al., 2009; Veyrier et al., 2011) in order to bring the Mn/Fe ratio back into balance (Veyrier et al., 2011).

which includes genes encoding a manganese import pump MntH and the DNA binding iron-scavenging protein Dps (Zheng et al., 2001), as well as catalases and peroxidases capable of reducing ROS or organic peroxides (**Figure 3**). In Gram-positive bacteria, PerR carries out essentially the same regulatory role as OxyR. Redox-cycling organic molecules (Gu and Imlay, 2011) and perhaps O2 − itself induce the SoxRS regulon, a major component of which is Mn-superoxide dismutase (SOD).

As a result of ROS stress, four major cellular adaptations result that collectively limit pro-oxidant Fe availability and upregulate Mn import to harness the antioxidant properties of Mn(II). First, increased Mn(II) availability allows for increased metallation of Mn-SOD which efficiently catalyzes the dismutation of the superoxide anion radical to H2O2 and O2, the former of which is cleared by catalase and related peroxidases which are also induced under these conditions (May and Dennis, 1989; Wintjens et al., 2004) (**Figure 2**). However, as early as 1981, a series of studies established that LMW manganese-metabolite complexes from extracts of *Lactobacillius plantarum* (see **Figure 1B**) were capable of scavenging superoxide from solution (Archibald and Fridovich, 1981a,b) (**Figure 2**). A number of diverse bacterial species (Inaoka et al., 1999; Tseng et al., 2001; Al-Maghrebi et al., 2002; Daly et al., 2010; Kehl-Fie et al., 2011) and the baker's yeast *Saccharomyces cerevisiae*(Chang and Kosman, 1989; McNaughton **FIGURE 3 | Superoxide disproportionation by LMW-Mn complexes (Barnese et al., 2012). (A)** Plausible noncatalytic mechanism of the reaction of O2 <sup>−</sup> with [Mn(II)-P2O7] <sup>2</sup><sup>−</sup> and Mn(II)-citrate. L, pyrophosphate or citrate. **(B)** Proposed catalytic mechanism of the reaction of O2 − with [Mn(II)-HPO4] and [Mn(II)-HCO3] + leading to the catalytic disproportionation of O2

<sup>−</sup>. L, phosphate or carbonate; Anion*n*<sup>−</sup> represents an additional bound anion to the form the intermediate. **(C)** Overall rate constants for the non-catalytic and catalytic disproportionation of O2 −, the latter of which incorporates the MnOO+ dependence of *k*5. Rate law simulations reveal that 91μM MnHPO4 (165μM Mn(II), 5 mM phosphate), and 25μM MnHCO+ <sup>3</sup> (formed by 36μM Mn(II) and 5 mM carbonate) gives rise to a steady-state [O2 −] (3μM) identical to 1μM CuZn-SOD following a 25μM burst of superoxide (Barnese et al., 2012). Thus, intracellular Mn(II) in the ≈ 100μM range is expected to be sufficient to resist the effects of superoxide stress, as found previously in yeast; these studies also physically document the presence of MnHPO4 species in whole cells (McNaughton et al., 2010). The same may well be true for manganese-centric bacterial pathogens vs. iron-centric *E. coli* (Aguirre et al., 2013; Sharma et al., 2013) (see **Figure 1B**). **(D)** Chemical structures of "layered" Mn-HPO4, where the *gray* box encompasses the next layer in the crystal lattice (*left*) (Krishnamohan Sharma et al., 2003) and a calculated model of frozen neutral [Mn(HCO3)2] from ENDOR studies (*right*) (Potapov and Goldfarb, 2008).

et al., 2010; Reddi and Culotta, 2011) are now known to posses this activity as well, although in most cases it is supplemented by SOD enzyme-catalyzed superoxide dismutation, with the prominent exceptions of *L. plantarum* and *Neisseria gonorrhoeae* (Tseng et al., 2001).

Simple Mn-phosphate (Pi) and Mn-carbonate complexes are efficient catalysts of superoxide disproportionation and the chemical mechanism of this reaction has recently been investigated in detail (Barnese et al., 2008, 2012) (**Figure 3**). This catalysis occurs at physiologically relevant rates and metabolite concentrations and may well-explain studies that connect oxidative stress resistance to phosphate accumulation and changes in phosphate metabolism (Tseng et al., 2001; Jensen et al., 2003; McNaughton et al., 2010; Rosenfeld et al., 2010; Wu et al., 2010). Additionally, manganese carbonate complexes have been shown to catalyze the decomposition of H2O2 (**Figure 2**) suggesting that other small molecule Mn-complexes can potentially function downstream of superoxide, although this reaction has not been thoroughly investigated (Stadtman et al., 1990; Liochev and Fridovich, 2004). Recent findings in *S. aureus* support the presence of manganesedependent, SOD-*independent* mechanisms to effectively scavenge superoxide (Kehl-Fie et al., 2011).

In addition to the chemical clearance of ROS mediated by LMW-Mn complexes, cofactor substitution (see **Figure 2**) of Fe(II) for Mn(II) in selected mononuclear iron enzymes has recently been proposed to protect these enzymes from redox chemistry at the Fe active site that accompanies H2O2 stress (Imlay, 2013) (**Figure 2**). Indeed, many mononuclear Fe enzymes are reversibly inactivated by H2O2, and continued exposure leads to irreversible inactivation (Sobota and Imlay, 2011; Anjem and Imlay, 2012). This Fe(II)-for-Mn(II) substitution is facilitated by the generally weak binding (rapid dissociation) of these two metals to enzymes (see **Figure 1A**) and is predicted to function well in enzymes that employ Fe(II) in Lewis acid catalysis, given the similar coordination preferences of Fe(II) and Mn(II); however, this process is projected to fail when the Fe(II) atom needs to undergo a change in oxidation state, given the very different redox potentials of these two metals (Cotruvo and Stubbe, 2012). In any case, Mn is known to protect these enzymes from inactivation *in vitro* and *in vivo* where both manganese import and iron sequestration were required for protection (Anjem and Imlay, 2012) (**Figure 2**). These studies reveal that *E. coli* is capable of shifting from a metabolism based on Fe(II) to one based on Mn(II) in order to protect key enzymes from inactivation by ROS. Another example of this type of cofactor replacement is the iron sequestration protein, Dps, in *Kineococcus radiotolerans* (**Figure 2**). Dps is a binuclear Fe-containing ferroxidase that binds Fe and precipitates iron oxide inside of the Dps dodecamer; however, the *K. radiotolerans* enzyme is also active as a mixed metal Mn-Fe enzyme, further evidence for the protective role of Mn(II) via cofactor substitution in the oxidative stress response (Ardini et al., 2013).

Thus, Mn(II) functions as an antioxidant through a combination of enzymatic degradation of oxidants by Mn-SOD and other Mn(II)-containing enzymes, nonenzymatic degradation of oxidants by LMW-Mn complexes, and metalloenzyme cofactor substitution to prevent Fe-induced peroxide chemistry and subsequent enzyme inactivation (**Figure 2**). The extent to which each process contributes likely varies from organism to organism and will be dependent on the prevailing microenvironment. However, a metabolism capable of utilizing Mn(II) and not absolutely dependent on Fe may well-represent a general strategy that nature has evolved to develop robust viability in the presence of significant or chronic ROS. The causative agent of Lyme disease, *Borrelia burgdorferi*, which completely lacks an Fe requirement and as a result is characterized by a very high Mn:Fe ratio (**Figure 1B**), may represent an extreme example of this evolutionary adaptation that protects the organism from host-mediated ROS (Posey and Gherardini, 2000; Aguirre et al., 2013).

### **STRUCTURAL STUDIES OF BACTERIAL MANGANESE IMPORT SYSTEMS**

The ability of a bacterial pathogen to obtain sufficient Mn(II) is critically important for pathogenesis and as such, Mn(II) dependent metal sensor proteins control the expression of operons that encode additional virulence factors unrelated to the acquisition of Mn(II) (Gold et al., 2001; Johnston et al., 2006; Rolerson et al., 2006; Hendriksen et al., 2009). This suggests that Mn(II) limitation may well be a generic signal that poises the invading pathogen to quickly adapt to a wide range of host immune defenses. Unlike the case for Fe siderophores, there is no known LMW, high affinity chelator that is secreted by bacteria to scavenge Mn from the environment. As a result, the capture and transport of manganese into the cell is facilitated directly by manganese import systems, which include MntH, a NRAMP1 (natural resistance-associated macrophage protein 1) family transporter and the Mn/Fe/Zn-specific cluster A-I ABC (ATP-Binding Cassette) transporters (Dintilhac and Calverys, 1997; Papp-Wallace and Maguire, 2006; Berntsson et al., 2010).

Although structural studies of bacterial NRAMP1 Mn/Fetransporters are limited, some molecular-level insights are available from extensive modeling studies for this large family of proteins (Cellier, 2012). Limited structural information is available for intact multisubunit cluster A-I ABC transporters as well, although the crystallographic structures of the distantly related bacterial cobalamin transporter BtuC2D2F (Locher et al., 2002; Korkhov et al., 2012) and the molybdate transporter (ModB2C2A) are known to modest resolution (Hollenstein et al., 2007). Significant structural data are, however, available for component metal-binding subunits of cluster A-I ABC transporters, termed the solute-binding proteins (SBPs) (**Figure 4A**). These include a number of Mn- and Zn-specific SBPs (Lawrence et al., 1998; Lee et al., 1999, 2002; Banerjee et al., 2003; Rukhman et al., 2005; Chandra et al., 2007; Li and Jogl, 2007; McDevitt et al., 2011; Zheng et al., 2011; Gribenko et al., 2013). It is generally believed that the SBP defines the metal specificity of transport, although precisely how this is accomplished is currently unknown. In addition, Zn is generally a poor substrate and in some cases, a competitive inhibitor, of Mn-specific ABC transporters (DeWitt et al., 2007) despite forming a very similar coordination complex to that of Mn(II) (see below).

The first of the cluster A-I SBPs (Berntsson et al., 2010) to be structurally characterized was PsaA from *Streptococcus pneumoniae* (Lawrence et al., 1998), solved as the Zn(II)-complex to 2.0 Å resolution, and lacking the N-terminal LXXC motif required to anchor PsaA to the lipid membrane. We use the recently determined structure of Mn(II)-bound *S. aureus* MntC to illustrate the fold of this subfamily of SBPs, described as a "Venus fly trap" containing two homologous mixed (βα*)*<sup>4</sup> sandwich domains linked via a ≈30 amino acid helix that resembles a backbone brace for this two-domain molecule (**Figure 4B**). The metal binding site is located in a deep cleft between the two domains of MntC, and metal ligands are contributed by both domains in roughly homologous positions (H50, H123 in the N-terminal domain; E189, D264 in the C-terminal domain). The structure of the apostate of MntC is unknown, but structural studies of ligand-free *Treponema* TroA (Lee et al., 2002) and the SBP specific for vitamin B12 (BtuF) reveal essentially closed, metal bound-like structures, with BtuF indicative of a slightly more open and conformationally dynamic structure that collapses around the Co(II)-ligand complex (Karpowich et al., 2003). The Mn(II) in MntC is bound to four protein-derived ligands in what is best described as a pentacoordinate distorted trigonal bipyramidal coordination geometry (**Figure 4C**) that is completely shielded from solvent (**Figure 4D**). The structure of the Mn(II)-bound form of PsaA has also recently been solved and compares very favorably with that of Mn-MntC (**Figures 4E,F**) (McDevitt et al., 2011). While the overall structure is virtually identical with that of Zn(II)-PsaA complex determined earlier (see **Figure 4E** for an overlay) (Lawrence et al., 1998), there are subtle differences in the metal coordination site, with the Zn(II) complex tending toward distorted tetrahedral as a result of monodentate coordination by each of the two carboxylate ligands (E205, D280) (**Figure 4F**). These trends in metal coordination geometry of Mn(II) vs. Zn(II) are consistent with expectations (Dudev and Lim, 2013), although the resolution of the structures precludes a stronger conclusion on this point. It is important to point out that some Zn-specific SBPs, e.g.,*E. coli* ZnuA, lack the bidentate Asp ligand of Mn-specific SBPs, e.g., D280 in **Figure 4F**, and recruit a solvent molecule to complete the tetrahedral coordination complex using the other three Mn-SBP ligands (Chandra et al., 2007). This may well have strong implications for metal specificity and the forward rate of cognate or native metal transport across the membrane. A recent paper provides new insights on molecular basis of functional discrimination of cognate Mn(II) vs. non-cognate Zn(II) by *S. pneumoniae* PsaA (Couñago et al., 2013).

The structures of other Mn(II)-specific cluster A-I SBPs have been reported including those from distant bacterial phyla such as cyanobacteria (Rukhman et al., 2005) and the spirochaete *Treponema*, the causative agent of syphilis (Lee et al., 1999). Each structure shares the same MntC/PsaA fold revealing that the (βα*)*<sup>4</sup> sandwich two-domain structure is evolutionarily conserved and is utilized for transition metal transport in both Gram-negative and Gram-positive bacteria and in nonpathogenic and pathogenic bacteria alike. Given the ubiquity of these proteins on the "outside" of Gram-positive organisms, they have been targeted for use in commercial vaccines. For example, improved serotype coverage and clearance of *Streptococcus pneumoniae* has been obtained with a vaccine containing adjuvant-conjugated PsaA, PiuA and PiaA, the latter two of which are involved in Fe-uptake in this organism (Brown et al., 2001; Whaley et al., 2010).

Since Mn(II) can only enter the cytoplasm efficiently through Mn(II)-specific transporters, elucidation of the affinity of each for Mn(II) vs. noncognate Zn(II) and the rates at which Mn(II) is transported across the plasma membrane takes on added significance when considered in the context of the discovery of

**FIGURE 4 | Structural studies of Mn-specific solute binding proteins (SBPs) from** *S. aureus* **MntC (panels B-D) (Gribenko et al., 2013) and** *S. pneumoniae* **PsaA (panels E,F) (Lawrence et al., 1998; McDevitt et al., 2011). (A)** Cartoon representation of a canonical ABC transporter, with the subunits and the direction of transport labeled. **(B)** Ribbon representation of the structure of Mn-bound MntC (pdb code 4K3V), with the four Mn(II)-coordinating ligands highlighted and shown in *stick*. This orientation is similar to that implied by the cartoon in panel **(A)**. **(C)** First coordination sphere of the Mn(II) complex in MntC; this orientation differs from that in panel B in order to highlight the distorted trigonal bipyramidal Mn(II) coordination geometry. E189 tends toward monodentate ligation, while D264 is bidentate. **(D)** Surface representation of MntC in the same orientation as in

panel B revealing that bound Mn(II) is buried from solvent. **(E)** Ribbon representation of a global superposition of PsaA in the Mn(II)-bound (3ZTT) and Zn(II)-bound (1PSZ) states, with metal ligands shown in *stick*. This orientation is from the docking surface that would interact with the transmembrane subunits (see panel **A**). **(F)** Coordination complexes of PsaA bound to Mn(II) (*top*) and Zn(II) (*bottom*) revealing that the same four protein-derived ligands are used to coordinate both cognate and noncognate metals. The Mn(II) complex is very similar to that observed for MntC, with E205 tending toward monodentate ligation (*r*Oe2••Mn = 2*.*55 Å). In contrast, the Zn(II) complex tends toward tetrahedral coordination with only one oxygen atom of each carboxylate group sufficiently close to directly coordinate the Zn(II). The overlay of these two chelates is shown in panel **(E)**.

calprotectin (see below). This is also true from the perspective of fundamental inorganic chemistry since Mn(II) complexes will tend to be far less thermodynamically stable than "isostructural" Zn(II) complexes (**Figure 1A**) (Waldron and Robinson, 2009). Metal transport studies have been carried out on bacterial NRAMP1 homologs in *S. typhimurium* and *E. coli* and generally show half maximal transport rates at 0.1 to 1μM total Mn(II) depending on the transporter (Kehres et al., 2000). For ABC transporters, the concentration of any metal that gives maximal rates of transport has not yet been measured to our knowledge; on the other hand, the Mn(II) and Zn(II) affinities of the component SBPs have been determined using chelator competition assays or via direct titration by isothermal titration calorimetry (ITC). We compile these values here (**Table 1**) with the caveat that in an ITC experiment the affinity (*K*<sup>d</sup> or *K*a) is often too tight to measure at the protein concentrations required to make the measurement (**Figure 5**), despite the ability to obtain a reliable measure of the stoichiometry and the heat of binding (*-H*cal). As can be seen, determined *K*<sup>d</sup> Mn values range from the low nM to several hundred nM, with some indication that Zn(II) may bind more weakly than Mn(II) (Desrosiers et al., 2007; McDevitt et al., 2011; Zheng et al., 2011; Gribenko et al., 2013). We note that the *K*<sup>d</sup> Mn obtained for *S. aureus* MntC of 4.0 ± 0.3 nM (50 mM citrate, 150 mM NaCl, pH 6.0, 20◦C) is robust since this value was extracted from a nonstoichiometric binding isotherm acquired in the presence of 50 mM citrate as a Mn(II) competitor chelator (Gribenko et al., 2013). It will be interesting to learn how *K*<sup>d</sup> Mn corresponds to *K*<sup>m</sup> for transport, since rapid dissociation of Mn(II) from the SBP into the transport cavity (Pinkett et al., 2007) upon productive association with the transmembrane domain of the transporter (see **Figure 4A**) could facilitate rapid movement of Mn(II) across the membrane *(Gribenko et al., 2013).*



*a20 mM NaH2PO4, pH 6.5, 25* ◦*C, 4.5–20*μ*M PsaA (McDevitt et al., 2011).*

*b100 mM sodium acetate, pH 6.5, 20* ◦*C, 40*μ*M TroA (Desrosiers et al., 2007).*

*c100 mM sodium acetate, pH 6.5, 20* ◦*C, 42*μ*M YfeA (Desrosiers et al., 2010).*

*<sup>d</sup> 20 mM sodium acetate, pH 6.5 at 25* ◦*C, 30 or 90*μ*M TroA (Zheng et al., 2011). e50 mM citrate, 150 mM NaCl, 10% glycerol, pH 6.0, 25* ◦*C, 36–43*μ*M MntC*

*<sup>f</sup> Mn parameters measured in 75 mM Hepes, 100 mM NaCl, 0.2 mM Ca(II), pH 7.5, 25* ◦*C. Values for metal site 1 (S1; His6) and metal site 2 (S2; His3Asp) are shown as Kd Mn1, Kd Mn2, Kd Zn1, and Kd Zn2 (Figure 5).*

*gZn parameters measured in 75 mM Hepes, 100 mM NaCl, 1 mM Ca(II), pH 7.5, room temperature.*

*hMeasured in 20 mM Tris, 100 mM NaCl, 22.5* μ*M Ca(II), 5 mM* β*mercaptoethanol, pH 7.5, 30* ◦*C, 7.5*μ*M CP (Kehl-Fie et al., 2011). A -S1 mutant CP does not bind Mn tightly in contrast to the -S2 mutant, revealing the His6 S1 site is the high affinity site for Mn (Damo et al., 2013).*

not fully reflected in the Mn(II)-binding thermodynamics (**Figure 5**).

#### **HOST SEQUESTRATION OF TRANSITION METAL IONS**

The mammalian host is a reservoir that is potentially rich in essential nutrients, including transition metals that must be acquired by bacterial pathogens (Versieck, 1985). In many cases, the host limits critical micronutrients such as iron (Weinberg, 1974) through both intracellular and extracellular complexation in an effort to withhold these metals from the invading pathogen (Weinberg, 1975; Hood and Skaar, 2012). For example, lipocalin 2 (Lcn2; siderochalin) binds Fe(III)-enterochelin, carboxymycobactin and bacillibactin complexes in direct competition with the bacterium (*E. coli*, *Mycobacterium tuberculosis,* or *Bacillus anthracis* in this case) that secretes these siderophores to capture bioavailable Fe from the host (Flo et al., 2004; Holmes et al., 2005; Sia et al., 2013). This establishes a competition between host and pathogen for the same metals (Bachman et al., 2009), and consistent with this model, Lcn2 expression and secretion is greatly elevated at sites of infection (Flo et al., 2004), and knockout mice lacking these and other host defenses are more susceptible to bacterial infection (Flo et al., 2004; Corbin et al., 2008; Hammer and Skaar, 2012).

Accumulating evidence assembled over the last several years reveals that a similar competitive strategy is used by the host to restrict the availability of both zinc and manganese in response to bacterial infection (Corbin et al., 2008; Kehl-Fie and Skaar, 2010; Kehl-Fie et al., 2011). This occurs in one of several ways. Macrophages and neutrophils are known to engulf intracellular pathogens in order to isolate them into a phagosomal compartment from which essential metals Mn and Fe are depleted by efflux, while Cu is concentrated (Wagner et al., 2005; White et al., 2009; Osman et al., 2010; Achard et al., 2012; Botella et al., 2012). Natural resistance-associated macrophage protein 1 (NRAMP1) (Cellier et al., 2007) and related H+-coupled transporters are known to efflux Mn(II) and Fe(II) from intracellular compartments of macrophages (Forbes and Gros, 2001), and knockout mice lacking NRAMP1 are susceptible to more virulent bacterial infections relative to wild-type mice (Skamene et al., 1982).

A number of S100 family proteins are now known to function extracellularly to chelate Mn(II) and Zn(II) to sequester these metals from the invading bacterium. For example, the S100A7 homodimer limits growth and invasion at epithelial surfaces through chelation of Zn(II) (Gläser et al., 2005), although the mechanistic details require further study. More recently it has been established that the heterotetrameric S100A8/S100A9 complex, also known as calprotectin (CP), binds both Mn(II) and Zn(II) (**Figure 6**) and is a major neutrophil-derived protein found in *Staphylococcus aureus*-induced tissue abscesses (Corbin et al., 2008). Laser ablation (LA)-ICP-MS was used to demonstrate that Mn(II) and Zn(II) were undetectable in abscesses relative to the surrounding uninfected tissue in a process dependent on host-encoded S100A8 and S100A9; furthermore, this chelation strategy is synergistic with neutrophil-mediated processes that sensitize these bacteria to superoxide stress by diminishing the effectiveness of Mn-SOD-dependent and independent antioxidant mechanisms (Kehl-Fie et al., 2011). These data support a model in which CP cripples bacterial defenses to both macrophage and neutrophil-mediated killing, and limits proliferation in tissue abscesses through Mn(II) chelation. More recent findings suggest that this general chelation strategy is likely operative in other tissues but is CP-independent, revealing that calprotectin may not be the only route that the host can use to limit Mn(II) from invading bacteria (Kehl-Fie et al., 2013). In addition, the degree to which Zn(II) chelation, relative to Mn(II), by CP limits bacterial growth is not fully understood, although a recent unbiased mutant screen carried out with the Gram-negative opportunistic respiratory pathogen *Acinetobacter baumannii* specifically identified components of the zinc acquisition and metabolism systems in that organism when challenged with CP (Hood et al., 2012; Moore et al., 2013).

#### **CALPROTECTIN: STRUCTURAL, METAL BINDING AND FUNCTIONAL PROPERTIES**

Recent studies reveal that CP has widespread antimicrobial activity against many Gram-positive and Gram–negative human pathogens grown in liquid culture, albeit to widely varying degrees, the explanation for which remains incompletely understood (Damo et al., 2013). We have recently shown that CP inhibits the growth of both wild-type and encapsulated strains of *Streptococcus pneumoniae* D39, extending the range of this broad spectrum antimicrobial activity (Lisher et al., unpublished). One simple explanation is that more resistant bacteria express Mn(II) uptake systems that possess a *higher* affinity than CP for Mn(II) and thus will compete more effectively with CP for extracellular

**FIGURE 5 | Simulated binding curves for ITC thermograms using the reported thermodynamics and injection volumes of the MnCl2 binding to the S***-***2 mutant of CP (Damo et al., 2013).** The simulated curve that corresponds to the reported *Kd* of 5.8 nM (at 10μM total CP heterodimer), *n* = 1*.*0 (*red* vertical arrow) and *-<sup>H</sup>*cal <sup>=</sup> <sup>26</sup>*.*2 kcal mol−<sup>1</sup> (*red* horizontal arrow) was converted to *<sup>K</sup>*<sup>a</sup> (1*.*<sup>7</sup> <sup>×</sup> <sup>10</sup>8M−1) to create the isotherm labeled 1•*K*<sup>a</sup> (*black* squares). Other simulated curves are shown for *K*<sup>a</sup> for 10– (10 ∗ *K*a) and 100-fold (100 ∗ *K*a) higher *K*a, and 10– (0.1 ∗ *K*a), 100– (0.01 ∗ *K*a) and 1000-fold (0.001 ∗ *K*a) lower *<sup>K</sup>*a. These simulations reveal that binding affinities greater than <sup>≈</sup> 108 <sup>M</sup>−<sup>1</sup> (*K*<sup>d</sup> <sup>≤</sup> 10 nM) can not be reliably measured under these conditions, and are indicative of essentially stoichiometric binding as evidenced by a paucity of data points in the transition region. At 5-10-fold-higher concentration of protein, which is more typical of the SBP-Mn(II) measurements in the literature (see **Table 1**), a *K*<sup>a</sup> *>* 107 M−<sup>1</sup> (*K*<sup>d</sup> *<* 100 nM) will not be reliably measured unless a chelator competitor, e.g., citrate for Mn(II), is used to measure *K*<sup>a</sup> Mn (Grossoehme and Giedroc, 2009; Gribenko et al., 2013).

two heterodimers is marked. The two intersubunit Mn ions per heterodimer

chains are shaded in *red* and *salmon*. The tetramer interface between the

in stick representation are labeled with residue number; one of the two S2 sites showed partial occupancy in the structure.

Mn(II). This requires knowledge of the structure and Mn(II) and Zn(II) binding affinities of CP. Initial studies established that a single S100A8/S100A9 heterodimer is capable of binding two molar equivalents of transition metal, designated S1 and S2; however, while Zn(II) could fill both sites, Mn(II) could fill only one with high affinity (S1) (Kehl-Fie et al., 2011). The same is true in the context of the heterotetramer (S100A82- S100A92) which is well-modeled in metal binding experiments as two functionally independent heterodimers (**Figure 6**). Estimates of the metal binding affinity obtained in the presence of calcium from ITC show essentially stoichiometric binding at pH 7.5 with reported values of *K*<sup>d</sup> Zn1 <sup>=</sup> <sup>1</sup>*.*4 nM and *<sup>K</sup>*<sup>d</sup> Zn2 <sup>=</sup> <sup>5</sup>*.*6 nM (**Table 1**). For Mn(II), reported values are *K*<sup>d</sup> Mn1 <sup>=</sup> <sup>1</sup>*.*3 nM and a *K*<sup>d</sup> Mn2 <sup>=</sup> <sup>3</sup>*.*7μM (**Table 1**). For reasons discussed above, nM values measured by ITC may well-reflect lower limits of *K*<sup>a</sup> and correspondingly upper limits on *K*<sup>d</sup> (see **Figure 5**). Indeed, subsequent metal competition experiments that employed a fluorescent sensor ZP4 (*K*Zn = 0*.*65 nM; measurable range in *K*Zn of 0.01–10 nM) as a competitor ligand for Zn(II) revealed *K*<sup>d</sup> Zn1 <sup>=</sup> 0*.*13 nM and *K*<sup>d</sup> Zn2 <sup>=</sup> 185 nM (–Ca) and *<sup>K</sup>*<sup>d</sup> Zn1 <sup>≤</sup> <sup>0</sup>*.*01 nM and *K*d Zn2 <sup>≤</sup> <sup>0</sup>*.*24 nM (+Ca) (Brophy et al., 2012), both significantly tighter than estimates from ITC. Room-temperature Mn(II) EPR titrations reveal *K*<sup>d</sup> Mn1 of 4.9μM and *K*<sup>d</sup> Mn2 <sup>=</sup> <sup>1</sup>*.*0 mM in the absence of calcium (Hayden et al., 2013) which shift to ≈200 nM and 21μM in the presence of calcium (**Table 1**). These studies taken collectively reveal that calcium binding "switches on" CP to become a high affinity Zn/Mn binding protein with the major antimicrobial form of CP likely the mixed Mn (S1)/Zn (S2) heterotetramer (Hayden et al., 2013).

Crystallographic and mutagenesis experiments establish an unprecedented hexahistidine-coordinated (His6) Mn(II) site conforming to octahedral coordination geometry (Hayden et al., 2013) as the high affinity (S1) Mn(II) site, with the Zn(II) site (S2) adopting a tetrahedral His3-Asp complex (Damo et al., 2013) (**Figure 6B**). Two of the six histidines in the His6 site are derived from the conserved His103 and His105 in the C-terminal tail of S100A9 both of which are essential for antimicrobial function (Damo et al., 2013). The close proximity of these tail ligands to one of the Ca(II) binding sites immediately explains the strong calcium-dependent increase in Mn(II) binding affinity (Brophy et al., 2012). The broad-spectrum antimicrobial activity CP is largely dependent on the integrity of this S1 His6 site rather than the S2 Zn(II) site, thus likely connecting Mn(II) sequestration to the biological activity of CP. Although CP may have a lower affinity for Mn(II) than bacterially encoded SBPs (**Table 1**), the degree to which Mn(II) is coordinated by CP vs. SBPs or other Mn(II) import transporters is of course dictated by mass action which is set by the relative effective concentrations of each "chelator" in the milieu. Subsequent findings from Skaar and coworkers are consistent with this direct competition model, in that CP-imposed Mn(II) starvation increases the expression of a constitutively expressed NRAMP1-like manganese transporter, MntH, as well as the ABC family importer, MntABC, of which MntC is the Mn(II)-binding subunit (Kehl-Fie et al., 2013) (see **Figure 4B**). Both uptake systems are required to fully resist the effects of CP-dependent metal limitation since the IC50 for CP of an *mntC/mntH* stain was ≈50% lower than a *S. aureus* wild-type strain; additionally, both importers were required for resistance to superoxide stress as a result of increased SOD activity, and the ability of *S. aureus* to establish a systemic infection (Kehl-Fie et al., 2013). This "tug-of war" used to tip the balance of mass action in favor of the pathogen relative to Mn(II) acquisition may well be a general one (Champion et al., 2011).

#### **THE IMPACT OF OTHER TRANSITION METALS ON MANGANESE ACQUISITION AND HOMEOSTASIS**

In addition to competition from host proteins of the innate immune response, there is some evidence that Zn(II) and Fe(II) can influence Mn(II) acquisition and intracellular Mn(II) dependent metalloregulation of transcription. For example, Zn(II) has been shown to inhibit Mn(II) uptake by binding irreversibly to *S. pneumoniae* PsaA and effectively blocks Mn(II) transport into the cytoplasm (McDevitt et al., 2011). This induces an intracellular Mn(II) deficiency leading to upregulation of the expression of the entire PsaR regulon, as part of an effort to scavenge Mn(II) from the environment (Kloosterman et al., 2008; Jacobsen et al., 2011). In addition, Zn(II) is capable of binding to the Mn(II) sensor PsaR, converting PsaR into a poorly active repressor (Lisher et al., 2013) thereby minimizing transcriptional repression of *psaBCA* under these conditions (Jacobsen et al., 2011). Both of these effects can be reversed by the addition of Mn(II) to the growth media, suggesting the possibility that the pneumococcus may use one or both mechanisms to maintain a favorable intracellular Zn(II):Mn(II) ratio under conditions of high extracellular zinc toxicity that might occur in the lung, for example (McDevitt et al., 2011). Like PsaR (Lisher et al., 2013), other structurally related Mn(II)-specific metalloregulatory proteins*,* e.g., *B. subtilis* MntR, also bind Zn(II) with significantly higher affinity (≥100-fold), a finding consistent with the Irving-Williams series (**Figure 1A**) (Golynskiy et al., 2006; Maret, 2010; Ma et al., 2012), yet the Zn(II)-bound repressor binds much more weakly to the DNA operator than the cognate Mn(II) bound repressor (Lieser et al., 2003). The degree to which this competition in the cell is a general strategy to modulate Mn(II) homeostasis under conditions of zinc toxicity is unknown.

Intracellular crosstalk between Fe(II) and Mn(II) homeostasis systems may well be more relevant to bacterial cell physiology and pathogenesis than is Zn(II)-Mn(II) crosstalk. These two metals lie at the same weakly competitive end of the Irving-Williams series and the Fe:Mn ratio might be considered a reporter of microbial lifestyle, capable of altering the altering the resistance of an organism to ROS (see **Figure 1**). As discussed above, a LMW-Mn(II) pool may well be present is most bacterial cells, albeit to differing degrees (Sharma et al., 2013), and some cells contain a chelatable pool of several hundred micromolar Fe(II) that is detectable by EPR spectroscopy of whole cells (Pericone et al., 2003). Thus, changes in the Mn:Fe ratio by upregulation of the Mn(II) acquisition system (**Figure 2**), or by crippling Fe(II) uptake repression in a *fur* mutant, for example, might be expected to change the metal specificity of an Fe(II)- or Mn(II)-specific metal-sensing repressor (Guedon and Helmann, 2003; Ma et al., 2011, 2012) and selected metalloenzymes (Whittaker, 2003; Anjem et al., 2009). This kind of Mn(II)-Fe(II) regulatory crosstalk is exemplified by recent work in *B. subtilis* on Mn(II)-MntR and two related Fur family members, the peroxide sensing, Fe(II)-binding PerR (see **Figure 2**) and the Fe(II)-sensing repressor Fur (Ma et al., 2011, 2012). It was reported that selected mutations of PerR introduced into a nonfunctional metal site (found in other Fur family proteins) close to the primary Fe(II) binding site altered the structure of PerR such that Mn(II) bound more tightly then Fe(II). Strains harboring these mutations were correspondingly more sensitive to peroxide stress since Mn(II)-PerR thus formed is unable to perform peroxide-catalyzed autooxidation which drives transcriptional derepression of the *perR* regulon. Remarkably, Fe-PerR-dependent H2O2-sensing was restored in this mutant in a *fur* mutant background, presumably allowing Fe(II) levels to rise to a level sufficient level to fill the mutant PerR regulatory site with cognate Fe(II) (Ma et al., 2011).

A second example concerns *B. subtilis* Fur itself (Ma et al., 2012). In this study, it was shown that cognate Fe(II) and noncognate metals Mn(II) and Zn(II) are equally effective in activating Fur to bind to its DNA operator *in vitro*; however, Fur is Fe(II) specific *in vivo*. Remarkably, this Fe(II)-specificity is lost in a *perR* mutant strain. Here, the combined impact of increasing Fur concentrations and intracellular Mn(II) levels relative to Fe(II), leads to conditions where Fur binds Mn(II), which in turn, leads to inappropriate Mn(II)-mediated repression of the *fur* regulon, including genes responsible for Fe(II) uptake. Thus, PerR may directly impact Fe(II) homeostasis by modulating Fur levels in response to a change in the Mn:Fe ratio, i.e., that which might occur under conditions of high Mn(II) and Fe(II)-deplete conditions (see **Figure 2**).

Although the degree to which Fe(II)-Mn(II) crosstalk influences bacterial pathogenesis is not firmly established, a number of recent studies suggest that maintenance of an optimal Mn(II):Fe(II) ratio can impact the virulence of pathogenic bacteria. For example, the ability of *S. pneumoniae* to maintain a high Mn(II):Fe(II) ratio (Jacobsen et al., 2011) (**Figure 1B**) may be relevant to resistance to oxidative stress important for pathogenesis of this organism (Ong et al., 2013). In *Yersinia pestis*, the manganese import systems Yfe and MntH are regulated by Fe-Fur and loss of these systems leads to reduced virulence in sepsis models (Perry et al., 2012). In *Neisseria meningitidis,* a novel efflux protein, MntX, that maintains optimal Mn(II):Fe(II) ratios under conditions of low iron is also critical to virulence in sepsis models (Veyrier et al., 2011); this ensures that Fe(II) will remain bioavailable following conditions of high Mn(II) import in response to oxidative stress (see **Figure 2**). Interestingly, *Streptococcus pneumoniae*, like *Neisseria meningitidis*, also encodes a Mn(II) effluxer, MntE, that is required for virulence (Rosch et al., 2009). It will be interesting to see if the presence of a dedicated manganese effluxer represents a general strategy of providing a "release valve" to avert the effects of high intracellular Mn(II) particularly in Mn-centric bacterial pathogens (see **Figure 1B**).

#### **CONCLUDING REMARKS**

Recent studies of bacterial transition metal physiology and crosstalk places manganese acquisition by human microbial pathogens on center stage of the host-pathogen "arms race" (Botella et al., 2012). Mn(II) functions to metallate key enzymes, notably Mn-SOD that are responsible for the long-appreciated antioxidant properties of this metal. Recent insights from the application of sophisticated spectroscopies capable of probing Mn(II)-speciation in whole cells (Sharma et al., 2013), coupled with chemical investigations, provide strong support for the proposal that specific LMW-Mn complexes are catalytically competent and functionally important in clearing superoxide from cells, in a way that supplements SOD-dependent mechanisms (Barnese et al., 2012). The discovery of host immune proteins that limit biologically available Mn(II) for both intracellular and extracellular pathogens in an effort to cripple the resistance of invading pathogens to ROS establishes this as a general strategy used by the host to curtail bacterial infections (Corbin et al., 2008; Damo et al., 2013; Kehl-Fie et al., 2013).

Although structural and biophysical studies provide general support for a simple competition model in which the extracellular chelator calprotectin and Mn(II)-specific uptake systems compete for the same metal on the basis of their respective affinities, there is much more to be learned about this process. This includes elucidation of the rates and rate-limiting steps of Mn(II) transport, and structural studies of intact bacterial ABC transporters positioned at the "front line" of Mn(II) acquisition. This is particularly interesting since Mn(II) is generally handicapped relative to other divalent metal ions, notably Zn(II), in chelate stability, and as a result, other factors including formation of kinetically trapped Mn(II) metalloenzyme complexes in the cell (Whittaker, 2003; Tottey et al., 2008), may well be operative. In this context, it is interesting to note the *Streptococcus pneumoniae* expresses polyhistidine triad proteins (Pht) proteins attached on the cell surface known to bind zinc (Riboldi-Tunnicliffe et al., 2005) and thus could be used to scavenge Zn(II) from the host milieu under conditions of zinc limitation (Reyes-Caballero et al., 2010; Shafeeq et al., 2011; Plumptre et al., 2012, 2013). A bonus role for these proteins is that they could be used sequester Zn(II) and thereby reduce competition at the manganese importer PsaBCA, allowing pneumococcus to efficiently obtain Mn(II) which is likely bioavailable at vanishingly small quantities relative to Zn(II) (Shafeeq et al., 2013).

Further insights into molecular mechanisms of host nutritional immunity against bacterial pathogens will continue to rely on concerted and collaborative efforts of microbiologists, coordination chemists and structural biologists in an effort to win the "tug-of-war" over transition metals at the host-pathogen interface through the development of intervention strategies based on metals in biology of infectious disease.

#### **AUTHOR CONTRIBUTIONS**

John P. Lisher and David P. Giedroc wrote the manuscript.

#### **ACKNOWLEDGMENTS**

Work in the authors laboratory on bacterial transition metal homeostasis is supported by a grant from the US National Institutes of Health (R01 GM042569 to David P. Giedroc). John P. Lisher acknowledges support from the Quantitative and Chemical Biology Training Program at Indiana University.

#### **REFERENCES**


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in cells expressing functional Nramp1. *Microbiology* 157, 1115–1122. doi: 10.1099/mic.0.045807-0


oxidase (SpxB) and avoidance of the toxic effects of the fenton reaction. *J. Bacteriol.* 185, 6815–6825. doi: 10.1128/JB.185.23.6815-6825.2003


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 01 October 2013; paper pending published: 17 October 2013; accepted: 18 November 2013; published online: 05 December 2013.*

*Citation: Lisher JP and Giedroc DP (2013) Manganese acquisition and homeostasis at the host-pathogen interface. Front. Cell. Infect. Microbiol. 3:91. doi: 10.3389/fcimb. 2013.00091*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2013 Lisher and Giedroc. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## *Salvatore Bozzaro\*, Simona Buracco and Barbara Peracino*

*Department of Clinical and Biological Sciences, University of Torino, Orbassano, Italy*

#### *Edited by:*

*Mathieu F. Cellier, Institut National de la Recherche Scientifique, Canada*

#### *Reviewed by:*

*Thierry Soldati, University of Geneva, Switzerland Michelle D. Snyder, Towson University, USA Ricardo Escalante, Consejo Superior de Investigaciones Científicas, Spain*

#### *\*Correspondence:*

*Salvatore Bozzaro, Department of Clinical and Biological Sciences, University of Torino, AOU S. Luigi, Reg. Gonzole 10, 10043 Orbassano (TO), Italy e-mail: salvatore.bozzaro@unito.it*

*Dictyostelium* cells are forest soil amoebae, which feed on bacteria and proliferate as solitary cells until bacteria are consumed. Starvation triggers a change in life style, forcing cells to gather into aggregates to form multicellular organisms capable of cell differentiation and morphogenesis. As a soil amoeba and a phagocyte that grazes on bacteria as the obligate source of food, *Dictyostelium* could be a natural host of pathogenic bacteria. Indeed, many pathogens that occasionally infect humans are hosted for most of their time in protozoa or free-living amoebae, where evolution of their virulence traits occurs. Due to these features and its amenability to genetic manipulation, *Dictyostelium* has become a valuable model organism for studying strategies of both the host to resist infection and the pathogen to escape the defense mechanisms. Similarly to higher eukaryotes, iron homeostasis is crucial for *Dictyostelium* resistance to invasive bacteria. Iron is essential for *Dictyostelium*, as both iron deficiency or overload inhibit cell growth. The *Dictyostelium* genome shares with mammals many genes regulating iron homeostasis. Iron transporters of the Nramp (Slc11A) family are represented with two genes, encoding Nramp1 and Nramp2. Like the mammalian ortholog, Nramp1 is recruited to phagosomes and macropinosomes, whereas Nramp2 is a membrane protein of the contractile vacuole network, which regulates osmolarity. Nramp1 and Nramp2 localization in distinct compartments suggests that both proteins synergistically regulate iron homeostasis. Rather than by absorption via membrane transporters, iron is likely gained by degradation of ingested bacteria and efflux via Nramp1 from phagosomes to the cytosol. *Nramp* gene disruption increases *Dictyostelium* sensitivity to infection, enhancing intracellular growth of *Legionella* or *Mycobacteria*. Generation of mutants in other "iron genes" will help identify genes essential for iron homeostasis and resistance to pathogens.

**Keywords:** *Dictyostelium***,** *Legionella***,** *Mycobacterium***, Nramp1, Nramp2, iron homeostasis, iron genes, hostpathogen interactions**

#### **INTRODUCTION**

*Dictyostelium discoideum* is a member of the *Amoebozoa* (Schilde and Schaap, 2013). The cells live as unicellular amoebae in deciduous forest soil, feeding on bacteria that are taken up by phagocytosis and dividing by binary fission. Exhaustion of the food supply triggers a shift from growth to development, resulting in cells gathering by chemotaxis into aggregates of several thousands of cells. The tight aggregates transform into elongated sausage-like multicellular organisms, called slugs, in which cells differentiate into pre-stalk and pre-spore subtypes. After extensive migration, the slug eventually culminates into a fruiting body, consisting of a slender stalk of vacuolated cells bearing on top a ball of fully differentiated spores (Kessin, 2001).

Due to their life cycle, easy handling and genetic tractability, *D. discoideum* (in the followings *Dictyostelium*) has long been a preferred model organism for studying basic processes, such as motility and chemotaxis, cell-cell communication and adhesion, cell differentiation, pattern formation and morphogenesis (Bozzaro, 2013). Their ability to phagocytose has been exploited, in the last decade, to investigate dynamics and regulatory pathways of phagocytosis as well as interactions with an increasing number of clinically relevant bacterial pathogens, including *Legionella pneumophila*, *Mycobacterium avium* or *marinum*, and *Pseudomonas aeruginosa* (Bozzaro et al., 2008, 2013; Cosson and Soldati, 2008; Clarke, 2010; Bozzaro and Eichinger, 2011; Steinert, 2011). In the light of these recent developments, in this review we will discuss the opportunities offered by *Dictyostelium* in investigating the role of divalent metal homeostasis, with a special emphasis on iron, for cell growth, and defense against pathogenic bacteria.

#### *Dictyostelium***: PROFESSIONAL PHAGOCYTE AND PATHOGEN HOST**

Wild type *Dictyostelium* strains are strictly dependent on bacteria for growth, though a few selected laboratory strains are able to grow in liquid axenic media by fluid-phase endocytosis, mostly macropinocytosis (Kessin, 2001; Maniak, 2001). Thousands of prokaryotic species are present in the forest soil; how many of them can serve as food for *Dictyostelium* is unknown, but the cells appear to be rather omnivorous. Soil bacteria which have been isolated in association with wild type strains include close relatives of *Burkholderia xenovorans*, *Stenotrophomonas maltophilia*, *Enterobacter sakazakii, Pseudomonas fluorescens*, and *Flavobacterium johnsoniae* (Brock et al., 2011). Under laboratory conditions, the cells are able to graze on a very large variety of Gram-negative and Gram-positive bacteria, including different species of *Enterobacter*, *Serratia*, *Salmonella*, *Yersinia*, *Proteus*, *Aeromonas*, *Alcaligenes*, *Acinetobacter*, *Staphylococcus*, *Listeria,* and *Bacillus* (Depraitere and Darmon, 1978). They are also capable of modulating their response to different types of bacteria by activating specific sets of gene transcripts (Farbrother et al., 2006; Carilla-Latorre et al., 2008; Sillo et al., 2008, 2011). In a recent paper, (Nasser et al., 2013) have studied global transcriptional response of wild type and selected mutant cells to a series of Gram-negative and Gram-positive bacteria, and they could show that cells respond differently to these two large families of bacteria. By analysing the transcriptional response to live, in contrast to dead bacteria, they were also able to identify selective gene pathways needed for defense, rather than growth, on either Gram-negative or Gram-positive bacteria, such as the activation of different sets of lysozymes or a set of glycoproteins apparently required for growth on Gram-positive bacteria.

Phagocytosis, both on agar plates or under shaking in simple salt solution, is very efficient. Only a handful of bacteria strains are not phagocytosed, but very few systematic studies have been published in this regard. *Legionella pneumophila* is taken up by macropinocytosis, thus its uptake by natural wild type strains is negligible (Peracino et al., 2010; Balest et al., 2011). *Bacillus* anthracis was reported not to be phagocytosed (Depraitere and Darmon, 1978), but a recent report described its uptake, though no details were offered on the efficiency of phagocytosis (Nasser et al., 2013). Human pathogenic Escherichia coli strains were found to be graze-resistant when co-cultured with *Dictyostelium* cells at high, but not low, density. Whether this was due to the high bacterial density inhibiting phagocytosis or killing the cells was not assessed, except for one strain (Adiba et al., 2010). A correlation between phagocytosis efficiency and bacterial density or previous growth conditions has been also described for *Salmonella typhimurium* (Sillo et al., 2011), *Aeromonas spp.* (Froquet et al., 2007) and for *Klebsiella pneumoniae* (March et al., 2013). Cells are also able to discriminate between edible and less appetizing bacteria. When co-cultured with *S. typhimurium* and *E. coli*, cells depleted *E. coli* from the medium leaving quite unaltered the *Salmonella* (Sillo et al., 2011). Co-culturing with live Gram-negative bacteria was also shown to prime the cells to grow on dead Gram-positive bacteria that otherwise were not utilized as food source (Nasser et al., 2013).

It is nowadays accepted that free-living amoebae and protozoa, by predating bacteria, can become a driving force for the evolution of pathogenic traits, paradigmatic cases being Acanthamoeba castellanii parasitic interaction with *Legionella pneumophila* (Barker and Brown, 1994; Levin, 1996; Steinert et al., 2000); or the variation of the rfb virulence locus in *Salmonella* enterica mediated by intestinal protozoan predation (Wildschutte et al., 2004). The ability of many pathogens to grow in macrophages and cause human diseases appears thus to be a consequence of their adaptation and survival in the normally hostile amoeboid niche (Steinert et al., 2000; Greub and Raoult, 2004; Casadevall, 2008).

Being a bacterial predator, it would be rather surprising that *Dictyostelium* would be an exception on this regard, though *Dictyostelium* parasites naturally occurring in the wild have not been described, likely due to lack of systematic studies. At least for the fungus *Cryptococcus neoformans* it was, however, shown that passage through *Dictyostelium* cells enhanced its virulence in mice (Steenbergen et al., 2003). Invasive pathogenic microbes which have been shown to be able to grow in *Dictyostelium* cells include, in addition to *Cryptococcus*, *Legionella* (Haegele et al., 2000; Solomon and Isberg, 2000), *Mycobacteria* (Solomon et al., 2003) and Burkholderia species (Hasselbring et al., 2011). *S. typhimurium co*-cultured under optimal nutrient conditions has been shown to enter wild type cells and kill the cells, but its intracellular replication has been documented in autophagic *Dictyostelium* mutants only (Jia et al., 2009; Sillo et al., 2011). Similarly, *K. pneumoniae* is pathogenic for some *Dictyostelium* mutants, not for the parental wild type strain (Benghezal et al., 2006). Capsulated *Neisseria meningitidis* (Colucci et al., 2008) as well as pathogenic *Escherichia coli* strains (Adiba et al., 2010) have been shown to resist degradation, but their intracellular growth has not been documented.

Since *Dictyostelium* can be easily grown in association with bacteria on agar plates, a plaque assay, and in some cases growth assay under shaking, have been used by several labs for screening microbial virulence genes, using either wild type or in some cases mutant cells. Virulence traits have thus been identified in *E. coli* (Adiba et al., 2010), *Pseudomonas aeruginosa* (Cosson et al., 2002; Pukatzki et al., 2002; Alibaud et al., 2008), *Vibrio cholerae* (Pukatzki et al., 2006; Miyata et al., 2011; Zheng et al., 2011), *Stenotrophomonas aeruginosa* and *S. malthophylia* (Alonso et al., 2004; Adamek et al., 2011), *K. pneumoniae*(Benghezal et al., 2006; Pan et al., 2011; March et al., 2013), *Burkholderia coenocepacia* and *B. pseudomallei* (Aubert et al., 2008; Hasselbring et al., 2011).

In contrast to these bacteria, *Legionella pneumophila*, *Mycobacterium avium*, or *M. marinum* grow rapidly intracellularly, and their interaction with *Dictyostelium* cells has been extensively studied [for recent reviews see (Bozzaro and Eichinger, 2011; Hilbi et al., 2011; Steinert, 2011)]. *In vivo* imaging of the dynamics of infection, favored by the large array of fluorescent probes against cytoskeletal and organelle proteins, has shown that the process is highly conserved between *Dictyostelium* and macrophages. Both *Legionella* and *Mycobacteria* manipulate the endocytic pathway to hinder fusion of the pathogen-containing phagosome with acidic and lysosomal vesicles, favoring association with other compartments, such as the endoplasmic reticulum (Fajardo et al., 2004; Lu and Clarke, 2005; Ragaz et al., 2008; Peracino et al., 2010) and mitochondria (Francione et al., 2009), generating a replication vacuole. Whereas massive intracellular growth of *Legionella* leads to cell lysis, *M. marinum* has been shown to escape intact cells by a novel non-lytic exocytic mechanism that may be active also in macrophages (Hagedorn et al., 2009). Genomic-wide transcriptional changes during infection and proteomic analysis of the *Legionella*-containing vacuole (LCV) have also helped in characterizing the dynamics of infection (Farbrother et al., 2006; Li et al., 2009; Shevchuk et al., 2009; Urwyler et al., 2009).

Infection assays have been exploited particularly with *Legionella* to identify and/or characterize virulence genes. Thus, it has been shown that *Legionella* mutants defective in sigmaS or its effector LqsR, which regulate expression of several genes involved in virulence, motility and transmission, are defective for growth in *Dictyostelium* and macrophages (Tiaden et al., 2007; Hovel-Miner et al., 2009). The Icm/Dot TFSS (Type Four Secretion System) substrate SdhA appears to hinder cell apoptosis, thus the mutant is defective for growth in macrophages and to a lower extent in *Dictyostelium* (Laguna et al., 2006). Mutants in the Icm/Dot effectors SidJ or SidjA (Liu and Luo, 2007), SidC or SidA (Ragaz et al., 2008) display impaired recruitment of endoplasmic reticulum to the LCV, with differential effects on intracellular growth, which was depressed in macrophages and *Dictyostelium* or *Dictyostelium* only for SidJ or SidjA, respectively, but unaltered in the SidC or SidA mutants. The Icm/Dot TFSS effectors LepA and LepB have been shown to regulate non-lytic exocytosis from the host (Chen et al., 2004), whereas Leg proteins regulate LCV traffic, by disrupting the endocytic vesicle traffic of the host (de Felipe et al., 2008). The envelope-associated protein EnhC was found to be required for bacterial growth in macrophages, but not in *Dictyostelium* (Liu et al., 2008). Among the ICM/Dot IV injected effectors, AnkyrinB (AnkB) was shown to be anchored via farnesylation to the LCV and be required for docking polyubiquinated host proteins, favoring intracellular growth, both in macrophages and in *Dictyostelium* (Al-Quadan and Kwaik, 2011).

From the site of the host, the large number of available *Dictyostelium* mutants has been exploited with *Legionella* or *Mycobacteria* to identify host resistance factors, whose genetic disruption leads to enhanced pathogen proliferation. They include cytoskeletal and signal transduction proteins, transcription factors, autophagy and mitochondrial proteins [for recent reviews see: (Bozzaro and Eichinger, 2011; Steinert, 2011)]. Among the several host cell factors so far identified, the iron transporters of the Nramp family have been investigated in detail.

## *Dictyostelium* **Nramp IRON TRANSPORTERS IN BACTERIAL INFECTION**

Since the discovery of an allelic form of the gene encoding natural resistance associated membrane protein (Nramp)1, which conferred susceptibility to various intracellular microbes (Vidal et al., 1995), the number of studies on this metal transporter family has increased dramatically (Cellier, 2012). The Nramp family is widely distributed, from bacteria to humans (Courville et al., 2006). In mammals, Nramp1 (Slc11A1) expression is restricted to macrophages, but a second Nramp protein, (Nramp2, Slc11A2, or DMT1), is localized in the plasma membrane of several tissues, and mutations have been linked to severe microcytic anemia and serum and hepatic overload (Courville et al., 2006; Shawki et al., 2012). Eukaryotic Nramp proteins are Fe2<sup>+</sup> and Mn2+, possibly Co2+, transporters (Forbes and Gros, 2001; Nevo and Nelson, 2006), whereas the bacterial homologs (MntH subfamily) transports mainly Mn2<sup>+</sup> (Papp-Wallace and Maguire, 2006; Cellier, 2012).

The *Dictyostelium* genome harbors two genes encoding members of this family, which have been named Nramp1 and Nramp2. Nramp1 is the ortholog of mammalian Nramp1, and like the macrophage counterpart, it is localized exclusively in phagosomes or macropinosomes. The protein is recruited from trans-Golgi to phago- or macropinosomes shortly after their closure, and is then retrieved from the vesicles during their post-lysosomal maturation (Peracino et al., 2006). The *nramp1* gene is expressed during growth, up-regulated upon incubation with bacteria and down-regulated upon starvation. In contrast, the Nramp2 protein is phylogenetically closer to α-proteobacteria MntH and to Nramp proteins from yeast, fungi and protists, and is exclusively localized in the membrane of the contractile vacuole (Peracino et al., 2013). The contractile vacuole is a bladder and tubular network, which in *Dictyostelium*, like other free-living amoebae and protozoa exposed to sudden environmental changes, regulates osmolarity. Under hypotonic conditions, water is pumped into the contractile vacuole, giving rise to large vacuoles that fuse with the plasma membrane, expelling their content. Under hypertonic conditions, bladder and tubules flatten, releasing water in the cytosol (Gerisch et al., 2002; Heuser, 2006).

The contractile vacuole membrane is studded with the V-H+ ATPase, which pumps protons inside the lumen (Heuser et al., 1993; Clarke et al., 2002). The V-H+ ATPase is also rapidly recruited to phagosomes or macropinosomes, shortly after their engulfment (Clarke et al., 2002; Peracino et al., 2006). Thus, both Nramp1 and Nramp2, though in two different compartments, co-localize with the vacuolar ATPase that can provide the electrogenic potential regulating their transport activity. Though still debated, it is likely that the proton gradient generated by the activity of the V-ATPase favors Nramp-dependent iron transport via a symport rather than antiport mechanism (Forbes and Gros, 2001; Courville et al., 2006; Nevo and Nelson, 2006). Experiments with purified phagosomes from *Dictyostelium* wild type and Nramp1 null cells supported this hypothesis, showing also that the protein was essential for iron transport (Peracino et al., 2006). It is likely that Nramp2 in the contractile vacuole acts similarly to Nramp1, but this has not been proven yet.

Both Nramp1 and Nramp2 are dispensable for phagocytosis or growth on non-pathogenic bacteria, but their disruption enhances intracellular growth of *Legionella pneumophila* and, at least for Nramp1, also *Mycobacterium avium* (Peracino et al., 2006, 2013). Constitutive Nramp1 overexpression depresses *Legionella*, not, however, *Mycobacterium*, growth. Interestingly, endogenous Nramp1 gene expression is down-regulated during *Legionella*, but not *Mycobacterium* infection (Peracino et al., 2006). These differences may be related to differences between both pathogens in the establishment of their replication vacuole. In *Mycobacteria* infection, the replication vacuole transiently recruits the vacuolar ATPase during the first 90 min post-infection, retrieves it between 2 and 12 h, avoiding recruitment of lysosomal enzymes while acquiring markers of a postlysosomal compartment (Hagedorn and Soldati, 2007). *Legionella* instead forms a replicative vacuole that avoids fusion with acidic vesicles, associates with mitochondria and recruits proteins of the endoplasmic reticulum and other trafficking routes, similarly to what occurs in macrophages (Fajardo et al., 2004; Lu and Clarke, 2005; Francione et al., 2009). Fusion with vesicles decorated with Nramp1 is not inhibited, but recruitment of the V-H+ ATPase, and thus vacuole acidification, is delayed of several hours, though eventually occurs after 12–24 h post-infection (Peracino et al., 2010). Down-regulating Nramp1 expression, could thus help maintaining in the long run a replication-friendly vacuole, if acidification would occur.

Iron is an essential element for virtually all cells, and pathogens such as *Legionella*, *Mycobacteria,* or *Salmonella* are known to assimilate significant amounts of iron for their metabolism and virulence (Pope et al., 1996; Robey and Cianciotto, 2002; Rodriguez, 2006; Pandey and Rodriguez, 2012; Soldati and Neyrolles, 2012). A systematic analysis of human pathogenic *Escherichia coli* strains showed that *E. coli* genes involved in iron metabolism, such as *irp*, *fyuA*, and *IroN*, favored resistance to predation by *Dictyostelium* cells (Adiba et al., 2010). Thus, depleting iron from the phagosome via Nramp1 can be an effective host defense strategy to starve the pathogen for iron. Conversely, hindering co-recruitment of the V-H+ ATPase by the pathogen, as shown for *Legionella*, not only avoids acidification of the vacuole, thus neutralizing Nramp1-dependent iron efflux, but could even favor iron influx in the vacuole via Nramp1, thus turning Nramp1 to *Legionella* advantage (Peracino et al., 2010) (**Figure 1**). Experiments with isolated phagosomes showed indeed that inactivating the vacuolar ATPase resulted in passive iron flux (Peracino et al., 2006).

**FIGURE 1 | Model for Nramp1 activity and its manipulation by** *Legionella***. (Left)** Nramp1 and the V-H+ ATPase are recruited to phagosomes shortly after their uptake. The activity of the vacuolar ATPase generates a proton gradient in the maturing phagosome that provides the electrogenic force necessary for Nramp1 to transport iron, and possibly other divalent metals, to the cytosol, thus depleting the bacteria from an essential nutrient element. **(Right)** *L. pneumophila* is taken up in *Dictyostelium* cells by macropinocytosis. Following uptake, the pathogen inhibits fusion of its vacuole with acidic vesicles bearing the V-H+ ATPase. Nramp1 recruitment is not affected, but lack of the electrogenic force neutralizes Nramp1-dependent iron efflux, and can even lead to passive influx of cytosolic labile iron to the advantage of the pathogen. For original data see: Peracino et al. (2010).

Intracellular growth of *L. pneumophila* is enhanced in Nramp1-null mutants and inhibited in cells overexpressing Nramp1, but the latter effect was found to be reversed by phosphatidylinositide-3 kinase (PI3K) inhibitors or by genetic ablation of PI3K and the phosphatase PTEN (Peracino et al., 2010). The PI(4,5)P2 or PI(3,4,5)P3 phosphatase Dd5P4 (OCRL1) also enhanced *Legionella* growth (Weber et al., 2009), and PI3K inhibitors stimulated *Legionella* infection also in macrophages (Peracino et al., 2010). Membrane phosphatidylinositides are important regulators of actin assembly/disassembly in the plasma membrane and in phago- or macropinosomes as well as phago- and macropinosome fusion with vesicles of the endo-lysosomal pathway. It was shown that altering phosphoinositide metabolism in *Legionella* infection resulted in even stronger inhibition of the *Legionella*-containing macropinosome with acidic vesicles, a process apparently stimulated by PI3P formation (Clarke et al., 2010; Peracino et al., 2010). This has led to the hypothesis that *Legionella* hinders fusion of the *Legionella*-containing macropinosome with acidic vesicles, but not vesicles bearing Nramp1, by inhibiting PI(3)P formation either by secreting a 3-P phosphatase or by anchoring a 3-P phosphatase of the host to the replication vacuole.

## **THE GENETIC BASIS OF IRON HOMEOSTASIS IN** *Dictyostelium*

*Dictyostelium* cell growth is sensitive to iron depletion as well as to iron loading above 0.2 mM. When axenic wild type cells are incubated in minimal medium without iron, growth decreases dramatically and after 4–5 generations stops completely. *Nramp1* or *nramp2* knockout mutants fail to duplicate already after 2 generations. High iron concentrations reduce the growth rate in the wild type, to a lower extent in the *nramp* single knockout mutants, but only minimally in the double KO mutant. These results suggest that inactivating *nramp1* and *nramp2* leads to a lower intracellular level of bioavailable cellular iron, independently of the total amount that may enter the cell (Peracino et al., 2013). *Dictyostelium* development occurs under starving conditions in simple salt solutions, or even in water; addition of divalent transition metals is not required. During development, the only source of energy and organelle recycling is the autophagic breakdown of cellular components, which is responsible for the observed decrease in cell size (Kessin, 2001). Autophagy could also be the major source of iron during development, unless excess labile iron is accumulated during growth in the contractile vacuole, and the latter acts as iron reserve. It is worth mentioning that Nramp2 gene expression is stimulated by starvation, with maximal mRNA accumulation reached during aggregation and slug formation (Peracino et al., 2013).

In addition to the Nramp iron transporters, the *Dictyostelium* genome encodes many proteins involved in cellular iron homeostasis (**Table 1**). Homologs of the mitochondrial iron transporter mitoferrin (Satre et al., 2007), Fe-S and heme ABCB transporters (Anjard et al., 2002), and the iron sensor frataxin as well as a cytosolic and a mitochondrial aconitases are present. The cytosolic aconitase (Aco1) is highly similar to mammalian Iron Regulatory Protein (IRP), raising the possibility that it may bind



*The listed ABCB transporters are homologs or orthologs of yeast and mammalian mitochondrial transporters (see Anjard et al., 2002). For mitoferrin see Satre et al., 2007. Genes in italics encode distantly-related proteins of the indicated families. For information on each gene see text and www.dictybase.org.*

iron regulatory elements (IREs), thus regulating iron-dependent genes (Anderson et al., 2012). Two distantly-related ferroportin (Slc40A1 or IREG1)-like proteins exist, but no homologs for trasferrin or transferrin receptors, the systemic iron traffic regulator hepcidin or the hepcidin inhibitor hemojuvelin are found, in agreement with the notion that iron is mainly derived from bacterial digestion. Ferritin or mitoferritin homologs are also not evident, though a putative ferritin-like protein, but with very low homology to other ferritin-like proteins, is encoded in the genome.

Bacterial digestion in phago-lysosomes will result in ferric ions that need to be reduced for transport via Nramp1 in the cytosol. In macrophages, where the major source of iron is represented by aged erythrocytes and bacteria, this is accomplished by ferric reductases of the STEAP family (Wang and Pantopoulos, 2011), which have no orthologs in *Dictyostelium* genome. Two putative ferric reductases of the domon-cytB561 family are, however, encoded in the genome, one of which is highly expressed during growth (**Table 1**). Whether any of them is localized in the phagosome, and may be responsible for ferric ion reduction, is under study. It is clear, in any case, that cells manage to reduce ferric ions, as ferric chloride added to minimal medium stimulates cell growth (Peracino et al., 2013). As summarized in the model in **Figure 2**, it can be hypothesized that following ferric ion reduction, iron is exported from the phagosomes via Nramp1 in a proton-gradient dependent co-transport. Most cytosolic iron will be transported to the mitochondria via mitoferrin, to be incorporated into Fe-S clusters and heme groups, a process likely regulated by frataxin, as in mammalian cells (Wang and Pantopoulos, 2011). Excess labile iron in the cytosol could accumulate in the contractile vacuole, either to be released following CV discharge upon fusion with the membrane or to be recycled to the cytosol via Nramp2, particularly under starving conditions, when the only

source of iron is intracellular. In this model, the contractile vacuole is proposed to act as homeostatic vesicle-bound iron store, taking over ferritin function. In principle, also a plasma membrane iron exporter, such as ferroportin, would be dispensable in this model. If the model is correct, new questions arise: how is iron transport in the contractile vacuole regulated? Is Nramp2 the only iron transporter and does it mediate both import and export? How does the electrogenic potential generated by the V-H+ ATPase in the CV membrane regulate Nramp2 activity, taking in consideration that protons are efficiently buffered in the contractile vacuole lumen (Clarke and Heuser, 1997), contrary to what occurs for Nramp1 in the phagolysosomes?

### **NOT ONLY IRON**

In addition to iron, major divalent transitions metals that have been involved in host-pathogen interactions are manganese, copper, and zinc (Kehl-Fie and Skaar, 2010; Botella et al., 2012; Soldati and Neyrolles, 2012). Whereas iron and manganese are depleted from mature phagosomes, copper, and possibly zinc have been suggested to be pumped in to intoxicate bacteria; increased concentrations of both metals have been reported in *mycobacteria*l infection after stimulation of infected macrophages with cytokines (Wagner et al., 2006).

Very little is known about these metals in *Dictyostelium*. In contrast to iron, cell growth is unaffected by both manganese depletion or millimolar addition to minimal medium, and manganese does not rescue cell growth in iron-depleted medium (Peracino, unpublished results). Manganese can, however, stimulate cell aggregation and cell differentiation, as it directly stimulates the activity of adenylyl cyclase (Loomis et al., 1978; Hagmann, 1985) and of a bi-functional glycosyltransferase that regulates O2-dependent development (Trinchera and Bozzaro, 1996; West et al., 2010). *Dictyostelium* development is also sensitive to heavy metals present in the soil. Hg in particular inhibits development at concentrations of 50 mg per kg dry soil, compared to about 6-, 16-, and 32-fold higher concentrations for Fe, Zn, and Cu, respectively (Ponte et al., 2000; Balbo and Bozzaro, 2009; Rodriguez-Ruiz et al., 2013).

The *Dictyostelium* genome encodes several putative copper and zinc transporters. A Menkes type Cu2<sup>+</sup> ATPase and a putative p80 copper transporter have been shown to be localized in the plasma membrane and in phagosomes. The unusual resistance to Cu has been linked to the high secretory efficiency of the Cu2<sup>+</sup> ATPase (Burlando et al., 2002), which in the phagosome could pump Cu2<sup>+</sup> ions in the lumen, favoring a potentially toxic effect of this metal on bacteria. A chemical assay for detection of metallothioneins, which could also account for copper resistance, failed to detect any activity, suggesting that *Dictyostelium* cells do not express these proteins (Burlando et al., 2002). The *Dictyostelium* genome harbors, however, one gene (DDB\_G0288281), which has been annotated as putative metallothionein. Metallothioneins are small cysteine-rich proteins capable of binding heavy metals through the thiol group of their cysteine residues. Unfortunately, their primary structure is extremely variable and their secondary and tertiary structures are highly heterogeneous, making it difficult to recognize homologs among phyla, sometimes even among species (Coyle et al., 2002). Thus, whether the DDB\_G0288281gene product is truly a metallothionein is open.

The p80 copper transporter is present in the plasma membrane and in endocytic vesicles. The protein is retrieved from the phagosome just after closure, but is then recruited again to mature phagosomes, probably by fusion with endocytic vesicles (Ravanel et al., 2001). In *M. marinum* infection, p80 has been shown to be recruited to the pathogen-replication vacuole at the onset of intravacuolar growth (Hagedorn and Soldati, 2007). Based on homology with other channel transporters, p80 is expected to transport copper outside of the vacuole, but no functional studies have been done, nor it is known whether disruption or overexpression of the gene affects host-pathogen interactions. The *Dictyostelium* zinc transporter family includes 12 members, most of which are expressed in late development and are assumed to be involved in cell differentiation (Sunaga et al., 2008). No data are available on potential localization of zinc transporters in phagosomes and their role during growth or host-pathogen interactions.

Recently, attention has been driven to a potential involvement of Mg2<sup>+</sup> in nutritional immunity, based on results showing that synthesis of the *M. tuberculosis* virulence factor isoTb, which is involved in phagosome maturation arrest, was up- or down-regulated by Mg2<sup>+</sup> overloading or depletion, respectively (Mann et al., 2011; Soldati and Neyrolles, 2012). In *Dictyostelium*, a V P-ATPase was identified as the tagged gene in the kil2 mutant, characterized by reduced phagosomal protease activity and defective growth on *Klebsiella pneumoniae* (Lelong et al., 2011). Addition of Mg2<sup>+</sup> rescued the mutant, suggesting that the V P-ATPase could be a magnesium pump responsible for optimal Mg2<sup>+</sup> concentration in the phagosome. Intriguingly, the kil2 mutant was able to grow on other bacteria species and did not display increased susceptibility to *M. marinum* infection.

#### **CONCLUSION**

As obligate phagocytes, at least during the growth phase of their life cycle, *Dictyostelium* cells resemble macrophages for their ability to engulf bacteria and dead cells, to discriminate between self and non-self and to fight potential pathogens. They also share with macrophages several "iron genes" regulating cellular iron metabolism, though lacking genes involved in systemic iron homeostasis. The organism is thus a useful model for investigating iron homeostasis at cellular, rather than systemic, level and the role of iron (and other divalent metals) in hostpathogen interactions. The potential absence of ferritin, and possibly also ferroportin, homologs raises the question of how labile iron is neutralized and stored in the cell. The possibility that the contractile vacuole acts as iron (divalent metal ions?) store and/or sink, thus taking over the functions of ferritin and ferroportin, is very suggestive, opening novel potential roles for this compartment also in free-living amoebae and protozoa, but needs to be proven. Investigating the mechanism of action of Nramp2 can be of help in this context as well as developing more sensitive probes for detecting iron in intracellular compartments. The highly conserved function of Nramp1 in the phago-lysosomal membrane, from *Dictyostelium* to macrophages, corroborates the central role of iron control for the host and the pathogen in this compartment. The finding that Nramp2 KO mutants are sensitive to infection almost as much as Nramp1 KO mutants raises the question of potential cross-talk between the phago-lysosome (or the pathogen-replication vacuole) and the contractile vacuole in iron homeostasis and in resistance to pathogens. The ease in generating and analysing mutants in *Dictyostelium* will help in the near future in dissecting the role of mitochondrial and cytosolic iron genes in these intracellular interactions and in extending these studies to genes regulating metabolism of other divalent metals relevant for host-pathogen interactions.

#### **ACKNOWLEDGMENTS**

Work in the lab was supported by research grants of the University of Torino and by the Compagnia San Paolo (12-CSP-C03-065).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 11 July 2013; accepted: 22 August 2013; published online: 19 September 2013.*

*Citation: Bozzaro S, Buracco S and Peracino B (2013) Iron metabolism and resistance to infection by invasive bacteria in the social amoeba Dictyostelium discoideum. Front. Cell. Infect. Microbiol. 3:50. doi: 10.3389/ fcimb.2013.00050*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2013 Bozzaro, Buracco and Peracino. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Iron in intracellular infection: to provide or to deprive?

## *Sandro Silva-Gomes 1,2\*†, Sílvia Vale-Costa1,2\*†, Rui Appelberg1,2\* and Maria S. Gomes 1,2\**

*<sup>1</sup> Infection and Immunity Unit, Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal*

*<sup>2</sup> Department of Molecular Biology, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal*

#### *Edited by:*

*Mathieu F. Cellier, Institut National de la Recherche Scientifique, Canada*

#### *Reviewed by:*

*Geneviève Milon, Institut Pasteur, France Michael Niederweis, University of*

*Alabama at Birmingham, USA*

#### *\*Correspondence:*

*equally to this work.*

*Sandro Silva-Gomes, Sílvia Vale-Costa, Rui Appelberg and Maria S. Gomes, Infection and Immunity Unit, Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal e-mail: sandro@ibmc.up.pt; svcosta@ibmc.up.pt; rappelb@ibmc.up.pt; sgomes@ibmc.up.pt †These authors have contributed*

Due to their chemical versatility, transition metals were incorporated as cofactors for several basic metabolic pathways in living organisms. This same characteristic makes them potentially harmful, since they can be engaged in deleterious reactions like Fenton chemistry. As such, organisms have evolved highly specialized mechanisms to supply their own metal needs while keeping their toxic potential in check. This dual character comes into play in host-pathogen interactions, given that the host can either deprive the pathogen of these key nutrients or exploit them to induce toxicity toward the invading agent. Iron stands as the prototypic example of how a metal can be used to limit the growth of pathogens by nutrient deprivation, a mechanism widely studied in *Mycobacterium* infections. However, the host can also take advantage of iron-induced toxicity to control pathogen proliferation, as observed in infections caused by *Leishmania*. Whether we may harness either of the two pathways for therapeutical purposes is still ill-defined. In this review, we discuss how modulation of the host iron availability impacts the course of infections, focusing on those caused by two relevant intracellular pathogens, *Mycobacterium* and *Leishmania*.

**Keywords: transition metal, immunity,** *Mycobacterium***,** *Leishmania***, infection**

#### **IRON IN BIOLOGIC SYSTEMS AND INFECTION PROCESSES**

Transition metals are elements that have an incomplete inner electron shell and can easily shift between different oxidation states. Cells took advantage of this property and included transition metals into proteins, gaining catalytic potential and protein stability. The function of transition metals in enzymatic catalysis can be divided into two groups, depending upon the metal acting as a redox center or not. Of the redox active metals, iron is the most prevalent, followed by copper and molybdenum, while zinc is the most common non-redox transition metal (Andreini et al., 2008). Iron is found in an unparalleled variety of sites and cofactors, such as haem groups and iron-sulphur clusters, and is involved in processes such as oxygen sensing and transport, energy metabolism and nucleic acid synthesis. The predominance of iron is presumably due to the large availability of water soluble ferrous iron during prebiotic times, before the rise of atmospheric oxygen levels caused by photosynthesis resulted in the precipitation of insoluble iron (III) (Crichton and Pierre, 2001). As a result, almost all living organisms from archaea to eukaryotes require iron in their metabolism. *Borrelia burgdorferi* is unique among pathogens in that it bypassed iron dependence by substituting zinc or manganese for iron in its metalloproteins (Posey and Gherardini, 2000; Nguyen et al., 2007).

#### **IRON METABOLISM IN THE HOST**

Vertebrates have evolved a complex network of proteins to acquire, transport and store iron, while maintaining free iron concentration at very low levels (reviewed in Kaplan and Ward, 2013). In mammals there is no regulated excretion of iron and the replenishment of losses from desquamation and minor bleeding, which account for the loss of less than 0.05% of body iron per day, occurs at the level of intestinal absorption. Dietary iron, either inorganic or bound to haem, is absorbed at the brush border of enterocytes lining the proximal portion of the duodenum (Andrews and Schmidt, 2007). Iron is transported as ferrous iron into circulation through ferroportin, the only cellular iron exporter. Transferrin is the physiological carrier of iron in plasma, binding two atoms of ferric iron. In humans, the normal saturation of transferrin is only 20–40% (Ganz and Nemeth, 2012b). Iron-loaded transferrin binds with high affinity to the transferrin receptor (TFR)-1, ubiquitously expressed at cell surfaces, and is internalized by clathrin-dependent endocytosis. Iron is then released intracellularly and becomes part of the labile iron pool (Correnti and Strong, 2012). Iron that is not needed for immediate use or export is stored in ferritin. In vertebrates, cytosolic ferritin is formed by the spontaneous assembly of 24 subunits of Heavy (H) and Light (L) chains, resulting in a hollow shell capable of accommodating up to 4500 iron atoms in an inert, non-toxic form (Harrison and Arosio, 1996). Erythropoiesis is the most avid consumer of iron in the mammal organism. Approximately 60–70% of the human adult body iron is bound within haemoglobin (∼2.5 g). With the erythrocyte lifespan of 120 days, the reutilization of iron recycled from

**Abbreviations:** ACD, anaemia of chronic disease; β2m, beta-2-microglobulin; DFO, desferrioxamine; HH, hereditary hemochromatosis; Lcn-2, Lipocalin 2; MHC-I, major histocompatibility complex class I; NO, nitric oxide; NOS2, nitric oxide synthase 2; ROS, reactive oxygen species.

senescent cells accounts for most of the iron flux (Ganz and Nemeth, 2012b). The hormone hepcidin is regarded as the central regulator of systemic iron homeostasis. Hepcidin is a 25 amino acid peptide that is mainly produced by hepatocytes. It binds to the iron exporter ferroportin, causing its internalization and degradation. Consequently, it reduces the absorption of dietary iron by enterocytes and the release of iron from intracellular stores (Ganz and Nemeth, 2012a).

As described, macrophages play a central role in regulating iron metabolism since they recycle haem iron and regulate its storage. However, since macrophages respond to other environmental cues such as signals from the immune system, they may undergo major and distinct physiological adaptations in different settings namely those that are triggered by infection. For example, it is known that classic activation of macrophages (M1 macrophages) e.g., by gamma interferon together with microbial molecules acting as Toll-like receptor ligands modulates a different program of iron handling as compared to the alternative activation (M2 macrophages) induced by cytokines such as interleukin (IL)-4 and IL-13 induced in certain types of infection or by cues from tissues in steady-state conditions (Recalcati et al., 2012). The ensuing consequences at the systemic level are distinct as classic activation promotes the sequestration of iron leading to systemic lowering of its availability whereas alternative activation, by promoting export of the metal by macrophages would lead to the opposite systemic effects.

#### **TRANSITION METALS DURING INFECTION**

Successful colonization of a host by pathogens requires that these must gain access to the required amounts of transition metals. Vertebrate hosts exploit this requirement by sequestering these elements (**Figure 1A**). Iron withholding is the most wellstudied example of metal deprivation. A complex network of host proteins renders this valuable nutrient largely inaccessible to pathogens, a concept usually known as nutritional immunity (Appelberg, 2006a; Hood and Skaar, 2012). However, recent evidences suggest that this mechanism is also used to sequester other transition metals, including manganese and zinc (Kehl-Fie and Skaar, 2010). The strict requirement for transition metals is due to their involvement in numerous processes ranging from microbial metabolism to accessory virulence factor function. Hence, without the required concentration of these nutrients, the invading agent is unable to proliferate and cause disease (Kehl-Fie and Skaar, 2010).

The host can capitalize on the toxicity of transition metals and increase their concentration in the compartment where pathogens proliferate (**Figure 1B**). By this mechanism, transition metals can potentiate pathogen killing together with the respiratory burst in phagocytes. The moderately reactive superoxide radical is rapidly reduced to hydrogen peroxide, either spontaneously or enzymatically. In turn, hydrogen peroxide may give rise to the highly reactive and short-lived hydroxyl radicals in the Fenton reaction, by reacting with a reduced transition metal, such as Fe(II) or Cu(I) (Galaris and Pantopoulos, 2008; Hodgkinson and Petris, 2012), resulting in oxidative damage to the pathogen. The toxicity of copper against *M. tuberculosis* has been reported (Wolschendorf et al., 2011) and a copper-transporting ATPase

**FIGURE 1 | Transition metals in immunity against intracellular pathogens.** Upon entry into the host cell, the pathogen is able to retrieve metals from its surroundings to incorporate them in mettaloproteins strictly required for its survival. The pathogen uses dedicated transporters and specialized proteins [e.g., siderophores (Hider and Kong, 2010), high-affinity iron chelating compounds] to acquire metals from the cytoplasmic pools or by hijacking the cell pathways of metal acquisition (for example, *Mycobacterium* and *Leishmania* have access to transferrin-bound iron in the endocytic pathway). One of the countermeasures employed by the host cell to reduce the proliferation of the pathogen is metal deprivation **(A)**. This is achieved by pumping out the metal from the phagosome, mediated by metal transporters such as SLC11A1, which is recruited to the phagosomal membrane, where it transports iron and manganese out of this compartment. The metal can then be diverted into storage [e.g., the iron-storage ferritin is induced during infection with *Salmonella* (Nairz et al., 2007) and *Mycobacterium* (Silva-Gomes et al., 2013b)] or exported [e.g., ferroportin, an iron exporter that localizes to the cellular membrane, is induced in macrophages infected with *Mycobacterium* (Van Zandt et al., 2008) and *Salmonella* (Nairz et al., 2007)]. On the other hand, the host cell can explore the toxicity of transition metal and direct them to the microbial invader **(B)**. During infection, metal transporters such as copper transport 1 (CTR1) (White et al., 2009) are induced, and transport the metal from the extracellular environment. Metals are also mobilized from intracellular storage [e.g., macrophages mobilize zinc from intracellular stores when infected with *M. tuberculosis* or *E. coli* (Botella et al., 2011)], which will then accumulate in the phagosome by the action of metal transporters, such as the copper transporter ATP7A (White et al., 2009).

has been identified in macrophages (White et al., 2009). Other mechanisms of metal intoxication independent of oxidative stress have also been proposed. For example, it was suggested that metal cofactor replacement may mediate copper toxicity against bacteria (Rowland and Niederweis, 2012) and that perturbation of a defined ratio of transition metals is detrimental to bacterial physiology, which may underlie zinc and manganese toxicity (Botella et al., 2011; Hood and Skaar, 2012).

This review focuses on the effect of availability of iron, the most abundant transition metal in the vertebrate host, during infection with two intracellular pathogens, *Mycobacterium* and *Leishmania*.

## **IRON METABOLISM IN** *Mycobacterium* **INFECTION**

Tuberculosis remains the most important bacterial infection worldwide. It is estimated that one third of the world population is infected with *M. tuberculosis*. However, in 90% of the cases immunity is able to prevent disease, leading in the majority of cases to latent infection. The advent of the AIDS epidemic and the introduction of immunosuppressive therapies dramatically increased the number of people at risk of infection not only with *M. tuberculosis* but also with other *Mycobacterium* species that otherwise would not cause disease. This is the case of *Mycobacterium avium*, a Non Tuberculous Mycobacteria (NTM). Whereas *M. tuberculosis* is a primary pathogen, *M. avium* is seldom identified as one. The two mycobacterial species differ in that opportunistic infection by *M. avium* occurs in advanced stages of AIDS when blood CD4+ T cell counts are lower than 50 per mm3, whereas infection of AIDS patients with *M. tuberculosis* is not limited to such late stages of the disease. *M. tuberculosis* has no known reservoir other than humans. The bacteria are almost exclusively transmitted by aerosolized droplets, generated by the cough or sneeze of a person with *M. tuberculosis* lung infection and are inhaled by an uninfected person. In contrast, *M. avium* does not seem to be transmitted between hosts and infection occurs usually through tap water. Although mycobacteria can infect several cell types (e.g., epithelial cells, eosinophils, neutrophils and dendritic cells) the macrophage has long been established as the central cell during the infection, being the primary player of cellular immunity as well as the main site of bacterial replication. Despite the differences that make *M. tuberculosis* a highly successful pathogen and *M. avium* an opportunistic infectious agent that mostly affects patients with compromised immunity, studies in the mouse model showed that immune response to both mycobacteria is similar, namely regarding the pivotal roles of CD4+ T cells, macrophages, and the IL12–IFNγ cytokine axis. This is also the case of iron availability, as it will be discussed below.

For a review regarding pathogenesis of infection with *M. tuberculosis* and *M. avium* we refer the readers to (Cooper, 2009) and (Appelberg, 2006b), respectively.

#### **HOST IRON STATE: EVIDENCES FROM HUMAN STUDIES**

Iron availability can be a determinant factor during infections with mycobacteria. In the nineteenth century, Armand Trousseau, a French physician, already documented the dangers of giving iron to patients suffering from tuberculosis. He observed that although tuberculosis patients developed some degree of anaemia, iron supplementation in their diet resulted in a poorer outcome than those patients who did not receive additional iron (Trousseau, 1872). Contemporary studies have corroborated this link between iron and tuberculosis. In 1996, Gordeuk et al. reanalyzed an autopsy study performed in South Africa in the 1920s, and found a relationship between death from tuberculosis and hepatic and splenic iron overload, notably with increased iron deposition in the mononuclear macrophage system (Gordeuk et al., 1996). Epidemiological studies in rural Zimbabwe have also shown a correlation between iron overload induced by dietary intake of iron (although genetic predisposition could also be a factor), and the risk of active pulmonary tuberculosis (Gangaidzo et al., 2001). Finally, a recent study has shown iron redistribution to macrophages in HIV infected patients to be a risk factor for the development of tuberculosis (McDermid et al., 2013).

Owing to the strict regulation of intestinal absorption, iron overload of mammalian organisms is rare. However, certain genetic diseases can lead to iron overload, such as hereditary hemochromatosis (HH), the most common inherited single gene disorder in people of Northern and Western Europe. This disease is most often associated with mutations in a molecule, HFE, homologous to class I major histocompatibility complex (MHC) alpha chains. These patients spontaneously develop hepatic iron overload, with iron accumulation in hepatocytes but not in macrophages. Given the reduced capacity of the macrophage to retain iron, human macrophages from HH patients where shown to be less permissive to *M. tuberculosis* growth (Olakanmi et al., 2007). However, whether this is true at an all-organism level is not known, as epidemiologic data from the incidence of tuberculosis in HH patients are not available. Furthermore, results from mouse models of HH indicated that the overall tissue iron overload contributes to susceptibility to mycobacteria (discussed below).

#### **HOST IRON STATE: EVIDENCES FROM THE MOUSE MODEL**

For the most part, the findings in humans described above have been very effectively reproduced in the mouse model of experimental infections with mycobacteria.

Our group has shown that an iron poor diet led to a reduced proliferation of *M. avium* in mice (Gomes et al., 1999a), while the parenteral administration of iron-dextran led to an increased proliferation of the bacilli (Gomes and Appelberg, 1998; Gomes et al., 2001). Likewise, others have shown that either the parenteral overload of iron in the form of polymaltose ferric hydroxide (Lounis et al., 1999, 2001) (Lounis et al., 2003) or the administration of iron citrate through the drinking water (Schaible et al., 2002) rendered the mice more susceptible to experimental infection with *M. tuberculosis*.

Mice deficient in HFE or in the HFE-binding protein beta-2-microglobulin (β2m−*/*−) spontaneously develop hepatic iron overload, similar to HH patients (De Sousa et al., 1994; Zhou et al., 1998). β2m−*/*<sup>−</sup> mice are known for a long time to be more susceptible to *M. tuberculosis*, an effect initially attributed to a lack of major histocompatibility complex class I (MHC-I) restricted cells (Flynn et al., 1992). However, MHC-I-KO animals are less susceptible to *M. tuberculosis* than β2m−*/*<sup>−</sup> mice (Rolph et al., 2001; Schaible et al., 2002). Schaible et al. have shown that correcting the iron overload in these mice, through the administration of lactoferrin, reduced their mycobacterial loads to levels comparable to MHC-I-KO animals, proving that iron availability is the major factor accounting for the increased susceptibility of β2m−*/*<sup>−</sup> mice to experimental tuberculosis (Schaible et al., 2002). In our group, we also found that the increased accumulation of iron in β2m−*/*<sup>−</sup> mice is associated with an increased susceptibility to *M. avium* experimental infection (Gomes-Pereira et al., 2008). Furthermore, we showed a similar effect in HFE−*/*<sup>−</sup> mice (Gomes-Pereira et al., 2008). Interestingly, although these models of iron overload tend to accumulate iron in hepatocytes and not in macrophages, we observed that during infection iron accumulates in macrophages within granulomas (Gomes-Pereira et al., 2008).

#### **THE FIGHT FOR IRON**

In order to successfully establish an infection, mycobacteria must gain access to iron and have therefore evolved strategies to acquire iron from the host. Mycobacteria are able to block phagosomal maturation, replicating in an intracellular compartment with access to iron-loaded transferrin (Clemens and Horwitz, 1996; Halaas et al., 2010). Interestingly, the ability of *M. avium* to prevent phagosome maturation was shown to be dependent on its capacity to acquire iron (Kelley and Schorey, 2003). In addition to transferrin, *M. tuberculosis* was also shown to be able to acquire iron from lactoferrin (Olakanmi et al., 2004) and from the macrophage cytoplasmic iron pools (Olakanmi et al., 2002). Furthermore, *M. tuberculosis* can use haem as an iron source (Jones and Niederweis, 2011; Tullius et al., 2011; Nambu et al., 2013). In line with the notion that access to iron is a requisite to cause a persistent infection, it was reported, using Xray microscopy, that iron concentration increased over time in the phagosomes of macrophages infected with *M. avium* or *M. tuberculosis*, while it decreased in those infected with the nonpathogenic *M. smegmatis* (Wagner et al., 2005). Several bacteria are able to produce siderophores, low molecular weight molecules with high affinity for iron. Pathogenic mycobacteria, such as *M. tuberculosis* and *M. avium*, synthetize siderophores that remain associated to the cell wall, termed mycobactins, and others that are secreted, named carboxymycobactins (Ratledge, 2004). These siderophores are able to remove iron from host iron-binding proteins, such as transferrin and lactoferrin (Gobin and Horwitz, 1996). In *M. tuberculosis* a gene cluster that encompasses 10 genes, designated mbtA-J, encodes the machinery responsible for siderophore biogenesis (Quadri et al., 1998). *mbtB* (De Voss et al., 2000), *mbtD* (Jones and Niederweis, 2011), and *mbtE* (Reddy et al., 2013) mutants show restricted growth in iron-deficient medium and in macrophages, suggesting that siderophore synthesis is required for *M. tuberculosis* virulence. Indeed, guinea pigs infected with the *mtbE* mutant exhibited reduced bacillary load in comparison with animals infected with the parental strain (Reddy et al., 2013). However, these latter data should be confirmed since this study showed that the mutant strain induced the same amount of moderate pathology in the lung as the wild type strain despite its virtually complete elimination by 4 weeks of infection. Whether that was due to the high inoculum used and the persistence of inflammatory material from the mycobacteria or to an underestimation of the bacterial loads given the difficulties in growing the mutant strain *in vitro* is unclear. Noteworthy, whereas pathology subsequently exacerbated in the hosts infected with the wild type strain, it did not in animals infected with the mutant strain. In addition to the synthesis of siderophores, proteins involved in their import or export are also critical for virulence. IrtAB, a transporter of iron-loaded carboxymycobactin, is required for the replication of *M. tuberculosis* in macrophages and *in vivo* in a mouse model of aerosol infection (Rodriguez and Smith, 2006). A siderophore export system was recently identified in *M. tuberculosis,* involving the proteins MmpS4 and Mmsp5 (Wells et al., 2013). Deletion of both *mmpS4* and *mmpS5* drastically decreases synthesis and secretion of siderophores and notably reduces *M. tuberculosis* virulence in mice (Wells et al., 2013).

The essentiality of iron acquisition systems for mycobacterial survival has led to the suggestion that this pathway could constitute a new drug target in anti-mycobacteria drug development (Appelberg, 2006a; Meyer, 2006). In fact, several compounds with the capacity to interfere with siderophore synthesis and/or function were shown to have strong inhibitory activity against *M. tuberculosis* (Ferreras et al., 2005, 2011). It should be noted that although some compounds that interfere with siderophore synthesis showed promising results in axenic cultures of *M. tuberculosis* (Ferreras et al., 2005), their effect in a mouse model of *M. tuberculosis* infection is very limited (Lun et al., 2013). These observations may indicate that (1) mycobacteria have access *in vivo* to others forms of iron independent of siderophore acquisition [e.g., haem iron (Jones and Niederweis, 2011)] or (2) the compound is not reaching the cells harboring mycobacteria. Alternative possible targets for new drug development may instead include the siderophore transport systems. We have also shown that the addition of iron chelators to *M. avium* cultures, either in axenic culture, in macrophage cultures, or *in vivo* led to significant decreases in mycobacterial growth (Gomes et al., 1999a; Fernandes et al., 2010). Furthermore, we have developed new molecules based on the 3-hydroxy-4 pyridinone iron chelating moiety, in which the inclusion of a rhodamine residue improved anti-mycobacterial activity, presumably through improved intracellular distribution and targeting for the mycobacteria-containing phagosome (Fernandes et al., 2010; Nunes et al., 2010; Moniz et al., 2013).

During an infection, the host uses several mechanisms to withhold iron access from pathogens. Anaemia often develops during acute or chronic infections, which is known as anaemia of chronic disease (ACD), and is thought to represent a mechanism to limit iron availability to the invading microorganisms (Weiss, 2009). ACD, the second most common type of anaemia (after anaemia of iron-deficiency) is characterized by hypoferremia (low serum iron) and increased iron retention within the mononuclear phagocyte system (Roy, 2010). Anaemia was described in the mouse model of infection with *M. avium* (Rodrigues et al., 2011) and *M. bovis* BCG (Marchal and Milon, 1981), and epidemiologic studies showed that tuberculosis is frequently associated with anaemia (Lee et al., 2006; Sahiratmadja et al., 2007). One of the putative links between immunity and the anaemia is hepcidin, a peptide that regulates iron homeostasis by mediating the degradation of ferroportin, an iron exporter protein. *In vitro*, infection of macrophages with *M. avium* and *M. tuberculosis* induce the hepcidin mRNA expression (Sow et al., 2007). However, using a microarray platform to analyse the iron-related genes regulated by *M. avium* infection, we did not find hepcidin to be induced in the liver (Rodrigues et al., 2011), questioning the role of hepcidin in the development of anaemia during mycobacterial infections. Nevertheless, hepcidin produced by infected macrophages may have a local effect, rather than a major role in the systemic iron regulation. Indeed, Sow et al. (2007) showed that hepcidin localizes to the mycobacteria-containing phagosomes and possesses direct antimicrobial activity against *M. tuberculosis*, causing structural damage to the mycobacteria. How anaemia develops in the context of mycobacterial infection remains to be determined. Anaemia independent of the expression of hepcidin has been observed in other situations, namely after LPS administration to TIR domain-containing adaptor protein (TRIF)-deficient mice (Layoun et al., 2012) and Hepcidin+*/*<sup>−</sup> mice (Deschemin and Vaulont, 2013), in a murine model of protracted peritonitis (Schubert et al., 2012) and notably following TNF administration to mice (Laftah et al., 2006).

There is experimental evidence that some degree of haemolysis and release of free haem may occur during severe infection (Larsen et al., 2010). Free haem contributes to an increased susceptibility to mycobacterial infection, most likely involving mechanisms that differ from those associated to other forms of iron, and haem-oxygenase-1, the enzyme responsible for haem detoxification is essential for host protection against these infections (Silva-Gomes et al., 2013a).

One of the ways by which the host can interfere with the pathogen acquisition of iron is through the action of lipocalin (Lcn)-2. Lcn-2, also known as siderocalin, neutrophil gelatinaseassociated lipocalin (NGAL) or 24p3, is capable of binding siderophores (Goetz et al., 2002) and transport them into cells by endocytosis, after interacting with a specific cell-surface receptor (Devireddy et al., 2005). Lcn-2 is able to bind carboxymycobactin from mycobacteria (Holmes et al., 2005) and inhibit the growth of *M. tuberculosis* and *M. bovis* BCG in liquid culture, in a dose-dependent manner reversible by the addition of iron or recombinant carboxymycobactin (Saiga et al., 2008). Furthermore, Lcn-2-deficient mice are highly susceptible to intratracheal infection with *M. tuberculosis*(Saiga et al., 2008). Of note, recent studies have shown that Lcn-2 can also function as an iron shuttle in basal metabolism of the host, either delivering iron to or stealing iron from specific cell types, implicating Lcn-2 in processes unrelated to its function in immunity (Correnti and Strong, 2012).

Inside infected macrophages, pathogen's access to iron may be limited by SLC11A1. SLC11A1 is a divalent metal transporter, recruited to the late endosomal and phagosomal membrane of macrophages and other professional phagocytes (Nevo and Nelson, 2006). Although SLC11A1 contributes to macrophages' efficiency in the recycling of erythrocyte-derived iron (Soe-Lin et al., 2009), the main function of SLC11A1 seems to be the protection against microbes. The *Slc11a1* gene is present in inbred strains of mice in two allelic forms which determine the resistance or susceptibility to several intracellular pathogens such as *Mycobacterium* spp., *Salmonella* spp and *Leishmania* spp (Vidal et al., 1993). Susceptible mice carry a glycine (G) to aspartic acid (D) substitution at position 169, resulting in a misfolding and loss of function of the protein (White et al., 2004). This mutation confers susceptibility to several mycobacterial species, such as *M. avium* (Appelberg and Sarmento, 1990; Gomes and Appelberg, 1998), *M. intracellulare* (Goto et al., 1989), *M. bovis* BCG (Gros et al., 1981) but not to *M. tuberculosis* (Medina and North, 1996; North et al., 1999). Although the G169D mutation has never been found in humans, polymorphic variations at or near human *SLC11A1* are associated with susceptibility to tuberculosis and leprosy in populations from areas of endemic disease (Fortier et al., 2005). The directionality of metal transport in the SLC11A1 is still not consensual. Some groups suggest that iron is transported via this protein into the pathogen-containing phagosome, causing the death of the pathogen by catalyzing the formation of reactive oxygen species (ROS) (Kuhn et al., 1999, 2001; Zwilling et al., 1999), while others argue for an iron efflux from the phagosome, restricting pathogen growth by iron deprivation (Gomes and Appelberg, 1998; Mulero et al., 2002). Studies of analogy with DMT1 (NRAMP2) (Forbes and Gros, 2003), the resistance of mycobacteria to ROS (Segal et al., 1999; Gomes and Appelberg, 2002) and the role of SLC11A1 in erythrocyte recycling (Soe-Lin et al., 2009) favor the second hypothesis, which is now seen as more consensual in the literature (Schaible and Kaufmann, 2004; Cellier et al., 2007; Wessling-Resnick, 2010).

Another iron transporter expressed in macrophages is ferroportin (SLC40A1). Ferroportin is present in the macrophage cytoplasmic membrane and is responsible for iron export (Knutson et al., 2005). Overexpression of ferroportin was reported to inhibit the intra-macrophagic growth of *M. tuberculosis* presumably through iron deprivation (Johnson et al., 2010). An interplay between the expression of ferroportin and the activation of the macrophage's inducible nitric oxide synthase (NOS2) seems to occur. While the overexpression of ferroportin decreased macrophages nitric oxide production (Johnson et al., 2010), the expression of NOS2 was reported by Nairz et al to be necessary to maintain ferroportin expression and iron efflux (Nairz et al., 2013). In this study, *Salmonella* residing within nitric oxide synthase deficient (*Nos2*−*/*−) macrophages acquired more iron than bacteria within wild-type macrophages and the authors concluded that iron deprivation is the main way through which NOS2 contributes to *Salmonella* control. However, while *M. avium* depends on access to iron to proliferate inside macrophages, *Nos2*−*/*<sup>−</sup> macrophages and mice are not more permissive to the growth of this mycobacterium (Gomes et al., 1999b), suggesting that the mechanism for the regulation of iron availability by NO during *Salmonella* infection does not apply to *M. avium* infections.

#### **IRON METABOLISM IN** *Leishmania* **INFECTION**

The leishmaniases are a complex of mammalian diseases characterized by distinct clinical manifestations: cutaneous, mucocutaneous and visceral leishmaniasis. Though considered to be neglected tropical diseases, their global incidence is a worrisome 2 million new cases per year. They are caused by protozoa of the genus *Leishmania*, whose transmission occurs through the bite of female sandfly. Transmission mediated by the insect may be zoonotic—between reservoir hosts (rodents and canines) and humans—or anthroponotic (Ready, 2010). Disease is not necessarily the final outcome in endemic areas where humans stably share the habitat with wild rodents, dogs and the sand fly populations as asymptomatic infections may occur.

*Leishmania* parasites have a digenetic life cycle, alternating between the promastigote stage in the insect gut and the amastigote stage in macrophages of mammalian hosts. Although *Leishmania* can infect diverse host cells (e.g., dendritic cells and neutrophils), there is only evidence for replication and long-term survival within mononuclear phagocytes (Kaye and Scott, 2011).

#### **HOST IRON STATE: EVIDENCES FROM HUMAN AND CANINE STUDIES**

A direct link between host iron status and human or canine susceptibility to *Leishmania* infection is not clearly found from available epidemiologic data. The fact that several studies found an association between susceptibility to leishmaniasis and polymorphisms in the *SLC11A1* gene, which codes for a transmembrane transporter of iron and other divalent metals, as mentioned before (Altet et al., 2002; Bucheton et al., 2003; Sanchez-Robert et al., 2008; Blackwell et al., 2009; Castellucci et al., 2010) and, on the other hand, the generalized association between leishmaniasis and malnutrition (Maciel et al., 2008; McCall et al., 2013) are indirect evidences that the host iron status may influence the outcome of the *Leishmania* infection. However, more direct correlations can only be made based on the studies performed in several infection models.

#### **HOST IRON STATE: EVIDENCES FROM RODENT MODELS**

Despite the lack of direct clinical evidences, we could expect iron to favor the growth of *Leishmania*, similarly to what is found for other pathogens (Weinberg, 2009). The fact that these parasites are equipped with diverse iron acquisition mechanisms and are capable of utilizing various iron sources suggested that iron acquisition was essential for pathogenicity and that iron deprivation could be an effective strategy to control leishmanial infections (Sutak et al., 2008; Taylor and Kelly, 2010). Such hypothesis is supported by the finding that the iron chelators desferrioxamine (DFO) and hydroxypyridin-4-ones moderately inhibit the multiplication of *L. major* and *L. infantum* promastigotes in culture medium (Soteriadou et al., 1995). However, DFO has shown either no effect (Murray et al., 1991) or an inhibitory effect on the intramacrophagic growth of *L. donovani* (Segovia et al., 1989; Das et al., 2009) and *L. amazonensis* (Borges et al., 1998). Moreover, treatment of mice with DFO does not affect the development of skin lesions caused by *L. major* (Bisti et al., 2000), but reduces the hepatic and splenic growth of *L. infantum* (Malafaia et al., 2011). Conversely, feeding mice with an iron-deficient diet did not influence *L. infantum* proliferation (Vale-Costa et al., 2013). Overall, the existing studies do not thoroughly support the notion that iron depletion contributes to the control of leishmanial infections.

Data obtained *in cellulo* concerning the impact of iron supplementation is also not consensual. Iron treatment either favored the intramacrophagic growth of *L. donovani* (Das et al., 2009) and *L. amazonensis* (Borges et al., 1998) and reversed the capacity of activated macrophages to eliminate *L. enriettii* (Mauel et al., 1991) or had no influence on both effects (Murray et al., 1991). Interestingly, the role of this nutrient on the *in vivo* growth of the parasite seems to be dependent on the host species. Iron given to hamsters prophylactically or therapeutically enhanced *L. donovani* replication (Garg et al., 2004). By contrast, *in vivo* iron administration to susceptible mice clearly leads to containment of *L. major* in the skin (Bisti et al., 2000, 2006; Bisti and Soteriadou, 2006) and *L. infantum* in the liver and spleen (Vale-Costa et al., 2013). The anti-leishmanial effect of iron is most likely due to its synergistic interaction with reactive oxygen and nitrogen species produced by the host's professional phagocytes (Bisti et al., 2006; Vale-Costa et al., 2013). Furthermore, the control of *L. major* infection, but not that of *L. infantum*, by iron also correlates with the development of a T helper 1 (Th1)-type immune response (Bisti et al., 2000), which is characterized by (i) an increased ability of splenic cells to present *L. major*-derived peptides, (ii) increased levels of IFNγ and NOS2 and decreased levels of IL-4 and IL-10 transcripts at the lesion site and (iii) reduced levels of serum immunoglobulin (Ig) E and IgG1 and increased levels of IgG2a (Bisti et al., 2000). Notably, iron overloaded mice are also resistant to re-infection with *L. major* (Bisti and Soteriadou, 2006). The iron-induced oxidative burst elicited during both primary and secondary infections with *L. major* is linked to the activation of the transcription factor NF-κB and with an enhanced proliferation of IFNγ-secreting CD4+ T cells in the draining lymph nodes (Bisti and Soteriadou, 2006). This is substantiated by the fact that iron and reactive oxygen and nitrogen species can modulate the activation of macrophagic NF-κB signaling pathways (Xiong et al., 2003; Leonard et al., 2004; Galaris and Pantopoulos, 2008) which are known to regulate numerous genes involved in immune and inflammatory responses (Bonizzi and Karin, 2004). Indeed, NF-κB regulates the development of IFNγ-secreting CD4+ T cells and concomitant resistance to *L. major* (Artis et al., 2003). Hence, iron not only seems to synergize with the host's oxidative mechanisms of defense, but also interacts with reactive oxygen and nitrogen species in order to activate signaling cascades that regulate the development of protective immunity against *Leishmania*.

The abovementioned reports clearly indicate that iron induces host protection against *Leishmania* infection. Future research should seek a confirmation of the inhibitory effect of iron on the *in vivo* growth of other *Leishmania* species and a better understanding of the molecular pathways involved in the iron-induced resistance against these protozoa.

### **THE FIGHT FOR IRON**

Like many other intracellular pathogens, *Leishmania* must be capable of acquiring iron from the host milieu in order to thrive. Besides holotransferrin (Borges et al., 1998), the growth and survival of *L. infantum* and *L. amazonensis* amastigotes can be supported by iron derived from haemoglobin and hemin (Carvalho et al., 2009). The uptake of haem by intramacrophagic *L. amazonensis* amastigotes is mediated by *Leishmania* haem response 1 (LHR1) protein (Huynh et al., 2012). Furthermore, intracellular *L. amazonensis* also possesses a ferric reductase, the *Leishmania* ferric iron reductase 1 (LFR1) (Flannery et al., 2011) which provides soluble ferrous iron for transport across the parasite plasma membrane by the ferrous iron transporter *Leishmania* iron transporter 1 (LIT1) (Huynh et al., 2006; Jacques et al., 2010). Moreover, LIT1-mediated iron acquisition seems to be essential for the differentiation of *L. amazonensis* parasites from the sandfly promastigote form to the macrophage-adapted amastigote form (Mittra et al., 2013).

Apart from the mechanisms of direct iron internalization, *Leishmania* parasites can also subvert the host's iron uptake systems to their own advantage. In fact, *L. amazonensis* amastigotes can obtain transferrin by forcing the fusion of transferrincontaining endosomes with the parasitophorous vacuole (Borges et al., 1998). Alternatively, *L. donovani* is capable of decreasing the macrophage labile iron pool, a process that triggers an increased surface expression of transferrin receptor 1 and internalization of transferrin, thus permitting a continuous provision of iron to the parasite (Das et al., 2009). This decrease in labile iron pool of activated macrophages has been recently proposed to be the result of the down-regulation of the expression of SLC11A1 by a *L. donovani*-secreted peroxidase (Singh et al., 2013). Also in line with these data, it has been reported that the expression of ferroportin is down-regulated in the spleen of *L. donovani*-infected mice, which may contribute to an increased accumulation of iron inside macrophages (Yang et al., 2002).

The existence of a parasitic strategy to counteract host SLC11A1 action reinforces the involvement of the latter in the *in vivo* control of infection by *Leishmania* (Vidal et al., 1995; Searle et al., 1998; White et al., 2005), as mentioned previously. This protein has also been implicated in the response to vaccination. Mice with functional SLC11A1 mount primarily a Th1 response to vaccination with the parasite metalloprotease Gp63 and display decreased skin lesions during a challenge infection with *L. major*. In striking contrast, mice with mutated SLC11A1 exhibit a Th2 response and an exacerbated lesion growth upon challenge (Soo et al., 1998).

Finally, we should acknowledge that the involvement of other host mechanisms of iron deprivation during leishmanial infections is largely unknown.

#### **IRON DEPRIVATION vs. IRON-INDUCED TOXICITY**

As the number of antibiotic resistant pathogens increases and the discovery of new antibiotics declines, the understanding of critical pathways in host-pathogen interaction emerges as a promising source of new approaches to fight infections. The modulation of iron availability may be one of such pathways. However, it should be noted that the choice of whether to provide or to deprive the pathogen of iron clearly depends on the microorganism in question.

The first widely used treatments for leishmaniasis relied on the administration of antimony complexes. Not excluding other possible mechanisms of action, strong evidences suggest that antimony compounds kill *Leishmania* parasites through the increased generation of ROS in the host (Ait-Oudhia et al., 2011) similarly to what we and others recently described for iron (Bisti et al., 2006; Vale-Costa et al., 2013). Of interest, other metal-containing drugs continue to be described as potential new therapies against *Leishmania* (Vale-Costa et al., 2012; Rocha et al., 2013). The main concern raised by these metal-providing tools is obviously the inherent toxicity of the metals associated with their propensity to induce oxidative stress. One of the possible ways to circumvent this problem will be to use new specific delivery systems to target the drug to infected macrophages.

In the case of mycobacterial infections, it is well-established that iron deprivation inhibits pathogen proliferation and iron depriving strategies seem the most promising in therapeutic terms. However, the administration of iron chelators is not exempt from risks to the host, especially in a context where the latter activates a series of iron withholding mechanisms which may lead to anaemia. The findings of the last decade on the players that control host iron metabolism, notably the identification of the iron exporter ferroportin and the hormone hepcidin, have opened new and exciting possibilities for the modulation of iron availability and localization inside cells which should provide ways of specifically depriving intracellular pathogens, without hampering the normal iron homeostasis of the host.

## **FUNDING**

Project "NORTE-07-0124-FEDER-000002-Host-Pathogen Interactions" co-funded by Programa Operacional Regional do Norte (ON.2—O Novo Norte), under the Quadro de Referência Estratégico Nacional (QREN), through the Fundo Europeu de Desenvolvimento Regional (FEDER) and by FCT (Fundação para a Ciência e Tecnologia).

## **REFERENCES**


Ready, P. D. (2010). Leishmaniasis emergence in Europe. *Euro Surveill.* 15, 19505


response is dependent on tumour necrosis factor activity in a murine model of protracted peritonitis. *Mol. Med. Rep.* 6, 838–842. doi: 10.3892/mmr.2012.1004


*Mycobacterium tuberculosis*. *Proc. Natl. Acad. Sci. U.S.A.* 108, 1621–1626. doi: 10.1073/pnas.1009261108


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 29 July 2013; accepted: 21 November 2013; published online: 09 December 2013.*

*Citation: Silva-Gomes S, Vale-Costa S, Appelberg R and Gomes MS (2013) Iron in intracellular infection: to provide or to deprive? Front. Cell. Infect. Microbiol. 3:96. doi: 10.3389/fcimb.2013.00096*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2013 Silva-Gomes, Vale-Costa, Appelberg and Gomes. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Zinc and copper toxicity in host defense against pathogens: *Mycobacterium tuberculosis* as a model example of an emerging paradigm

## *Olivier Neyrolles 1,2\*, Elisabeth Mintz 3,4,5 and Patrice Catty3,4,5*

*<sup>1</sup> Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France*

*<sup>2</sup> Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, Université Paul Sabatier, Toulouse, France*

*<sup>3</sup> Laboratoire de Chimie et Biologie des Métaux, Commissariat à l'Energie Atomique, Institut de Recherches en Technologies et Sciences pour le Vivant, Grenoble,*

*France <sup>4</sup> Centre National de la Recherche Scientifique, UMR 5249, Grenoble, France*

*<sup>5</sup> UMR 5249, Université Grenoble-Alpes, Grenoble, France*

*\*Correspondence: olivier.neyrolles@ipbs.fr*

#### *Edited by:*

*Frédéric J. Veyrier, Institut Pasteur, France*

#### *Reviewed by:*

*Heran Darwin, New York University, USA*

**Keywords:** *Mycobacterium tuberculosis***, macrophage, P-type ATPase, zinc, copper**

Microbial killing inside macrophages and other phagocytes involves a variety of mechanisms, including, for instance, acidification of the phagocytosis vacuole—or phagosome—and the production of toxic oxygen and nitrogen radicals (Flannagan et al., 2009). In addition, the immune modulation of nutrients available for microbial development in infected cells and tissues is a re-emerging concept referred to as "nutritional immunity" (Weinberg, 1975). This concept mostly developed from knowledge of the intracellular microbial starvation mechanism involving phagosomal iron and manganese depletion through the metal transporter NRAMP (Hood and Skaar, 2012). Growing evidence suggests that immune defense against microorganisms also involves microbial killing by transition metals, such as zinc and copper, present in excess in the microbial environment, and a set of recent reports showed that several bacterial pathogens, such as the tuberculosis (TB) bacillus, *Mycobacterium tuberculosis*, require transition metal efflux and detoxification systems to thrive inside their host.

Zinc and copper play key functions in all biological systems. Bioavailable levels of zinc are sufficiently low that most microbes have evolved high affinity transport systems to capture this metal. Bacterial zinc transporters are usually ABC transporters consisting of a periplasmicbinding protein, a membrane permease, and an ATPase (Hantke, 2005). Proteins involved in zinc import in mycobacteria have yet to be discovered. Regarding copper, as in most bacterial species, uptake systems for this metal have not been identified in *M. tuberculosis*.

Metallobiology of zinc and copper in *M. tuberculosis* recently provided insights into novel host defense mechanisms against bacterial infection involving intoxication by these transition metals. To resist potential intoxication by metal ions (Nies, 1999; Mergeay et al., 2003; Silver and Phung, 2005), microbes express a range of metal efflux pumps and transporters belonging to three main families: heavy metal efflux members of the resistance–nodulation–cell division superfamily (HME-RND), the cation diffusion facilitators (CDF) family, and the P-type ATPase family (Nies, 2003). A set of recent studies strikingly reported that some of these efflux systems are required for microbial virulence in various bacterial species, including the TB bacillus, in order to resist newly described immune mechanisms relying on metal poisoning of microbes inside host cells. The *M. tuberculosis* genome (Cole et al., 1998; Nies, 2003) contains no member of the HME-RND family and only one putative CDF transporter (Rv2025c). In addition, it contains no member of the recently discovered MntX family involved in Mn2<sup>+</sup> efflux (Veyrier et al., 2011) and no close homolog of ZntB, a member of the CorA family shown to mediate Zn2<sup>+</sup> efflux in *Salmonella* (Worlock and Smith, 2002). However, the *M. tuberculosis* genome codes for the striking amount of 12 P-type ATPases, whose substrate specificity is still partially unknown and that might, at least for some of them, result from gene duplication and gene loss from common gene ancestors (Botella et al., 2012). In addition, mycobacteria possess a Cu+-binding metallothionein, MymT (Gold et al., 2008) that is part of a copper-regulated gene cluster, or regulon, regulated by the transcriptional repressor RicR (Festa et al., 2011), and that is involved in resistance to copper toxicity. MymT binds up to six Cu+ ions and may contribute to resistance to copper overload by either or both chelating copper inside the bacterial cytoplasm, or/and extruding copper through a yet to be identified transport system that might involve the LpqS and Rv2963 proteins (Festa et al., 2011).

Inference on selectivity of P-type ATPases for various metal ion species can be drawn from similarity to known transporters and from the presence of conserved metal-binding motifs. From a general viewpoint, metal specificity of a protein mostly follows the HSAB rule (Pearson, 1963). Briefly, hard acids like Na+, K+, Ca2+, or Mn2<sup>+</sup> bind strongly to hard bases like carboxyl or hydroxyl groups whereas soft acids like Cu+ or Cd2<sup>+</sup> prefer coordination with soft bases like thiol groups. This rule applies for metals transporters and is particularly well-illustrated with the ion selectivity of P-ATPases. Ion transport mechanism by P-ATPases consists in coupling between ATP hydrolysis occurring in the cytoplasmic domain of the transporter, and ion motion from one side to the other side of the bilayer, through the transmembrane (TM) domain of the transporter. This mechanism is very well-conserved throughout evolution. Ion selectivity of P-ATPases follows the HSAB rule and is determined by conserved amino acids, located at conserved positions in the TM domain of the transporter. For instance, Ca2+-ATPase binds Ca2<sup>+</sup> thanks to Asp, Glu, Thr, and Asn residues located in TM helices 4, 5, 6, and 8. The same residues are found in the K+- and Na+-binding sites of the Na+/K+-ATPase (Bublitz et al., 2010). Metal transporting PIB-ATPases have been classified into five subfamilies on the basis of sequence homology (Arguello, 2003). Interestingly, this study reveals that each subfamily possesses conserved amino acids in TM helices 6, 7, and 8, likely to be involved in metal coordination, and these data, collected from *in silico* analysis of 249 sequences, corroborate biochemical data obtained on PIB-ATPases (Supplementary Table 1).

One of the *M. tuberculosis* P-type ATPases is KdpB, the ubiquitous PIA-type K+-ATPase (**Figure 1A**). Among the others, CtpA-D, CtpG, CtpJ, and CtpV display features of soft metal PIB-type ATPases (Arguello, 2003), i.e., a 4/2/2 arrangement of membrane spanning helices and specific sequence signatures in TM6, TM8, and the (N-P) domain (**Figure 1B**). Some of these sequences coincide with those determined from multiple alignments and allow assigning metal specificity. CtpA, CtpB, and maybe CtpV, would belong to the PIB1-type subfamily of Cu+-ATPases, while CtpD and CtpJ would belong to the PIB4-type subfamily of Co<sup>2</sup>+-ATPases. Less conserved motifs are found in CtpC

tree using TreeView; ∗, indicates the presence of a gene that could encode a metallochaperone, upstream the P-type ATPase encoding gene. **(B)** Hydrophobicity profiles are shown for the 12 P-type *M. tuberculosis* ATPases. Sequences are aligned on the phosphorylation motif (encircled P in red); black squares represent membrane spanning helices. Ion-binding motifs in P1B-type ATPases: 1, Cys-X2-Cys; 2,

Pro-Glu-Gly-(Leu/Met)-Pro; 5, Leu-Trp-X-Asn-X3-Asp. A domain: actuator domain; N domain: nucleotide-binding domain; P domain: phosphorylation motif. The method to identify transmembrane domains has been previously described (Kyte and Doolittle, 1982), and is accessible on the ExPASy website: http://web.expasy.org/protscale/. The analysis was performed using a 21-amino acid window.

and CtpG, preventing any prediction to be made about their metal selectivity. CtpE, CtpF, CtpH, and CtpI all exhibit a Pro-Glu-Gly-Leu-(Pro/Val) motif in the membrane spanning helix located upstream the phosphorylation site. This motif is found in all Ca2+-ATPases where it participates to the calcium transport site. In *M. tuberculosis*, only CtpF looks like a canonical Ca2+-ATPase, with a 2/2/6 arrangement of membrane spanning helices and a conserved motif in TM6.

The recent findings that *M. tuberculosis* mutants inactivated in *ctpV* and *ctpC*, are highly sensitive to copper and zinc, respectively (Ward et al., 2010; Botella et al., 2011; Padilla-Benavides et al., 2013), strongly suggest these two P-type ATPases transport these metal ions, but such a suggestion is not a formal proof of the metal selectivity. Biochemical characterization of these transporters in recombinant biological systems and in reconstituted liposomal fractions will be required to understand their exact function. In this context, a striking feature of three P-type ATPase members in *M. tuberculosis*, namely CtpC, CtpG, and CtpV, is the presence of a putative metallochaperone-encoding gene, namely and respectively Rv3269, Rv1993c, and Rv0968, upstream of the P-type ATPase-encoding genes that may play a part in metal selectivity and transport mechanism of their cognate P-type ATPase, as recently demonstrated for a similar transport system in *Streptococcus pneumoniae* (Fu et al., 2013).

A role for P-type ATPase-mediated metal detoxification in *M. tuberculosis* has been recently suggested by several independent reports. In particular *M. tuberculosis* mutants inactivated in the P-type ATPase-encoding genes *ctpV* and *ctpC* were shown to be impaired in their ability to proliferate in model animals and/or host macrophages (Ward et al., 2010; Botella et al., 2011). These results suggesting that *M. tuberculosis* is facing copper intoxication *in vivo* during infection were further strengthened by another report where it was shown that the outer membrane channel protein Rv1698/MctB is also required for both copper detoxification *in vitro* and for full virulence *in vivo* in guinea pigs (Wolschendorf et al., 2011). It was thus proposed that copper accumulation inside the mycobacterial phagosome might account for the phenotype of the *ctpV* and *mctB* mutants *in vivo* (Botella et al., 2012; Rowland and Niederweis, 2012; Samanovic et al., 2012). Several mechanisms have been proposed to explain copper ion toxicity, and the exact mechanism(s) of copper toxicity in *M. tuberculosis* remain to be identified (Rowland and Niederweis, 2012).

Regarding CtpC, we reported that genetic inactivation of this P-type ATPase dramatically increases *M. tuberculosis* sensitivity to Zn2+, which strongly suggested CtpC might be involved in zinc efflux (Botella et al., 2011). However, a recent report suggested that CtpC may transport Mn2<sup>+</sup> over Zn2+, and that the hypersensitivity of the *ctpC* mutant to zinc may be due to an increased sensitivity to oxidative stress following impaired Mn2<sup>+</sup> loading of the Fe2+-, and maybe Mn2+-, cofactored superoxide dismutase (SOD) SodA, and possibly other detoxification systems (Padilla-Benavides et al., 2013). In line with this hypothesis, it was shown recently that P-type ATPase-mediated copper export is required for copper supply to periplasmic Cu,Zn-SOD and resistance to oxidative stress in *Salmonella enterica* (Osman et al., 2013). Whether copper and zinc export through CtpV, CtpC, and possibly other P-type ATPases contributes to activation of the periplasmic Cu,Zn-SOD SodC in *M. tuberculosis* remains to be evaluated. Inside macrophages, we showed that zinc accumulates within *E. coli*- or *M. tuberculosis*-containing phagosomes, and that bacterial strains impaired in resistance to zinc (i.e., a *zntA* mutant in *E. coli* or a *ctpC* mutant in *M. tuberculosis*) are impaired in intracellular survival. *In vivo* attenuation of the *M. tuberculosis ctpC* mutant still has to be clearly established (Botella et al., 2011; Padilla-Benavides et al., 2013). The apparent discrepancy between our results suggesting that CtpC transports zinc (Botella et al., 2011) and the results reported by Padilla-Benavides and colleagues, suggesting that this P-ATPase transports manganese over zinc and other metal ions (Padilla-Benavides et al., 2013), might be explained by the fact that these authors did not include the putative CtpC metallochaperone Rv3269 in their *in vitro* systems. Rv3269 contains a clear putative zinc-binding motif (DLHDHDH) in its C-terminus end, which might confer zincspecificity to CtpC; this remains to be evaluated. Finally, the mechanism(s) of zinc ion toxicity in *M. tuberculosis* have yet to be discovered, and may include inactivation of iron-sulfur clusters, and inhibition of manganese uptake through transport competition in the bacterial periplasm (McDevitt et al., 2011; Xu and Imlay, 2012).

In summary, it is clear that *M. tuberculosis* uses P-type ATPases, such as CtpC and CtpV, and other systems, such as the metallothionein MymT, to resist poisoning by metal ions, such as Zn2<sup>+</sup> and Cu+, and thrive inside its host. The function of the other *M. tuberculosis* P-type ATPases, and their possible implication in mycobacterial virulence, remains to be understood. Equally important will be to understand the function of the putative metallochaperones associated to CtpC, CtpG, and CtpV, which may open novel venues for the development of new therapeutic strategies.

### **ACKNOWLEDGMENTS**

The authors received no specific funding for this work. The laboratory of Olivier Neyrolles is supported by the Centre National de la Recherche Scientifique (CNRS), the Fondation pour la Recherche Médicale (FRM), the Agence Nationale de la Recherche, the European Union, and the Fondation Mérieux. The funders had no role in the decision to publish this article or in its preparation.

#### **SUPPLEMENTARY MATERIAL**

The Supplementary Material for this article can be found online at: http:// www.frontiersin.org/journal/10.3389/fcimb. 2013.00089/abstract

## **REFERENCES**


*Received: 25 September 2013; accepted: 12 November 2013; published online: 27 November 2013.*

*Citation: Neyrolles O, Mintz E and Catty P (2013) Zinc and copper toxicity in host defense against pathogens: Mycobacterium tuberculosis as a model example of an emerging paradigm. Front. Cell. Infect. Microbiol. 3:89. doi: 10.3389/fcimb.2013.00089*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2013 Neyrolles, Mintz and Catty. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Pathogenic adaptations to host-derived antibacterial copper

## *Kaveri S. Chaturvedi and Jeffrey P. Henderson\**

*Division of Infectious Diseases, Department of Internal Medicine,Center for Women's Infectious Diseases Research, Washington University School of Medicine, St. Louis, MO, USA*

#### *Edited by:*

*Frédéric J. Veyrier, Institut Pasteur, France*

#### *Reviewed by:*

*Dennis J. Thiele, Duke University School of Medicine, USA James Imlay, University of Illinois at Urbana-Champaign, USA*

#### *\*Correspondence:*

*Jeffrey P. Henderson, Center for Women's Infectious Disease Research, Washington University School of Medicine, Box 8051, 660 S. Euclid Ave., St. Louis, MO 63110, USA*

*e-mail: jhenderson@dom.wustl.edu*

Recent findings suggest that both host and pathogen manipulate copper content in infected host niches during infections. In this review, we summarize recent developments that implicate copper resistance as an important determinant of bacterial fitness at the host-pathogen interface. An essential mammalian nutrient, copper cycles between copper (I) (Cu+) in its reduced form and copper (II) (Cu2+) in its oxidized form under physiologic conditions. Cu<sup>+</sup> is significantly more bactericidal than Cu2<sup>+</sup> due to its ability to freely penetrate bacterial membranes and inactivate intracellular iron-sulfur clusters. Copper ions can also catalyze reactive oxygen species (ROS) generation, which may further contribute to their toxicity. Transporters, chaperones, redox proteins, receptors and transcription factors and even siderophores affect copper accumulation and distribution in both pathogenic microbes and their human hosts. This review will briefly cover evidence for copper as a mammalian antibacterial effector, the possible reasons for this toxicity, and pathogenic resistance mechanisms directed against it.

**Keywords: copper, pathogenesis, yersiniabactin, copper tolerance, metal biology, copper resistance**

#### **INTRODUCTION**

Copper is both an essential mammalian micronutrient and a potent antibacterial agent. The Smith Papyrus, an ancient Egyptian medical text dated at 2400 BC, is the earliest medicinal archive to recommend copper sulfate to sterilize water and treat infections (Dollwet and Sorenson, 1985). Mesoamerican and Hellenistic civilizations used copper and copper salts to treat a broad variety of physical ailments, including microbial and parasitic infections. In 400 BC, Hippocrates prescribed copper salts to treat leg ulcers. In the nineteenth century, Victor Burq observed that copper workers in Paris appeared immune to recurrent cholera epidemics (Burq, 1867). He also noted that while neighboring towns were ravaged by frequent cholera epidemics, the pottery-making town of Aubagne was protected from these outbreaks. He attributed this protection to "*...* a rampart of copper dust" generated by copper-rich clay used by the city's potters. These observations led to rapid developments in the field of metallotherapy, and medically employed copper salts, amulets, and belts were widely used to treat dermatologic, gastrointestinal, and tubercular infections (Dollwet and Sorenson, 1985; Borkow, 2005) until the advent of commercially available antibiotics in 1932.

Human and animal studies now suggest a parallel between ancient medicinal copper use and antibacterial immune function. In this review, we summarize copper homeostasis mechanisms in the human host, and the means by which the host deploys the metal to combat infections. We describe the chemical and biochemical principles that define copper's toxicity, and how these toxic properties serve as potent leverage against invading pathogens. Finally, we discuss the pathogenic molecular, cellular, and biochemical responses that counter copper toxicity at host interface.

#### **COPPER AS NUTRIENT OR TOXIN**

With photosynthesis and dioxygen release in the atmosphere 2.7 billion years ago, the sulfides that sequestered copper were oxidized to sulfates, leading to increased copper bioavailability (Frausto da Silva and Williams, 1993). Copper-containing proteins appeared relatively late in an evolutionary timescale, likely in response to increasing need to use oxygen and oxygen containing molecules (Dupont et al., 2011; Nevitt et al., 2012). These enzymes are critical to cellular, biochemical and regulatory functions in the human host, leading to a nutritional requirement for sufficient copper levels. The most prominent examples include cytochrome c oxidase, the respiratory chain terminal electron acceptor, and Cu-Zn superoxide dismutase, required for defense against oxidative damage (Karlin, 1993). Putative copper binding proteins compose ∼1% of the total eukaryotic proteome, suggesting that known cuproproteins represent only a minor fraction of the total (Andreini et al., 2008). Copper's role in host biology and defense is better understood by examining its chemistry.

#### *Copper chemistry*

Copper is the 26th most abundant in the earth's crust and exists as 2 stable and 9 radioactive isotopes. A transition metal, copper primarily exists as one of two stable oxidation states: Cu2<sup>+</sup> in the oxidized cupric form, and Cu+ in the reduced cuprous form. Cu+ is a closed shell 3d10 transition metal ion with diamagnetic properties (Frausto da Silva and Williams, 1993). A soft Lewis acid, it favors tetrahedral coordination with soft bases such as hydrides, alkyl groups, cyanide, phosphines, and thiols from cysteine and thioether bonds with methionine (Crichton and Pierre, 2001). Cu2<sup>+</sup> has a 3d9 configuration, is paramagnetic, and is an intermediate Lewis acid. In addition to ligands bound by Cu+, Cu2<sup>+</sup> forms square planar complexes with sulphates, nitrates, nitrogen donors such as histidine, and oxygen donors like glutamate and aspartate (Bertini et al., 2007). Different ligand combinations, oxygenation levels, pH, organic matter, sulfates and carbonates, generate differential metal speciation and distinct metal coordination environments. Copper's value as a bioelement lies mainly in its unique electrochemical properties. The Cu+/Cu2<sup>+</sup> couple has a high redox potential, which allows it to act as an electron donor/acceptor in redox reactions (Crichton and Pierre, 2001). Most copper enzymes span a range of +200 to +800 mV, enabling them to directly oxidize substrates such as ascorbate, catechol, and phenolates. The same electrochemical properties contribute to copper's toxic effects through several mechanisms, outlined below.

#### *Copper as a Fenton reagent*

Within superoxide and hydrogen peroxide-rich environments such as the phagosome, copper may propagate toxic hydroxyl radical formation by Fenton-like chemistry [Equation (1)] (Liochev, 1999).

$$\mathrm{Cu}^+ + \mathrm{H}\_2\mathrm{O}\_2 \rightarrow \mathrm{Cu}^{2+} + \mathrm{OH}^- + \mathrm{OH}\bullet \tag{1}$$

Hydroxyl radicals are extremely reactive, cannot be scavenged by enzymatic reaction, and have a diffusion controlled halflife of <sup>∼</sup>10−<sup>9</sup> s before reacting with organic molecules *in vivo* (Freinbichler et al., 2011), suggesting that hydroxyl radical damage would occur in close spatial proximity to copper ions. Extensive work has implicated reactive oxygen species (ROS) derived from metal-catalyzed oxidation in lipid, protein, and DNA oxidation (Yoshida et al., 1993; Liochev, 1999; Stadtman, 2006). Copper ions can also oxidize sulfhydryls such as cysteine or glutathione in a cycle between reactions [Equations (2), (3a,b) or (4a,b), followed by (**5**)]:

$$\text{RSH} + \text{Cu}^{2+} \rightarrow \text{RS} \bullet + \text{Cu}^{+} + \text{H}^{+} \quad \text{(2)} \quad \text{and}$$

$$\text{RS} \bullet + \text{O}\_{2} \rightarrow \text{RS}^{+} + \text{O}\_{2}^{-} \qquad \text{(3a)}$$

$$\text{RS}^{+} + \text{RSH} \rightarrow \text{RSSR} + \text{H}^{+} \quad \text{(3b)} \quad \text{or}$$

$$\text{RS} \bullet + \text{RSH} \rightarrow \text{RSSR} + \text{H}^{+} \quad \text{(4a)}$$

$$\text{RSSR} \bullet + \text{O}\_{2} \rightarrow \text{RSSR} + \text{O}\_{2}^{-} \quad \text{(4b)} \quad \text{followed by}$$

$$\text{O}\_{2}^{-} + \text{H}^{+} \rightarrow 1/2 \text{H}\_{2}\text{O}\_{2} \qquad \text{(5)}$$

Hydrogen peroxide can in turn participate in reaction 1 and may further propagate radical formation.

Attempts to understand copper toxicity through classic copper-catalyzed Fenton chemistry to copper toxicity have produced contrary results. Macomber et al. exposed an *Escherichia coli* mutant with multiple copper efflux deficiencies to hydrogen peroxide (Macomber et al., 2007). Rather than exhibiting greater peroxide sensitivity [through Equation (1)], copper-loaded *E. coli* were instead more resistant to hydrogen peroxide. Furthermore, copper loading was associated with fewer, not more, oxidative DNA lesions. Lastly, EPR spectroscopy revealed no change in hydroxyl radical generation with copper addition. Most of the copper in overloaded strains was localized to the periplasm, where any hydroxyl radical generated would react locally before reaching DNA in the cytoplasm. This spatial compartmentalization may explain the lack of DNA damage. While there may exist circumstances in which copper propagates cytotoxic Fenton chemistry *in vivo*, this work suggests the existence of an alternative copper toxicity mechanism in *E. coli*.

#### *Non-Fenton destruction of iron-sulfur complexes by copper*

Recent evidence suggests a non-Fenton chemistry copper toxicity mechanism in which the reduced Cu+ ion is instrumental. Multiple investigators note that copper toxicity to bacteria is sustained or even enhanced in anoxic conditions (Beswick et al., 1976; Outten et al., 2001; Macomber and Imlay, 2009) where peroxide formation is minimal. Increased copper toxicity under anoxic conditions may reflect higher Cu+ prevalence. *E. coli* EPR spectroscopy indicates that considerable Cu2<sup>+</sup> is converted to non-paramagnetic Cu+ under anoxic conditions (Beswick et al., 1976). Macomber et al. show that intracellular copper in overloaded *E. coli* is in the reduced Cu+ valence, likely due to cytosolic reduction and its ability to enter bacteria by traversing bacterial membranes (Macomber et al., 2007). Cu+ toxicity in the *E. coli* cytosol can be explained by its intense thiophilicity, which is sufficient to competitively disrupt key cytoplasmic iron-sulfur enzymes both *in vitro* and *in vivo* (Macomber and Imlay, 2009). Indeed, other "soft" thiophilic metal ions that do not act as Fenton reagents have been found to exert comparable toxicity (Jozefczak et al., 2012; Xu and Imlay, 2012). Together, these data provide compelling evidence linking copper toxicity to iron displacement from solvent-exposed dehydratase iron-sulfur clusters, resulting in metabolic disruption and branched chain amino acid auxotrophy.

#### **COPPER AT THE HOST-PATHOGEN INTERFACE**

Copper homeostasis is essential for human growth and development. Average daily human dietary copper intake varies from 0.6 to 1.6 mg/dL, with a free copper ion concentration of 10−<sup>13</sup> M in human blood plasma (Linder and Hazegh-Azam, 1996). In mammalian cells, cytoplasmic metallothioneins, glutathione based redox maintenance, and the Cu/Zn superoxide dismutase mitigate copper toxicity (Fridovich, 1974; Babula et al., 2012; Hatori et al., 2012). This section reviews the basic characteristics of human copper transporters together with data that may speak to their functions during infection and inflammation.

#### *Human copper physiology*

Unlike antimicrobial peptides, proteolytic enzymes, or ROS, copper cannot be synthesized *in situ* during infections and so must be absorbed from the diet or mobilized from tissue depots for use by immune cells (see a more complete review Pena et al., 1999). Once dietary copper is absorbed from the intestinal lumen it is delivered to the liver, which exports it to the peripheral circulation or excretes it into the bile (Crampton et al., 1965; Vancampen and Mitchell, 1965). The liver incorporates copper into multiple proteins, including the secreted glycosylated multi-copper ferroxidase ceruloplasmin (Holmberg and Laurell, 1948). Ceruloplasmin-copper complexes bind Ctr1, an integral membrane protein that is structurally and functionally conserved from yeast to humans (Zhou and Gitschier, 1997). Ctr1 transports 60–70% of the total copper in flux. Ctr1 is responsive to copper levels: copper depletion increases Ctr1 expression at the plasma membrane through the recruitment from the intracellular pools, whereas elevated copper induces rapid transporter endocytosis from the plasma membrane to vesicles (Zhou and Gitschier, 1997; Petris et al., 2003; Guo et al., 2004). Following internalization by Ctr1, copper is shuttled to the trans-Golgi network by ATOX1/HAH1 in secretory compartments (Klomp et al., 1997). Atox1 gene deletion in mice results in perinatal lethality, reflecting its crucial role in normal cellular metabolism (Hamza et al., 2001). Copper is transferred directly from ATOX1 to the N-terminus of two homologous P1B-type ATPase Cu<sup>+</sup> transporters, ATP7A (Chelly et al., 1993; Mercer et al., 1993; Vulpe et al., 1999) and ATP7B (Bull et al., 1993; Tanzi et al., 1993; Vulpe et al., 1993), located in the trans-Golgi network. Macrophages infected with *Salmonella typhimurium* exhibit increased Ctr1, ATP7A and ceruloplasmin gene expression, indicating that they play a role in restricting infection by professional intracellular pathogens (Achard et al., 2012).

Copper fills varied roles in mammalian biology, and it is notable that copper-deficiency is associated with numerous deficiencies in host defense (Kaim and Rall, 1996). Mutations in ATP7A result in a severe copper-deficiency known as Menkes disease (Kaler, 2011). Infants with Menkes' disease are more susceptible to Gram-negative infections, consistent with copper's role in restricting microbial growth (Menkes et al., 1962; Danks et al., 1972; Gunn et al., 1984). Conversely, Wilson's disease is characterized by excess copper accumulation in brain and liver tissues, resulting in cirrhosis and neurodegeneration that may manifest well after infancy. Other human copper deficiency studies reveal impaired phagocytic indices, decreased antibody response, impaired peripheral mononuclear cell proliferation, lower early T-cell activation and proliferation, and lower cytokine expression (Sullivan and Ochs, 1978; Prohaska and Lukasewycz, 1990). While these conditions suggest a specialized role for copper in antibacterial immunity, caution must be taken to differentiate this from a less specific, more general nutritional role in the host (Newberne et al., 1968; Sullivan and Ochs, 1978; Boyne and Arthur, 1981; Jones and Suttle, 1983; Koller et al., 1987; Prohaska and Lukasewycz, 1990; Crocker et al., 1992; Smith et al., 2008).

### *Copper physiology during infections*

Although incompletely understood, there are indications that a coordinated physiologic response may increase both systemic and local copper availability during infections. Compared to normal controls, copper levels increase two- to ten-fold in the serum, livers and spleens of animals infected with a range of pathogens, including viruses, bacteria, and trypanosomes (Tufft et al., 1988; Crocker et al., 1992; Matousek De Abel De La Cruz et al., 1993; Ilback et al., 2003). Increased circulating copper may be selectively imported into infected sites, as indicated by two- to five-fold increase in copper-carrier proteins (Natesha et al., 1992; Chiarla et al., 2008). X-ray microprobe analyses indicate that copper's absolute atomic concentration in area density increases a hundred-fold to several hundred micromolar within granulomatous lesions of lungs infected with *Mycobacterium tuberculosis*, and high copper concentrations are selectively redistributed to the exudates of wounds and burns (Beveridge et al., 1985; Jones et al., 2001; Voruganti et al., 2005; Wagner et al., 2005). Whether this accumulation reflects uptake by myeloid cells alone or includes a tissue-wide response remains unclear.

## *Copper as a white blood cell antibacterial agent*

In 2009, White et al. published findings from cultured macrophage-like RAW264.7 cells that are consistent with a copper-specific bactericidal system directed against phagocytosed *E. coli* (White et al., 2009). Phagosomal killing of K12 *E. coli* was greatly affected by copper content of the cell culture media. Microscopy and posttranscriptional silencing investigations linked this copper-dependent activity to ATP7A-mediated copper trafficking from the Golgi apparatus to *E. coli*-containing phagolysosomes. These studies suggest that in addition to its role in physiologic copper absorption, ATP7A fills a host defense function by transporting antibacterial quantities of copper ions to phagolysosomal compartments containing engulfed bacteria. Consistent with this finding, low-density lipoprotein (LDL) oxidation by macrophage-like THP-1 cells was found to be ATP7A-dependent, suggesting metal catalyzed oxidation by secreted copper ions (Qin et al., 2010). ATP7A is expressed in a broad range of both myeloid and non-myeloid cell types (La Fontaine et al., 2010; Wang et al., 2011), raising the possibility that a variety of cell types may similarly direct the copper payloads to kill internalized bacteria. These observations suggest a specific functional rationale for the array of mammalian copper transport genes upregulated by proinflammatory stimuli such as interferon-gamma and lipopolysaccharide and for the altered copper physiology noted above in section Copper as a Fenton Reagent. (Achard et al., 2012). Studies to identify macrophage lineages or even non-professional phagocytes that use copper-mediated antibacterial activity would be of great interest in the area of infection biology. To date, copper-dependent uropathogenic *E. coli* killing has been observed in both RAW264.7 cells and mouse peritoneal macrophages (Chaturvedi et al., 2013). Altogether, these findings suggest an intriguing parallel between ancient medicinal copper use and innate immune function.

Phagosomal copper may add to, and perhaps synergize with, the diverse cellular microbial killing strategies described since Elie Metchnikoff's pioneering work on phagocytosis (Gordon, 2008). These strategies are often functionally redundant and have been broadly grouped into oxidative killing mechanisms exemplified by the macrophage respiratory burst and non-oxidative killing mechanisms such as antimicrobial peptides and hydrolytic enzymes. Interactions between copper and more established antibacterial effectors within the phagosome's restricted space are likely. Membrane permeabilizing defenses may facilitate copper entry into bacteria, while high concentrations of respiratory burst-derived oxidants are likely to modulate redox active copper ions. These interactions may be spatially and temporally governed during and after the respiratory burst. One recent finding in *E. coli* suggests that copper's interactions with phagosomal superoxide may greatly impact intracellular bacterial survival (see section Superoxide Dismutation).

Copper-mediated killing by vertebrate immune systems would be expected exert selective pressure on copper resistance in pathogenic bacteria. Below, we review the virulence-associated copper resistance systems described in several human pathogens. The classic intracellular pathogen *M. tuberculosis* upregulates genes encoding copper efflux-associated P1B-type ATPases during macrophage infection (Ward et al., 2008; Rowland and Niederweis, 2012). Urinary *E. coli* isolates collected from patients with urinary tract infections (UTIs) exhibit higher growth than concomitant rectal isolates in a medium containing an inhibitory concentration of copper (Chaturvedi et al., 2012). Copper resistance genes are often observed in virulence-associated mobile genetic elements carried by *E. coli* as well as *Legionella pneumophila*, *Klebsiella pneumoniae*, and methicillin resistant *Staphylococcus aureus* (Sandegren et al., 2012; Shoeb et al., 2012; Gomez-Sanz et al., 2013; Trigui et al., 2013). *E. coli* and *M. tuberculosis* strains with engineered deficiencies in copper resistance genes exhibit impaired intracellular survival in phagocytic cells (White et al., 2009; Wolschendorf et al., 2011; Chaturvedi et al., 2013). To date, these observations suggest that resistance to copper-mediated killing among pathogens may be a virulenceassociated property driven by host innate immunity.

#### **MECHANISMS OF MICROBIAL COPPER TOLERANCE**

Copper's direct and indirect toxicity can alter enzyme specificity, disrupt cellular functions, and damage nucleic acid structure. Changes in copper concentrations during infection suggest that the host harnesses the metal's toxic properties to combat microbial growth. In response, pathogenic bacteria have evolved a series of protein- and small-molecule based defenses against copper toxicity. Unlike eukaryotic cells, most known bacterial cuproproteins are located within the cytoplasmic membrane or in the periplasmic space, perhaps to compartmentalize a potentially toxic metal species. Microbes use this copper sparingly in metabolism, and for electron transport in respiratory pathways. Given this, copper's cytoplasmic availability is tightly controlled, and data indicate that there are fewer than 10<sup>4</sup> free copper atoms per bacterial cell, reflecting cytoplasmic copper-responsive transcriptional regulators' high copper sensitivity (Outten and O'Halloran, 2001; Changela et al., 2003; Finney and O'Halloran, 2003).

Both Cu<sup>+</sup> and Cu2+can permeate the outer membrane of *E. coli* and enter the periplasm, but only Cu+ is able to cross the inner membrane and reaches the cytoplasm by a currently unknown mechanism. While no copper uptake genes have yet been identified in *E. coli*, the outer-membrane protein ComC (under transcriptional control of the TetR-like regulator ComR) may reduce the outer membrane's copper permeability (Mermod et al., 2012). It is speculated that cytoplasmic Cu+ is largely complexed by millimolar quantities of thiols such as glutathione. Interestingly, glutathione biosynthesis gene deletion has little effect on microbial copper response, indicating that its role in detoxifying copper in bacterial cells may either be limited or redundant (Helbig et al., 2008). In this regard, qualitative and quantitative analyses of cytosolic copper binding sites in bacteria would aid our understanding of copper toxicity.

Microbial copper-resistance systems span copper efflux (*cue*, *cus*, and extrachromosomal efflux systems), copper sequestration (CusF and siderophores), and copper oxidation (mixed copper oxidases and superoxide dismutase mimics). For the sake of brevity, the following sections primarily discuss Cu2<sup>+</sup> detection and resistance proteins that have been described in *E. coli* (**Figure 1**). Their functional homologs in other microbial species are tabulated in **Table 1** (see a more complete review Rademacher and Masepohl, 2012).

## *Copper efflux*

*The cue system.* In *E. coli*, two chromosomal systems remove excess Cu+ from the cytosol (Outten et al., 2001). The *cue* system (for *Cu e*fflux) transcriptionally activates both plasmid- and chromosomally-encoded copper homeostatic systems in response to intracellular Cu+ sensing through CueR, a MerR-family metalloregulatory transcriptional activator (Petersen and Moller, 2000; Stoyanov et al., 2001). CueR coordinates one Cu+ ion per monomer in an unusual and distinctive linear S–Cu+–S center encompassing two cysteine residues (C112 and C120) located at the dimer interface (Changela et al., 2003; Chen et al., 2003). Both *holo*- and *apo*-CueR bind to dyad-symmetric sequences at target promoters, but only *holo*-CueR activates transcription (Yamamoto and Ishihama, 2005; Andoy et al., 2009). A genomewide transcriptional array study of the *E. coli* chromosome has identified 197 putative CueR-binding sites, which largely await experimental confirmation. Other bacteria that possess CueR-like copper-tolerance systems include *Pseudomonas aeruginosa* and *S. typhimurium* (Espariz et al., 2007; Pontel and Soncini, 2009; Thaden et al., 2010).

CueR is a copper-selective ortholog from multifunctional protein families that respond to a wide range of effector ligands (the MecI/BlaI-family repressors that mediate resistance to β-lactam antibiotics and the MerR family, respectively) (Brown et al., 2003; Portmann et al., 2006). While CueR is not widely distributed in

**FIGURE 1 | Copper resistance strategies across pathogenic** *E. coli* **membranes.** The virulence-associated siderophore yersiniabactin sequesters Cu2<sup>+</sup> outside the cell and prevents its reduction to the more toxic Cu+. Copper ions that reach the cytosol are subject to chelation by glutathione and export by two ATPases. The CusCBA ATPase complex exports Cu+ from both the cytoplasm and the periplasm (*via* CusF) to the extracellular space. Alternatively, the CopA ATPase exports cytoplasmic copper across the inner membrane. Periplasmic Cu+ can bind the proteins CusF and PcoE or be oxidized by the mixed copper oxidases CueO or PcoA to less toxic Cu2+. PcoB has a putative function of exporting Cu2<sup>+</sup> across the outer membrane. The systems are oriented to minimize free cytosolic copper ions by directing these to the periplasmic or extracellular spaces.

#### **Table 1 | Species-wide distribution of copper-resistance proteins.**


*\*Confers additional protection from gold toxicity (Espariz et al., 2007; Pontel et al., 2007).*

bacterial genomes, Liu et al. describe one such copper-specific ubiquitous regulator (Liu et al., 2007). The intracellular copper sensor CsoR from *M. tuberculosis* is the founding member of what appears to be a large family of bacterial Cu+-responsive repressors, with greater than 170 projected members in archaeal, bacterial, and cyanobacterial genomes (Liu et al., 2007). Upon copper binding, CsoR is deactivated, leading to copper-resistance gene expression.

CueR upregulates *copA* and *cueO* gene expression (Outten et al., 2000; Stoyanov et al., 2001). These genes are associated with copper efflux and oxidation, respectively. CopA is a copperexporting P1B-type ATPase active under high extracellular copper stress (Outten et al., 2000; Petersen and Moller, 2000; Fan and Rosen, 2002; Stoyanov et al., 2003). Mammalian and microbial P1B-type ATPases thus perform opposing functions that determine infection outcomes. Appropriate copper import and trafficking by mammalian ATPases is required to restrict microbial growth, while copper export by microbial ATPases is necessary to withstand this toxicity. CopA traverses the inner membrane and exports Cu+ from the cytosol in both oxic and anoxic conditions (Fan and Rosen, 2002; Kuhlbrandt, 2004; Arguello et al., 2007; Osman and Cavet, 2008). This efflux pump couples ATP hydrolysis to form an acylphosphate intermediate in the presence of Cu<sup>+</sup> but not Cu2+. It is speculated that two amino-terminal metal binding domains with a CXXC motif confer metal binding specificity. *copA* mutants in *E. coli*, *Streptococcus pneumoniae*, and *Neisseria gonorrhoeae* all demonstrate impaired copper efflux, intracellular metal accumulation, and increased copper sensitivity in both oxic and anoxic conditions (Rensing et al., 2000; Outten et al., 2001; Shafeeq et al., 2011; Djoko et al., 2012).

*The cus system.* An independent copper efflux system, the *cus* (for *Cus* ensing) system confers copper-tolerance under moderate to high copper levels in oxic conditions (Outten et al., 2001). *cusRSCFBA* products are believed to form a multiunit transport complex that spans the periplasmic space and is anchored in both the inner and outer membranes (Mealman et al., 2012). While CopA exports excess Cu+ from the cytoplasm to the periplasm, CusRSCFBA effluxes Cu+ from the periplasm (Outten et al., 2001; Franke et al., 2003; Long et al., 2010).

CusRS is a two-component regulatory system that monitors copper stress in the cell envelope and is particularly active in anoxic copper stress conditions (Munson et al., 2000). In addition to CusRS, CpxRA, and YedWV are two other previously described copper-responsive *E. coli* two-component regulatory systems (Yamamoto and Ishihama, 2005, 2006). CusR and CusS exhibit homology with other plasmid-borne two-component systems that are also involved in metal responsive gene regulation. Membrane bound CusS senses periplasmic Cu+, which leads to protein autophosphorylation. CusS then donates the phosphoryl group to CusR, which activates the transcription of the *cusCFBA* and *cusRS* operons. CusA belongs to the resistance-nodulationcell division (RND) proton antiporter family, CusB belongs to the membrane fusion protein family which anchor into the cytoplasmic membrane with a long periplasm-spanning domain, and CusC is an outermembrane protein with homology to the TolCstress response protein (Franke et al., 2003; Delmar et al., 2013). CusF is a periplasmic metallochaperone that binds a single atom of Cu+ and participates in metal efflux by delivering the metal to CusC and CusB (Xue et al., 2008; Mealman et al., 2011).

Other prominent RND proton antiporters include the multidrug efflux systems AcrB and AcrF from *E. coli*, MexB from *P. aeruginosa*, and MtrD from *N. gonorrhoeae* (Nies and Silver, 1995; Paulsen et al., 1996). Interestingly, *Cupriavidus metallidurans* CH34 resistance to copper is attributed to RND protein expression (von Rozycki and Nies, 2009).

*Extrachromosomally-encoded copper efflux systems.* In environments where copper concentrations would overwhelm chromosomally encoded copper metabolic systems, microbes contain extrachromosomal loci that confer copper resistance. These loci are present in copper-resistant *E. coli*, *Pseudomonas syringae*, and *Xanthomonas campestris* pv. vesicatoria isolates (Tetaz and Luke, 1983; Bender and Cooksey, 1987; Brown et al., 1992; Voloudakis et al., 1993; Williams et al., 1993). All copper-resistant strains were isolated from agricultural areas characterized by repeated copper salt application as a feed additive, bactericidal agent, or antifungal agent. In these strains, the plasmid borne *pco* and *cop* operons confer copper resistance. These operons carry four related genes, *pcoABCDRSE* and *copABCDRS,* which are expressed from chromosomal copper-inducible promoters regulated by CusRS (Brown et al., 1995; Adaikkalam and Swarup, 2005). The genes *copABCDRS* are arranged in two operons, *copABCD* and *copRS*, respectively. This arrangement is also found in the *pco* determinant but with an additional gene, *pcoE*, further downstream. Extrachromosomal systems encode two-component regulators similar to CusRS, including PcoR and PcoS from the *pco* operon of *E. coli*; CopR and CopS from the *cop* operon, which provides copper resistance to *P. syringae*; and SilR and SilS from the *sil* locus, which provides silver ion resistance to *Salmonella enterica* serovar Typhimurium (Gupta et al., 1999). Similar to these copper efflux systems, extrachromosomal *pco* system encodes PcoB and PcoD, two copper pumps that are incorporated in the outer and inner membranes, respectively (Lee et al., 2002).

Extrachromosomal resistance systems are metal oxidation state selective. Recently published PcoC spectroscopic and crystallographic data and nuclear magnetic resonance (NMR) studies of the closely related *P. syringae* protein, CopC, reveal a biologically unprecedented thioether ligation (Arnesano et al., 2003a,b; Peariso et al., 2003). PcoC can bind both Cu2+and Cu+: the protein exhibits a cupredoxin fold that binds Cu+ through two Met sulfur atoms and one nitrogen or oxygen ligand in a hydrophobic Metrich loop that is exposed to solvent on the protein surface. Cu2<sup>+</sup> can bind a separate site in the same protein, where it coordinates water, as well as two histidine imidazoles and two other nitrogen or oxygen ligands. Following copper sensing, microbes respond to microenvironments that contain high concentrations of unligated copper by upregulating systems associated with copper efflux, oxidation, or sequestration.

### *Copper sequestration*

In addition to copper oxidation and efflux systems, recent studies suggest that bacteria deploy both low molecular weight proteins and small molecules to bind and sequester intracellular copper. In *E. coli*, the periplasmic chaperone CusF binds copper, ultimately delivering it to CusCBA for export (Franke et al., 2003; Bagai et al., 2008; Xue et al., 2008; Mealman et al., 2012). Evidence indicates that PcoE acts as a soluble copper binder in the periplasm (Zimmermann et al., 2012). Across kingdoms, metallotheioneines sequester cytoplasmic copper (Leszczyszyn et al., 2011; Thirumoorthy et al., 2011; Gumulec et al., 2012). Recent work in *M. tuberculosis* shows that a five-locus regulon for copper resistance is upregulated during copper stress (Festa et al., 2011). This regulon includes MymT, a cytoplasmic metallothionein that binds Cu+ and attenuates copper toxicity (Gold et al., 2008). Although a native *E. coli* metallothionein has not yet been identified, data suggest that glutathione may exert similar cytoprotective effects by forming stable Cu+ complexes (Osterberg et al., 1979; Helbig et al., 2008; Macomber and Imlay, 2009).

Some microbial siderophores, low-molecular-weight iron chelating agents, sequester copper extracellularly and protect bacteria by minimizing intracellular copper penetration. There is precedent for this among environmental bacteria that express Cu+-binding compounds (those originally identified as copper binders are called chalkophores) such as methanobactin and phytochelatin (Cervantes and Gutierrez-Corona, 1994; Rauser, 1999; Kenney and Rosenzweig, 2012). In *E. coli*, chemically distinct siderophore types are observed to exert opposing copper phenotypes. Specifically, the catecholate siderophore enterobactin sensitizes *E. coli* to copper, likely through its ability to reduce cupric ion to the more toxic cuprous ion (Grass et al., 2004). Although known as a cuprous oxidase, CueO prevents this interaction by directly oxidizing catechols such as dihydroxybenzoic acid, an enterobactin biosynthetic precursor (Grass et al., 2004). Conversely, phenolate siderophores such as yersiniabactin bind Cu2<sup>+</sup> in complexes that prevent reductive free Cu+release (Chaturvedi et al., 2012). Uropathogenic *E. coli* strains that express yersiniabactin are protected from copper's toxic effects, suggesting that a strain's small molecule repertoire may affect its ability to survive and persist in a copper-rich environment. It is notable that yersiniabactin can protect bacteria with and without FyuA (the outer membrane ferric yersiniabactin importer) from copper toxicity, suggesting that yersiniabactin's iron uptake function does not contribute to this phenotype. Copper oxidation state selectivity among microbial small molecules is also observed in pyoverdin and pyochelin, two major siderophore types expressed by *P. aeruginosa* (Brandel et al., 2012). While both siderophores can bind Cu2+, Cu2+supplementation upregulates genes involved in the synthesis of pyoverdin but downregulates those for pyochelin (Frangipani et al., 2008; Brandel et al., 2012). Data indicate that both siderophores prevent Cu2<sup>+</sup> accumulation in the bacterial cell by 80% (Teitzel et al., 2006). Pyoverdin's selective expression indicates that it may play a direct role in copper tolerance, possibly by sequestering copper in reduction-resistant complexes like yersiniabactin. The chemical basis of pyoverdin's transcriptional selectivity is unclear, and response regulation is unknown. It is possible that ferric- and cupric siderophore complexes govern differential transcriptional responses.

It also remains unclear whether siderophore transport systems can discriminate between different metal bound forms. While sequestration by siderophores can attenuate copper toxicity, bacterial proteins that import siderophore-metal complexes may also play a role. The siderophore schizokinen eliminates copper's toxic effects on *Anabaena* (Clarke et al., 1987) but exacerbates copper toxicity in *Bacillus megaterium* (Arceneaux et al., 1984). It is possible that these differences arise from fundamental differences in metabolic and transport machinery between the two organisms. Copper schizokinen-mediated toxicity in *Bacillus* can be alleviated by the exogenous desferrioxamine, raising the possibility that cells transport iron to repair copper-mediated damage. This observation could be further explained by differences in each organism's ability to use its iron-uptake machinery to discriminate between cupric- and ferric-siderophore complexes. It is possible that copper indirectly affects siderophore expression by competitively inhibiting iron import or liberating intracellular iron, altering intracellular metal accumulation, and affecting a downstream biosynthetic feedback loop.

#### *Copper oxidation*

*Mixed copper oxidases (MCO).* Cu+is more toxic than Cu2+when applied under anoxic conditions, as demonstrated by Macomber and Imlay (2009). Consistent with this observation, *E. coli* cultures treated with both Cu2<sup>+</sup> and reductants such as ascorbate or catechols demonstrate lower viability than those treated with Cu2<sup>+</sup> alone (Chaturvedi et al., 2012). To detoxify extracytoplasmic Cu+, *E. coli* use the CueR-regulated multi-copper oxidase CueO to oxidize toxic cuprous copper to its less toxic cupric form (Grass and Rensing, 2001; Roberts et al., 2002; Singh et al., 2004). *E. coli* and *S. typhimurium* mutants lacking CueO exhibit extreme copper sensitivity in oxic conditions. CueO contributes to *S. typhimurium* virulence in a systemic murine infection model (Achard et al., 2010). A second, plasmidborne 605 amino acid MCO called PcoA has also been described in *E. coli*. Periplasmic extracts containing PcoA exhibit copperinducible oxidase activity, indicating that PcoA might similarly oxidize Cu+ to prevent toxicity (Huffman et al., 2002; Djoko et al., 2008). PcoA can functionally substitute for CueO in *E. coli,* indicating that these proteins have redundant function.

*E. coli* CueO is among the best-characterized bacterial multicopper oxidases (MCOs). CueO is structurally similar to the large, cross-Kingdom family of MCOs [including ascorbate oxidase and the ferroxidases Fet3 and ceruloplasmin (Outten et al., 2000)] that oxidize substrates using oxidizing equivalents in molecular oxygen. This oxygen requirement renders oxidases inactive under anoxic conditions. CueO's active site consists of a trinuclear copper center MCO active site in which a fourth copper atom mediates electron transfer from the substrate (Roberts et al., 2002; Grass et al., 2004). The enzyme couples Cu+ oxidation with four-electron oxygen oxidation to water through the hydroxide-bridged fourth copper atom. Reactive oxygen intermediates generated during the reaction remain coordinated and are not released from the protein. It is curious that despite low cytoplasmic copper levels, CueO and PcoA exhibit a twin-arginine motif in their leader sequences, suggesting that they are translocated from the cytoplasm by the twin arginine translocation (Tat) pathway with copper-bound active sites (Huffman et al., 2002). *Holo-*protein translocation from the cytoplasm means that some amount of chaperonebound copper must be delivered to these *apo*-proteins intracellularly. This indicates that intracellular copper may serve a biosynthetic role in this specific process. If MCOs ultimately evolved to prevent copper entry to the cytosol, it is possible that metallation by cytosolic copper is a form of feedback regulation in which higher cytosolic copper levels lead to higher MCO secretion. Further studies are necessary to discern this, and other, possibilities.

In addition to oxidizing periplasmic Cu+, *E. coli* CueO can also oxidize 2,3 dihydrobenzoic acid (DHB) (Grass et al., 2004). 2,3-DHB is the biosynthetic precursor to enterobactin, a catecholate siderophore, secreted during iron limitation. As enterobactin can reduce Cu2<sup>+</sup> to Cu+, it has been hypothesized that CueO's 2,3-DHB oxidation activity is a strategy to prevent toxic Cu+ accumulation. While it may seem paradoxical to both synthesize and destroy a siderophore, an intracellular copper requirement for CueO secretion may ensure that it's siderophore destructive activity is only relevant in the presence of high copper levels. Together, these findings suggest that MCO's such as CueO help protect bacteria from copper stress by controlling copper ion oxidation states in oxic environments.

*Superoxide dismutation.* Recent work shows that yersiniabactin expression greatly facilitates pathogen survival within phagocytic cells in a copper- and NADPH oxidase system-dependent manner (Chaturvedi et al., 2013). In the presence of copper- and NADPH oxidase-derived superoxide, yersiniabactin production protects urinary pathogenic *E. coli* within cultured macrophagelike cell phagosomes. Superoxide's contribution to this phenotype suggests that yersiniabactin's cytoprotective effects may not be attributable to copper sequestration alone. Subsequent biochemical characterizations reveal that the copper-yersiniabactin complexes catalyze superoxide dismutation according to [Equations (6) and (7)]:

O2**•**<sup>−</sup> <sup>+</sup> Cu2<sup>+</sup> <sup>−</sup> Ybt <sup>→</sup> Cu<sup>+</sup> <sup>−</sup> Ybt <sup>+</sup> O2 *(*6*)* O2**•**<sup>−</sup> <sup>+</sup> Cu<sup>+</sup> <sup>−</sup> Ybt <sup>+</sup> 2H<sup>+</sup> <sup>→</sup> Cu2<sup>+</sup> <sup>−</sup> Ybt <sup>+</sup> H2O2 *(*7*)*

Copper-yersiniabactin confined within the phagolysosome may thus greatly diminish concentrations of superoxide (a reductant), while maintaining or increasing production of hydrogen peroxide (an oxidant). This may have the effect of minimizing reduced Cu+ concentrations while increasing oxidized—and less toxic—Cu2<sup>+</sup> ion concentrations. Periplasmic Cu,Zn-SOD may similarly protect against copper stress, although there are distinctive pathogenic advantages to deploying a nonprotein catalyst such as copper-yersiniabactin in the phagosomal microenvironment (Chaturvedi et al., 2013). Yersiniabactin may synergize with CueO and other mixed copper oxidases by binding Cu2<sup>+</sup> product ions generated by these enzymes to form catalytic copper-yersiniabactin. While interactions such as these will require further experimental validation, they fit with an overall paradigm in which pathogens appear able to convert hostsupplied copper into catalysts (mixed copper oxidases, copperyersiniabactin, Cu,Zn-SOD) that help resist copper toxicity. SOD activity may promote bacterial survival in several pathologically important host niches and its connection with copper suggests new insights into host defense mechanisms that are critical to infection pathogenesis.

## **PROSPECTS**

Much remains to be understood about the mechanisms by which mammalian hosts deploy copper to resist infection, and how pathogenic bacteria respond to these strategies. ATP7A's emerging role in direct antibacterial immunity warrants its detailed study in mammalian cells that encounter bacterial pathogens. Cell type, pathogen, and regulatory activity may result in unforeseen interactions between copper and other innate immune effector molecules. Possible cooperation with mammalian copper absorption and trafficking may suggest routes by which copper-based immunity could be therapeutically supported. Both basic and translational research efforts will be necessary to understand these details.

The mechanisms by which pathogenic bacteria resist copper during mammalian infections merits further investigation. Studies conducted in bacterial cultures with environmental and pathogenic isolates provide an excellent starting point for infection models that may provide additional insights. The recent finding that yersiniabactin, a virulence-associated siderophore in *E. coli* binds copper during humans infections (Chaturvedi et al., 2012) and promotes microbial survival in phagocytic cells suggests that host microenvironments may reveal new copper resistance strategies (Chaturvedi et al., 2013). Yersiniabactin exemplifies the rich array of microbial secondary compounds that may include other copper-detoxifying microbial products. Metabolomic approaches, which are sensitive to the end products of multi-gene biosynthetic units, are well suited to discover additional copper-binding secondary compounds.

Copper's inherent toxicity has renewed interest in its use as an antimicrobial. Three hundred different copper and copper alloy surfaces are registered with the U.S. Environmental Protection Agency as antimicrobials and trials are underway to determine whether copper treated surfaces can significantly reduce nosocomial infections (http://www*.*epa*.*gov/pesticides/factsheets/ copper-alloy-products*.*htm) (Grass et al., 2011). While these approaches may be useful in limiting nosocomial infections, it is worth noting that environmental copper-resistance loci have been isolated from Gram-negative bacteria that colonize agricultural areas repeatedly treated with copper salts. Given the linkage between copper resistance and virulence, it would be worth knowing whether sublethal copper exposures might effectively select for increased virulence in bacteria. Improved insight into bacterial copper resistance mechanisms *in vivo* and in environmental settings will be necessary to optimize antimicrobial uses of copper.

#### **REFERENCES**


interactions using engineered Holliday junctions. *Biophys. J.* 97, 844–852. doi: 10.1016/j.bpj.2009.05.027


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 01 October 2013; accepted: 06 January 2014; published online: 03 February 2014.*

*Citation: Chaturvedi KS and Henderson JP (2014) Pathogenic adaptations to hostderived antibacterial copper. Front. Cell. Infect. Microbiol. 4:3. doi: 10.3389/fcimb. 2014.00003*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2014 Chaturvedi and Henderson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Mechanisms of copper homeostasis in bacteria

#### *José M. Argüello1 \*, Daniel Raimunda2 and Teresita Padilla-Benavides <sup>1</sup>*

*<sup>1</sup> Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, MA, USA*

*<sup>2</sup> Instituto de Investigación Médica M. y M. Ferreyra, INIMEC-CONICET, Universidad Nacional de Córdoba, Córdoba, Argentina*

#### *Edited by:*

*Mathieu F. Cellier, Institut National de la Recherche Scientifique, Canada*

#### *Reviewed by:*

*Fernando C. Soncini, CONICET, Argentina James Imlay, University of Illinois at Urbana-Champaign, USA Gregor Grass, Bundeswehr Institute of Microbiology, Germany*

#### *\*Correspondence:*

*José M. Argüello, Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, 100 Institute Road, Worcester, MA 01609-2280, USA e-mail: arguello@wpi.edu*

Copper is an important micronutrient required as a redox co-factor in the catalytic centers of enzymes. However, free copper is a potential hazard because of its high chemical reactivity. Consequently, organisms exert a tight control on Cu+ transport (entry-exit) and traffic through different compartments, ensuring the homeostasis required for cuproprotein synthesis and prevention of toxic effects. Recent studies based on biochemical, bioinformatics, and metalloproteomics approaches, reveal a highly regulated system of transcriptional regulators, soluble chaperones, membrane transporters, and target cuproproteins distributed in the various bacterial compartments. As a result, new questions have emerged regarding the diversity and apparent redundancies of these components, their irregular presence in different organisms, functional interactions, and resulting system architectures.

**Keywords: copper, homeostasis, transmembrane transport, metalloenzymes, metallochaperones, Cu+-ATPases**

#### **INTRODUCTION**

Copper (Cu) is a micronutrient required as a co-factor in multiple proteins. It participates in redox reactions (electron transport, oxidative respiration, denitrification, etc.) (Silva and Williams, 2001; Cobine et al., 2006; Tavares et al., 2006), and in some cases is also a structural element (Adman, 1991; Kaim and Rall, 1996). Cu homeostatic mechanisms were initially uncovered by phenotypic analysis of bacterial strains carrying mutations in genes participating in Cu tolerance (Odermatt et al., 1993; Outten et al., 2000; Rensing et al., 2000). Perhaps because of the simplicity of experiments measuring Cu tolerance and intracellular Cu accumulation, these mechanisms were the focus of much of the early research in the field. However, cell physiological fitness requires Cu homeostasis mechanisms that primarily address how this metal is distributed and targeted to cuproenzymes. Cells strive to supply Cu to these proteins via compartmentalization involving both, transport across membranes and trafficking within a given compartment (Robinson and Winge, 2010; Argüello et al., 2012). In addition, Cu homeostasis requires chelation by high affinity binding molecules and Cu+ sensing by transcriptional regulators to maintain low levels of free Cu, as the metal might participate in a number of deleterious reactions. These include the production of highly reactive radical oxygen species via the Fenton reaction and the interference with [Fe-S] cluster protein assembly (Gaetke and Chow, 2003; Macomber and Imlay, 2009; Dupont et al., 2011). In this review, we focus on these homeostatic mechanisms: sensors, transporters, chaperones, and chelators that distribute the ion to cuproproteins while maintaining beneficial Cu levels (**Figure 1**).

### **BACTERIAL CUPROENZYMES**

The evolutionary driving force for copper usage in living organisms is mainly represented by the increase of diooxygen on Earth approximately 2 billion years ago (Boal and Rosenzweig, 2009). In parallel, Fe use increased as Fe2+/Fe3<sup>+</sup> equilibrium shifted toward the more oxidized species, leaving the insoluble Fe3<sup>+</sup> out of the scene. Instead, Cu emerged as a readily available biological redox factor because of the higher solubility of Cu2+. Additional determinants for Cu selection over other transition metals are found in its high polarizability and preferential geometries of coordination. Linear and trigonal coordination with S/N-ligands (soft bases), and to a lesser extent with O-ligands (hard base), result in high stability constants of Cu adducts suitable for traffic and transport. On the other hand, static/catalytic Cu sites present higher coordination numbers and more complex geometries with similar stability constants (Boal and Rosenzweig, 2009; Argüello et al., 2012). Most cuproenzymes described in this section harbor one or more Cu sites with tetrahedral or higher geometries. A thorough examination of coordination geometries is beyond the scope of this review.

Only a small repertoire of bacterial cuproenzymes is known (**Table 1**) (Garcia-Horsman et al., 1994; Claus, 2003; Nakamura and Go, 2005; Claus and Decker, 2006; Ridge et al., 2008; Boal and Rosenzweig, 2009; Rensing and McDevitt, 2013). However, as suggested by bioinformatics studies and in some cases shown by metalloproteomics approaches, bacteria are likely to have many yet unidentified cuproproteins (Ridge et al., 2008; Cvetkovic et al., 2010; Osman et al., 2010; Gladyshev and Zhang, 2013). Early analysis assumed that since most cuproproteins were localized in the bacterial periplasm or plasma membrane (**Table 1**), there was little need for cytoplasmic cuproproteins metallation. However, consideration of secretion systems might lead to alternative hypothesis. For instance, secretion of cuproenzymes via the Tat system implies that these likely acquire their metal in the cytoplasm. On the other hand, membrane cuproenzymes, as well as soluble enzymes secreted by the Sec system, fold and acquire Cu+ from the periplasmic compartment. It can be proposed that this is not a random process. Since considerable energy

is spent during protein synthesis, the possibility of wrong metallation must be prevented. That is, highly regulated Cu transport and delivery systems should have co-evolved with the extracytoplasmic cuproenzymes. The participation of cytoplasmic influx and efflux systems in the eventual periplasmic metallation of *cbb*<sup>3</sup> cytochrome *c* oxidase (*cbb3*-COX) provides an example of the necessary passage of Cu+ through a controlled delivery system (**Figure 1**) (González-Guerrero et al., 2010; Ekici et al., 2012b; Lohmeyer et al., 2012).

Heme-Cu respiratory oxidases constitute a large superfamily of cuproenzymes present in most bacteria (Garcia-Horsman et al., 1994; Richter and Ludwig, 2003). They are responsible for the reduction of O2 and the generation of the H<sup>+</sup> electrochemical gradient. The O2 reduction takes place in the binuclear center of subunit I (also referred as subunit N). This reduction center is composed of a heme iron juxtaposed to a Cu site (Cu*B*). In addition, some *ba*3, *caa*<sup>3</sup> and *aa*<sup>3</sup> oxidases, contain a CuA center in subunit II (Garcia-Horsman et al., 1994). Terminal oxidases acquire their Cu cofactors from periplasmic Cu+-chaperones. Homologs of the Cu+-chaperones ScoI/SenC have been proposed to perform this function in *Pseudomonas putida*, *Rubrivibax gelatinosus*, and *Rhodobacter capsulatus* as the corresponding deletion mutants lack *cbb3*- COX activity (Banci et al., 2011; Ekici et al., 2012a; Lohmeyer et al., 2012). Studies in *Pseudomonas aeruginosa* revealed the requirement of cytosolic Cu+ efflux through CopA2-like ATPases for Cu+ insertion into *cbb3*-COX (González-Guerrero et al., 2010). Thus, it is likely that ScoI/SenC chaperones receive the metal from the ATPase. The identification of *R. capsulatus* CcoA has further supported the idea that cytoplasmic Cu+ is channeled to the periplasm for COX metallation (Ekici et al., **Table 1 | Bacterial Cuproenzymes∗.**


*\*References are in the text. \*\*Based on the presence of signal sequences. \*\*\*Based on experimental evidence.*

2012b). CcoA is a member of the major facilitator superfamily (MFS) of transport proteins. It has been proposed that it imports Cu into the cytoplasm and is required for *cbb*<sup>3</sup> assembly. Regarding oxidases containing a CuA site, characterization of the *Bradyrhizobium japonicum aa*<sup>3</sup> oxidase suggests that the metal binding PcuC acts as the periplasmic Cu+-chaperone (Serventi et al., 2012). The source of Cu for PcuC loading has not been identified.

Three enzymes involved in the bacterial denitrification pathway (i.e., the nitrite reduction to dinitrogen) are periplasmic cuproenzymes: nitrite reductase (Nir), nitric oxide reductase (qCuNOR), and nitrous oxide reductase (N2OR) (Messerschmidt et al., 1993; Brown et al., 2000; Zumft, 2005). Nir and N2OR are attached to the inner membrane. Nir is homotrimeric with each subunit containing type 1 and 2 Cu centers. N2OR is homodimeric with a binuclear CuA site related to electron transfer and a multinuclear CuZ related to catalysis (Brown et al., 2000). It is assumed that these three enzymes are exported via the Tat secretion system (Saunders et al., 2000; Berks et al., 2003). However, Nir is apparently secreted via Tat in some organisms and via Sec in others (Berks et al., 2000b). In any case, it has been proposed that Nir proteins present one Cu+ binding site at the subunits interface with metal ligands located in two separate polypeptides. Cu would bind these sites during or after oligomer formation in the periplasm (Godden et al., 1991; Berks et al., 2003). Similarly, it has been reported that assembly of the CuA and CuZ center of the *Pseudomonas stutzeri* N2OR occurs in the periplasm and *nos* genes are required for metallation (Wunsch et al., 2003).

Members of the superoxide dismutase (Sod) harbor different active redox metallic co-factors in their catalytic centers. Two major groups have been described in bacteria, SodA containing usually Mn2<sup>+</sup> (and in some cases Fe2+), and SodC carrying Cu<sup>+</sup> and Zn2+. Most bacterial Cu,Zn-SodC are periplasmic proteins. These are secreted unfolded by the Sec secretion system and likely acquire the metals in the periplasm (Kroll et al., 1995; Imlay and Imlay, 1996). Recent studies of *Salmonella enterica sv.* Typhimirium Cu,Zn-Sod have identified some of the proteins involved in periplasmic metallation. *Salmonella* virulent strains contain SodCI and SodCII (Fang et al., 1999; Krishnakumar et al., 2007; Rushing and Slauch, 2011). These are both periplasmic proteins. SodCI appears required for virulence. *Salmonella* mutant strains lacking functional Cu+-ATPases, CopA, and GolT, have inactive SodCII (Osman et al., 2013). Mutation of the periplasmic Cu+-chaperone CueP also results in inactive SodCII forms. Consequently, it was hypothesized that Cu+ might be transferred from either CopA or GolT to CueP to be inserted in SodCII. Further studies are necessary to understand the apparent redundancy of both ATPases, as well as the Cu transfer mechanism among the involved proteins. Conversely, in *Mycobacterium tuberculosis* and *Mycobacerium smegmatis,* the Cu,Zn-Sod is located in the cytosol whereas the Fe/Mn-SodA is secreted via the SecA2 pathway and is metallated outside the cell (Braunstein et al., 2003; Padilla-Benavides et al., 2013a). Therefore, these organisms require a cytoplasmic Cu-loading mechanism to produce functional Cu,Zn-Sod.

The superfamily of cupredoxins is characterized by an antiparallel β-barrel structure that presents a type 1 Cu binding site. This group includes plastocyanin located in the thylakoid, and a variety of periplasmic proteins such as azurin, multicopper oxidases (MCO), laccases, and nitrosocyanin (Redinbo et al., 1994; Donaire et al., 2002; Berks et al., 2003; Zaballa et al., 2012). In cyanobacteria, plastocyanin participates in the electron shuttling to photosystem I (Redinbo et al., 1994) and also confers protection against Cu+ stress (Tottey et al., 2012). Mutant strains lacking Cu+-ATPases PacS or CtaA have impaired photosynthetic electron transport via plastocyanin and cytochrome oxidase activity, suggesting that these transporters are required for the metallation of these proteins (Tottey et al., 2001). Periplasmic azurins participate in electron shuttling for deamination and denitrification processes by donating electrons to nitrite reductases (De Rienzo et al., 2000). *P. aeruginosa* azurin participates in the cellular response to Cu stress. For instance, mutation of *P. aeruginosa cinA,* an azurin/plastocyanin-like protein, leads to increased sensitivity to Cu2<sup>+</sup> in the media (Elguindi et al., 2009). Deletion of either *P. aeruginosa* Cu+-ATPase, CopA1, or CopA2, induces an increase in azurin transcription as a cellular response to Cu+-derived oxidative stress (Raimunda et al., 2013). However, whether azurins are metallated by the transporters or participate in Cu+ oxidation has not been established.

Ascorbate oxidases, laccases, and ceruloplasmin are homologous small blue MCO (Messerschmidt et al., 1993; Nakamura et al., 2003). Laccases, for instance, are involved in the oxidation of phenolic compounds (Claus, 2003). Laccases and ascorbate oxidases present a mononuclear type 1 Cu site -homologous to other cupredoxins- and a trinuclear Cu center. Ceruloplasmin has three mononuclear Cu sites and a trinuclear Cu domain (Nakamura et al., 2003). CueO and PcoA are MCO up-regulated by high Cu+ concentrations through the *cueR* regulon (Outten et al., 2000). It has been observed that *Escherichia coli* CueO is folded in the cytoplasm and exported via the Tat system (Outten et al., 2000; Grass and Rensing, 2001). Based on differences in Cu+ amounts relative to the binding capacity of CueO, it was proposed that they contribute to periplasmic metal tolerance by oxidizing the ion to the less toxic form Cu2<sup>+</sup> (Roberts et al., 2002; Singh et al., 2004). However, *S. enterica sv*. Typhimurium CueO seems to also play a significant role in Cu+ homeostasis under anaerobiosis (Espariz et al., 2007).

NADH dehydrogenase-2 (NDH-2), part of the electron transport chain, is a membrane bound protein that catalyzes the electron transfer from NADH to quinone (Jaworowski et al., 1981; Bjorklof et al., 2000). NDH-2 Cu2+-reductase activity was described in *E. coli* (Rodríguez-Montelongo et al., 1995; Rapisarda et al., 1999). Although not essential for Cu metabolism, NDH-2 is likely important for cell growth under Cu stress (Rodríguez-Montelongo et al., 2006). Moreover, *E. coli* NDH-2 presents four domains. Domain IV likely anchors the protein to the membrane. Domain III faces the cytosolic side near the FAD-binding site, shares homology with the cytosolic metal binding domains (MBD) of Cu+-ATPases and might be relevant for cytoplasmic Cu+ sensing (Rapisarda et al., 2002). The biological role for the Cu+-reducing enzymatic activity, as well as the characterization of the secretion mechanism and metallation of this enzyme, requires further investigation.

In eukaryotic cells, tyrosinase is required for synthesizing melanin. Some bacteria from the genus *Streptomyces* produce a melanin-like pigment (Katz et al., 1983; Hintermann et al., 1985; Huber et al., 1985; Ikeda et al., 1996). Tyrosinases catalyze the orthohydroxylation of monophenol and the subsequent oxidation of the diphenolic product to the resulting quinone. The quinone product is a reactive precursor for the synthesis of melanin pigments. Tyrosinases contain a flexible dinuclear Cu center required for catalysis (Matoba et al., 2006). Bacterial tyrosinase is encoded in a bicistronic operon composed by genes coding for a "caddie" chaperone protein (*melC1*) and tyrosinase (*melC2*) (Katz et al., 1983; Ikeda et al., 1996). Tyrosinases are secreted via the Tat pathway (Berks et al., 2003). MelC1 appears to function as a Cu+-chaperone, forming a transient complex with the apo-MelC2. This would facilitate the incorporation of Cu+ and the subsequent secretion of functional tyrosinase (Chen et al., 1992, 1993; Matoba et al., 2006).

Methane monooxygenases (MMO) are Cu+-containing enzymes present in methanotropic bacteria. There are two MMO forms, membrane-bound (pMMO) and cytosolic (sMMO). Most organisms present the pMMO form; however, in some bacteria both types are present. Interestingly, the expression of both proteins is regulated by the availability of Cu (Nielsen et al., 1997). The *Methylococcus capsulatus* (Bath) pMMO comprises three subunits, encoded by the *pmoB*, *pmoA*, and *pmoC* genes (Semrau et al., 1995; Stolyar et al., 1999). Copper stoichiometries ranging from 2 to 15 ions per αβγ complex have been reported (Nguyen et al., 1998; Basu et al., 2003). Ions appear organized in multiple trinuclear clusters composed of one Cu2<sup>+</sup> and two Cu<sup>+</sup> ions (Nguyen et al., 1996, 1998). These sites are hypothesized to function for catalysis and electron transfer. Interestingly, the uptake of methanobactin-bound Cu via TonB dependent transport appears critical for pMMO metallation (Balasubramanian and Rosenzweig, 2008).

Cu+-dependent amine oxidases (CuAO) are rare in bacteria but they provide the ability to obtain carbon and nitrogen from primary amines by oxidative deamination (Wilmot et al., 1997). The enzyme is induced in conditions where biogenic primary amine substrate is the sole source of carbon (Gladyshev and Zhang, 2013). Bacterial Cu+-dependent polysaccharide oxygenases (AA10) are secreted proteins involved in breaking internal linkages in plant cellulose (Levasseur et al., 2013). AA10 binds one Cu+ atom with high affinity. The metal ion is coordinated in a T-shaped configuration by three N atoms from two His side chains and the amino terminus (Hemsworth et al., 2013). Although these enzymes are secreted via the Tat pathway (Berks et al., 2003), there is no information regarding their metallation mechanisms.

This assessment of various cuproenzymes makes clear that they fulfill various roles essential for bacterial survival. It is also apparent that well-regulated mechanisms should be responsible for delivering the metal to final targets. Relevant questions immediately emerge when considering Cu delivery models. Are chaperones promiscuous in their interactions with various targets? If they are specific, how is the allocation of Cu to the various targets regulated? Moreover, the existence of novel uncharacterized cuproproteins must be also considered. Recent efforts using metalloproteomic approaches support this idea. For instance, liquid chromatography, high-throughput tandem mass spectrometry (HT-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) were combined to identify cytoplasmic metalloproteins in the extremophile *Pyrococcus furiosus* (Cvetkovic et al., 2010). Chromatography fractions revealed 343 metal peaks. Among these, 158 did not correspond to any predicted metalloprotein, supporting the likelihood of numerous novel cuproproteins.

### **MAINTAINING THE Cu+ QUOTA: Cu+-SENSING AND TRANSCRIPTIONAL REGULATION OF HOMEOSTATIC SYSTEMS**

Cells control alkali ion levels regulating transmembrane transport through various mechanisms: (a) modulating transport turnover rates through chemical modification and allosteric ligand binding; (b) managing the incorporation and removal of transporter proteins at the required membrane (eukaryotes); and (c) transcriptional regulation of transporter abundance. The high affinity binding of transition metals to their transporters, as well as the inability of apo-chaperones to accept Cu+ back from the exporting ATPase, makes the transmembrane transport functionally irreversible. Consequently, bacterial cells regulate this process mainly through transcriptional control by modifying the transporter abundance. This strategy facilitates the co-regulation of chaperone and chelating metallothioneins. Four families of Cu+-sensing homodimeric transcriptional regulators have been identified. MerR is a transcriptional activator, while CsoR, CopY, and ArsR are transcriptional repressors (Ma et al., 2009c).

CueR-like proteins, members of the MerR family, are present in most proteobacteria (Ma et al., 2009c). These activate the transcription of Cu+-ATPases (CopA) and Cu+ chaperones (CopZ, CueP, etc.) in response to high concentrations of Cu+ (Outten et al., 2000; Grass and Rensing, 2001; Pontel and Soncini, 2009). In the presence of low metal levels, the metalloregulator binds to DNA in a conformation that prevents the DNA-RNA polymerase interaction, therefore repressing transcription (Ma et al., 2009c; Reyes-Caballero et al., 2011). When cytosolic Cu+ level increases, metal binding to the sensor induces changes in the DNA binding region. The long-range communication between metal and DNA binding sites appears mediated by a hydrogen bond network. Once the cytosolic Cu+ levels return to normal, the metal-resistance genes are down regulated to basal levels. It is possible that the metal is released from the sensor-DNA complex followed by disassociation of the regulatory complex. However, this is an unlikely phenomenon considering the high Cu+ binding affinity of CueR (10−<sup>21</sup> M) (Changela et al., 2003). Rather, the exchange of apo- and holo-forms appears possible since both forms of MerR proteins interact with DNA with similar affinities (Brown et al., 2003; Joshi et al., 2012). Although CueR and other Cu+ sensing regulators bind the metal with high affinity, metal selectivity appears to be conferred by the singular coordination geometry of binding sites and the capability to induce the required allosteric changes to influence DNA conformations (Ma et al., 2009c; Reyes-Caballero et al., 2011). CueR-like sensors bind Cu+ with two Cys in two symmetrical loops in the periphery of the dimer. Nevertheless, the detail structure of the loop and the influence of the second coordination sphere are critical as shown by selectivity changes associated with minimal modifications in the region (Checa et al., 2007).

CsoR was identified as the Cu+ sensor in *M. tuberculosis* (Liu et al., 2007). This Cu+-responsive repressor controls the expression of the *cso* operon (*csoR, Rv0968,* the Cu+-ATPase *ctpV*, and *Rv0970*). Members of this family are widely distributed in most bacterial species (Smaldone and Helmann, 2007; Ma et al., 2009a; Sakamoto et al., 2010; Corbett et al., 2011). In CsoR, Cu+ is bound in a trigonal coordination by two Cys and one His (Liu et al., 2007; Ma et al., 2009b). These sites might also bind Ni2+, Zn2+, or Co2+, but these place the sensors in non-active conformations (Ma et al., 2009a). Thus, as in the case of CueR–like proteins, binding geometry appears critical for selectivity.

Members of the CopY family are present in Firmicutes. *Enterococcus hirae* CopY regulates the *copYZBA* operon, where *copZ* encodes a Cu+-chaperone, *copA* a CopA1-like ATPases (see below) and *copB* is a Cu2+-ATPase (Argüello et al., 2007; Solioz et al., 2010). Both ATPases mediate the efflux of cytoplasmic Cu+*/*2<sup>+</sup> (Raimunda et al., 2011). A conserved CXCXXXCXC motif appears to mediate the binding of two Cu+ per CopY monomer. CopY is also interesting because its capability to exchange Cu+ with the cytoplasmic Cu+-chaperone CopZ has been demonstrated (Cobine et al., 1999). This has not been shown for other Cu+ sensors.

*Oscillatoria brevis* BxmR is the only identified Cu+ sensor member of the ArsR family (Liu et al., 2004, 2008). This binds Ag<sup>+</sup> and Cu<sup>+</sup> through formation of a binuclear Cu2S4 cluster similar to that of *E. hirae* CopY. Like other described sensors, it regulates the expression of a metallothionein and a Cu+-ATPase.

Less is known about the role of bacterial two-component systems in the regulation of Cu+ homeostatic systems. The twocomponent system CusRS regulates the *cusCFBA* system under anaerobic conditions (Outten et al., 2001; Rensing and Grass, 2003; Yamamoto and Ishihama, 2005; Gudipaty et al., 2012). This is a three-component channel/pore that controls periplasmic Cu+ (Outten et al., 2001; Rensing and Grass, 2003). The Cus complex is composed by a plasma membrane energy-providing channel, CusA; an outer membrane pore, CusC; CusB, a periplasmic protein linking CusA and CusC; and a soluble periplasmic Cu+-chaperone, CusF (Outten et al., 2001; Rensing and Grass, 2003). In the case of CusRS, the periplasmic sensor domain of the transmembrane histidine kinase CusS binds Cu+. This drives the subsequent activation of the cytoplasm regulator CusR. The *pcoABCDE* cluster also appears to be regulated by a twocomponent system, PcoRS (Rouch and Brown, 1997; Munson et al., 2000). The Pco proteins, whose function is not fully understood, appear to contribute to the control of periplasmic Cu+ in *E. coli* and other Gram-negative bacteria (Brown et al., 1995; Rouch and Brown, 1997; Lee et al., 2002; Rensing and Grass, 2003; Djoko et al., 2008; Hernández-Montes et al., 2012). Sequence analysis and experimental evidence suggest that PcoA is a periplasmic MCO. PcoB may function as the outer membrane transporter, while PcoD appears to be the inner membrane transporter that drives Cu+ entry from the periplasm to the cytoplasm. PcoC is a periplasmic Cu+-chaperone, and PcoE is an additional putative chaperone.

### **Cu+ TRANSPORT SYSTEMS**

#### **Cu+-TRANSPORTING P-TYPE ATPASES**

The prevailing Cu+ transmembrane transporters throughout the bacterial kingdom are PIB1-type ATPases (Ridge et al., 2008; Hernández-Montes et al., 2012). These are polytopic membrane transporters that couple the unidirectional Cu+ efflux to the hydrolysis of ATP. Initial functional characterization of Cu+- ATPases showed that they are responsible for maintaining cytosolic Cu+ levels (Rensing et al., 2000; Argüello et al., 2007; Osman and Cavet, 2008; Solioz et al., 2010). The mechanism coupling the ATP hydrolysis to Cu+ translocation appears to follow the classical features of the Post-Albers catalytic cycle. This describes solute transport by well-characterized P-type ATPases (Palmgren and Nissen, 2011; Raimunda et al., 2011). Perhaps the most significant differences from alkali metal transport ATPases are those related to the substrate access to transport sites (**Figure 2**). That is, free Cu+ is absent in the cytoplasm and reaches the transmembrane sites after delivery by Cu+-chaperones. This substrate transfer is mediated by ligand exchange following protein-protein interactions (González-Guerrero and Argüello, 2008; González-Guerrero et al., 2009; Raimunda et al., 2011). The ATPase transmembrane metal binding sites (TM-MBS) bind two Cu+ with extremely high affinities (González-Guerrero et al., 2008). This high affinity, together with inability of the apo-chaperone to remove Cu+ from the TM-MBS prevents the backward release of Cu+ into the cytoplasm. It is hypothesized that the enzyme releases Cu+ to the extra-cytoplasmic compartments in a similar fashion; i.e., delivering the metal to an appropriate chaperone (Raimunda et al., 2011). Additional unanswered aspects of transport is whether another ion, like H+, is counter transported and

**FIGURE 2 | Structures and interaction between cytosolic Cu+-chaperones and Cu+-ATPases.** *Archeoglobus fulgidus* C-terminal Cu+ binding domain of CopZ modeled using *Enterococcus hirae* CopZ (Protein Data Bank code 1CPZ) in light *blue*. *A. fulgidus* CopA modeled using *Legionella pneumophila* CopA (Protein Data Bank code 3RFU) as a template. The transmembrane segments (TM) are in *gray*, the electropositive platform helix is highlighted in *green*, the Cu+ entrance site aminoacids are in *yellow*, the transmembrane metal binding sites (TM-MBS) in *dark blue*, the actuator (A) domain in *orange*, the nucleotide (N) binding and (P) phosphorylation domain in *red.*

whether Cu+ transport is electrogenic. However, considering the general low Cu+ transport rate of Cu+-ATPases the exchanged mass of the counter ion might not be significant enough to alter any metabolic process.

The crystal structure of *Legionella pneumophila* CopA shows the arrangement of the distinct elements of Cu+-ATPases (Gourdon et al., 2011) (**Figure 2**). The cytoplasmic ATP-binding domain (ATP-BD) is comprised of the phosphorylation (P-), nucleotide binding (N-), and actuator (A-) domains. Within the P-domain is the conserved DKTGT sequence. Phosphorylation of the aspartyl residue during the enzyme catalytic cycle is the hallmark of the P-type ATPase family of transporters. Importantly, six invariant residues in the TM region constitute the TM-MBS and are the determinants of Cu+ specificity (Argüello, 2003; González-Guerrero et al., 2008). Besides bacterial Cu+- ATPases contain one or two cytosolic N-terminal metal binding domains (N-MBD) (Argüello, 2003). N-MBDs are structurally similar to the Atx1-like family of Cu+-chaperones (Boal and Rosenzweig, 2009). These domains exchange Cu+ with cytoplasmic chaperones with Keq ≈ 1 (Argüello et al., 2007; Boal and Rosenzweig, 2009; Banci et al., 2010a,b; Robinson and Winge, 2010). Therefore, they sense the levels of cytoplasmic Cu+and regulate the ATPase turnover rate through a Cu+-dependent interaction with the catalytic soluble domains (Mandal and Argüello, 2003; González-Guerrero et al., 2009). The structure of *L. pneumophila* CopA also showed a unique feature on the cytosolic side of the second TM helix: a Gly-Gly kink that exposes an electropositive platform to the cytoplasm, as well as three conserved residues that constitute the Cu+ "entrance" site (Met, Glu, Asp) (Gourdon et al., 2011). The platform is where Cu+-loaded chaperones interact with the transporter while delivering the ion for transport (Padilla-Benavides et al., 2013b). These conserved "entrance" residues are required to take Cu+ from the chaperone via ligand exchange. Cu+ ions only transiently interacts with these residues during transfer to the TM-MBS, where they reside until ATP hydrolysis drives the necessary E1PE2 transition associated with ion translocation.

#### **Cus SYSTEM**

The Cus system, first identified in *E. coli*, is proposed to transport cytosolic Cu+ across the cell membrane toward the extracellular milieu (**Figure 1**). This efflux system is comprised of a proton-substrate carrier (CusA) and an outer membrane pore (CusC), which are joined in the periplasm by a linker protein, CusB (Zgurskaya and Nikaido, 1999; Su et al., 2009; Kim et al., 2011). A forth component, CusF is a periplasmic Cu+-chaperone found in some organisms (Kim et al., 2011; Mealman et al., 2011; Hernández-Montes et al., 2012).

Comparative studies of CopA and Cus proteins expression have suggested that Cus is not the principal Cu+ efflux system in cells growing under aerobiosis (Franke et al., 2001; Outten et al., 2001). This model considers that CopA's role is in fact supported by the periplasmic oxidase CueO. Thus, under anaerobic conditions where CueO would be inactive, Cus would become relevant in Cu+ detoxification (Outten et al., 2001). In agreement with this idea, bioinformatics analyses showed that the Cus system coexists with CopA in 44% of the organisms of the γ-proteobacteria group (Hernández-Montes et al., 2012). Among these, the pair CusA/CusC seems to be essential for the assembly of the efflux system, since CusB is absent in most members of this class. Then, CusB may be a dispensable accessory protein for the formation of the CusA-CusC channel, or homologous proteins from other RND complexes may fulfill its function (Hernández-Montes et al., 2012). The periplasmic chaperone CusF is the least conserved element of the Cus system (Hernández-Montes et al., 2012). CusF is a small (10 kDa) protein which binds one Cu+ atom in a tetragonal coordination with two Met, one His, and a conserved Trp. The Trp residue stabilizes the Cu+ binding, thus regulating metal transference and preventing redox reactions (Xue et al., 2008). Yeast two-hybrid assays and NMR analysis have shown that CusF interacts with both CusB and CusC (Franke et al., 2003; Mealman et al., 2011). Consequently, a proposed transport mechanism is that CusF delivers periplasmic Cu+ to CusABC for extracellular transport (Kim et al., 2011; Mealman et al., 2011). However, the transport of cytoplasmic Cu+ through the CusA antiporter has also been postulated based upon the CusA structure (Fu et al., 2013). Transport experiments are clearly necessary to discriminate among these alternative hypothetical Cus transport mechanisms.

#### **Pco SYSTEM**

Pco systems were the first characterized genetic determinants of bacterial Cu resistance (Tetaz and Luke, 1983; Bender and Cooksey, 1986; Cha and Cooksey, 1991). An *E. coli* strain showing a 7-fold increase in Cu resistance was isolated from a piggery that fed its animals a high Cu diet to promote growth. The phenotype was associated with the plasmid pRJ1004. This enabled the *E. coli* transconjugants to thrive in media containing up to 20 mM Cu2<sup>+</sup> (Tetaz and Luke, 1983). Similarly, increased Cu resistance in *Pseudomonas syringae* pv. *tomato* strains was shown to be determined by the plasmid pPT23D (Cooksey, 1987, 1990). Both plasmids contain 6–7 clustered genes, *pcoABCDRSE* in *E. coli* and *copABCDRS* in *P. syringae* (Mellano and Cooksey, 1988b; Brown et al., 1995). PcoB/CopB and PcoD/CopD are membrane proteins located in the outer and inner membranes respectively (**Figure 1**), participating in Cu transport. PcoA/CopA and PcoC/CopC are soluble periplasmic Cu+-binding proteins (Cha and Cooksey, 1991, 1993; Lee et al., 2002). Initial characterization in *P. syringae* pv. *tomato* pointed that *copA* and *copB* are necessary to confer partial Cu+ resistance, whereas *copC* and *copD* genes are required for full resistance (Mellano and Cooksey, 1988a). However, indirect evidence provided by phenotypic characterization of *copC* and *copD* mutant strains suggests that these genes are required for Cu-uptake across the cell membrane. Cells expressing both proteins were hypersensitive toward Cu, and the presence of any of these two genes leads to slight Cu accumulation (Cha and Cooksey, 1993). Direct metal transport determinations are necessary to establish the role of these proteins in Cu homeostasis. The PcoA sequence shows homology to MCO, binds Cu, and has MCO activity (Djoko et al., 2008) and is predicted to be translocated into the periplasm via the Tat pathway (Berks et al., 2000a; Lee et al., 2002). The PcoC structure shows 2 solventexposed Cu binding sites and its participation in Cu handling and transferring in the periplasm has been suggested, although its interacting client proteins remain unknown (Arnesano et al., 2002b, 2003). *PcoE* is homologous to the *Salmonella* silver resistance gene *silE* (Cha and Cooksey, 1993; Gupta et al., 2001). It has been postulated that PcoE might provide initial sequestration of Cu in the periplasm before the remaining genes of the *pco* system are fully induced (Lee et al., 2002). *Bacillus subtilis ycnJ* encodes a protein with high homology to domains found in PcoC and PcoD (Chillappagari et al., 2009). The presence of the Cu+-chelator BCA in the media led to reduced cytoplasmic Cu, induction of *ycnJ* expression, and impaired growth of an *ycnJ* deletion mutant strain, suggesting a role for YcnJ in Cu import. Importantly, amino acids involved in the Cu2<sup>+</sup> binding of CopC are conserved in YcnJ sequence (Arnesano et al., 2003; Chillappagari et al., 2009).

Although counterintuitive when considering a Cu resistance mechanism, the biological function of Pco systems seems to be related to periplasmic, and probably, intracellular Cu storage pools (**Figure 1**) (Cooksey, 1993). The latter function is probably mediated by CopC/CopD pair. However, the *pco* system is not able to rescue the Cu sensitive phenotype in the *copA* mutant of *E. coli*, suggesting that cooperation between CopC/CopD-dependent Cu pools and CopA1-like ATPases is necessary to attain high Cu resistance (Lee et al., 2002).

#### **PUTATIVE OUTER MEMBRANE CHANNEL: MctB**

MtcB (*Rv1698*) was first characterized as a high conductance channel located in the complex outer membrane of *M. tuberculosis* (Siroy et al., 2008). Expression of MctB in *M. smegmatis* resulted in an increased uptake of carbon source nutrients. While MctB structure is unknown, a functional analogy with Gram-negative porins has been suggested (Siroy et al., 2008). Analysis of the *M. tuberculosis* deletion mutant *mctB* showed decreased Cu+resistance and intracellular Cu+ accumulation (Wolschendorf et al., 2011; Rowland and Niederweis, 2012). Importantly, infection experiments in mice and guinea pigs support the hypothesis that MctB is required for *M. tuberculosis* to attain maximal virulence (Wolschendorf et al., 2011). This is likely due to MctB's ability to counteract the bactericidal effect produced by the Cu+ overload in activated macrophage phagosomes. Supplementary structural and biochemical studies might define the role of MctB in *M. tuberculosis* Cu+ homeostasis and its interrelation with other Cu+ efflux systems.

#### **CYTOPLASMIC Cu+ INFLUX**

Although the major Cu+ efflux systems have been identified and well characterized, Cu+ influx mechanisms are poorly understood. Early characterization of the *E. coli ompB* porin mutant showed a Cu-resistant phenotype, suggesting that Cu+ may enter the cells through these outer membrane proteins (Lutkenhaus, 1977). Only recently has the participation of members of the MFS and TonB-dependent transport system in Cu import been proposed (see below). Based on phenotypic analysis, it was previously suggested that some Cu+-ATPases might drive metal influx (Odermatt et al., 1993; Koch et al., 2000; Tottey et al., 2001; Lewinson et al., 2009; Hassani et al., 2010). However, direct transport experiments and a better understanding of the Cu+-ATPase structure and transport mechanism have provided solid evidence that all PIB1-ATPases mediate Cu+ efflux (González-Guerrero et al., 2010; Raimunda et al., 2011).

#### *Import of copper-chelating molecules*

Methane-oxidizing bacteria rely on two MMOs to achieve methanotrophic metabolism (Balasubramanian and Rosenzweig, 2008). The particulate pMMO is located in intracellular membranes and requires Cu for function. Thus, these organisms present an opportunity to identify specific Cu+-import mechanisms. Two pathways for Cu entry have been described in the methanotroph *Methylosinus trichosporium* (Balasubramanian et al., 2011). One of these, likely involved in Cu-handling to pMMO, requires the production of the siderophore-like methanobactin (Hakemian et al., 2005). The chalkophore structure is known and the Cu+ binding capability extends to Cu removal from minerals (Hakemian et al., 2005; Knapp et al., 2007). Mechanistically, the holo-form is postulated to be taken up via an active TonB dependent transport mechanism (Balasubramanian et al., 2011). A less specific import pathway involves stripped or unchelated Cu+ import via an outer membrane porin. However, this mechanism is not required for metallation of pMMO.

#### *Copper import catalyzed by secondary carriers*

Recently, a novel transporter has been identified based on its requirement for the metallation of *cbb3*-COX. *R. capsulatus* CcoA, a member of the MFS family, has been proposed to function as Cu importer (Ekici et al., 2012b). The MFS family consists of seventy-three sub-families of transporters that catalyze the symport, antiport or uniport of a wide variety of substrates, dissipating chemical or electrochemical gradients (Reddy et al., 2012). Like other MFS, CcoA is predicted to have twelve TM helices divided into two subdomains of six helices each and separated by a large cytoplasmic loop. Similar to the eukaryotic CTR-type Cu+-importers, CcoA contains several transmembrane Met rich motifs associated with Cu+ binding and transport (Puig et al., 2002; Eisses and Kaplan, 2005; Ekici et al., 2012b). Importantly, mutation of a highly conserved tyrosine in the transmembrane YFLMLIFMT motif of the yeast CTR-type Cu+ importer leads to a decrease in Cu+ transport (Eisses and Kaplan, 2005). In the CcoA transporter, a similar sequence (Y230ALMNLVMT) is also present on the TM7 (Ekici et al., 2012b). Phenotypic characterization of *R. capsulatus* cells lacking a functional CcoA has implicated this transporter in Cu+ import pathways, as well as in Cu+ acquisition by *cbb3*-COX. Mutation of *R. capsulatus ccoA* leads to a decrease in the total Cu+ content of *R. capsulatus* and a decrease in the assembly and stability of the subunits of *cbb3*-COX without inactivating the periplasmic MCO (CutO) (Ekici et al., 2012b). The similar phenotypic characteristics of the *P. aeruginosa* mutant *copA2* (missing the FixI/CopA2-like Cu+-ATPase) (González-Guerrero et al., 2010) and the *R. capsulatus ccoA* mutant strains, together with their opposed direction of Cu+ transport, support a model with COX metallation depending on the efflux of cytosolic Cu+ (González-Guerrero et al., 2010; Ekici et al., 2012a).

## **CHAPERONES AND CHELATORS**

#### **CYTOPLASMIC CHAPERONES**

Bacterial Cu+-chaperones, CopZs, are involved in cytoplasmic Cu+ trafficking. Structurally, these proteins present a classic βαββαβ ferredoxin-like folding with an invariant GXXCXXC Cu+-binding motif (Banci et al., 2004, 2010a,b; Boal and Rosenzweig, 2009). Extensive studies have shown their interaction with the regulatory N-MBD of Cu+-ATPases, exchanging Cu<sup>+</sup> with Keq ≈ 1 (Argüello et al., 2007; Boal and Rosenzweig, 2009; Banci et al., 2010a,b; Robinson and Winge, 2010). Similarly, they exchange the metal with Cu+ sensors (Cobine et al., 1999), and probably can receive the metal from bacterial Cu+ importers. These interactions appear mediated by metal-dependent electrostatic interactions (Arnesano et al., 2001, 2002a; Boal and Rosenzweig, 2009; Banci et al., 2010a,b). An example of the importance of electrostatic interactions is the mechanism by which the CopZ loads Cu+ substrates into transmembrane transport sites of Cu+-ATPases (González-Guerrero and Argüello, 2008; González-Guerrero et al., 2009; Argüello et al., 2011; Padilla-Benavides et al., 2013b). In this case, the electrostatic docking between the negatively charged surface of CopZ with the electropositive platform region of CopA directs the chaperone Cu+-binding residues toward the "entrance" of the TM-MBS (Gourdon et al., 2011; Padilla-Benavides et al., 2013b). As the ion moves within the enzyme to the TM-MBS, the chaperone is unable to receive the transported metal back from the enzyme.

A new role for Cu+-chaperones has been proposed in *Halobacterium salinarum* (Pang et al., 2013). In this organism, high Cu+ induces temporal changes on transcription rates of two Cu+-chaperones and one Cu+-transporting ATPase through the activation of a Mer-like transcription factor. As observed previously in *P. aeruginosa*, the Cu+ responsive metalloregulator is not up-regulated by Cu+ (Teitzel et al., 2006; Raimunda et al., 2013). This, plus the fact that pools of small Cu+ sequestering molecules like glutathione do not compete with the metalloregulator for Cu+ (Changela et al., 2003), implies that a regulatory feedback involving physical interaction and Cu+ transfer between the chaperones and the regulator might exist. The study proposed that one CopZ would play this role limiting Cu+ access to the metalloregulator. This would allow the repression of the Cu+-transporting ATPase transcription, maintaining a hypothetically required intracellular Cu+ quota. Accordingly, the cytosolic Cu+ increase observed in Cu stressed chaperone mutant cells, points to a dual role of the chaperone participating in the sensing mechanisms (interaction with pools and metalloregulator) and delivering Cu+ to the CopA1-like ATPase.

Finally, CupA, a novel membrane-bound Cu+ chaperone was identified in *Streptococcus pneumoniae* (Fu et al., 2013). This pathogenic bacterium lacks the "classical" CopZ-type Cu+ chaperone. The soluble domain of CupA is able to interact with and deliver Cu+ to CopA N-MBD. Consequently, it was suggested to function as a membrane-bound Cu+-chaperone contributing to Cu+ homeostasis. Surprisingly, CupA adopts a cupredoxin-like folding with a binuclear Cu site accessible and flexible enough to allow Cu+ exchange. The finding of a folding more suited for electron transfer functions, instead of the classical ferredoxinlike observed in previously described bacterial Cu+-chaperones, is an intriguing variation that opens the possibility of novel functions.

#### **PERIPLASMIC CHAPERONES**

Cu distribution and dynamics in the periplasmic space are complex. The presence of different cuproenzymes such as azurin (Raimunda et al., 2013), CueO (Roberts et al., 2002), Cu/Zn-Sod (Gort et al., 1999), laccases (Claus, 2003), cytochrome *c* oxidases (Richter and Ludwig, 2003), and tyrosinases (Claus and Decker, 2006) among other cuproproteins, exemplifies the vast diversity of periplasmic Cu+-binding proteins in different bacterial species (**Figure 1**). Although the function of these enzymes is fairly understood, the mechanisms underlying their metallation and Cu+ traffic in this compartment require further studies. Experimental evidence has shown that both Cu+-ATPases and periplasmic chaperones, such as *Thermus thermophilus* PCu(A)C and *R. capsulatus* Sco/SenC, are required for the assembly of functional cytochrome *c* oxidases (Swem et al., 2005; Abriata et al., 2008; González-Guerrero et al., 2010; Lohmeyer et al., 2012). However, the metal trafficking pathways from either the ATPases or the chaperones to the target proteins are not known. A similar novel example of Cu+ trafficking within the periplasm is the metallation of *S. enterica* sv. Typhimurium SodCII (Osman et al., 2013). Here, the participation of the periplasmic chaperone CueP appears necessary. In this case, two Cu+-ATPases, CopA and GolT, seem able to provide Cu+ for metallation of SodCII.

The role of the periplasmic chaperone CusF, associated with the CusCFBA efflux system, in the efflux of periplasmic Cu+ has also been described. CusF interacts with and delivers Cu+ to CusB, which will further translocate the metal to the extracellular milieu (Mealman et al., 2011). Less is known about the periplasmic Cu+-chaperones from the Pco system. Bioinformatics analysis suggests the existence of two periplasmic chaperones: PcoA and PcoC. PcoA is a MCO (Djoko et al., 2008) that may functionally replace CueO in some bacterial systems where CueO is not present (Hernández-Montes et al., 2012) or under microaerobiosis, when this enzyme is not active (Outten et al., 2001; Rensing and Grass, 2003). Double mutation of*cueO* and *cusCFBA* in *E. coli* GR10 cells rendered a hypersensitive response to Cu+; complementation of this strain with a plasmid encoding for *pcoA* restored Cu+ tolerance of these cells (Lee et al., 2002). No specific function has been proposed for PcoC, though this putative periplasmic protein binds one Cu+-equivalent (Lee et al., 2002). Whether or not these chaperones interact with the putative transporting components of the Pco system (PcoB and PcoD) remains to be elucidated. It is evident that within the bacterial periplasm, a vast diversity of periplasmic chaperones and enzymes have fundamental roles in maintaining Cu+ homeostasis in coordination with the different efflux and influx systems. Thus, highlighting the necessity of further experimental and metalloproteomics integrative studies.

#### **OTHER CYTOPLASMIC CHELATORS AND UNCHARACTERIZED BACTERIAL COPPER POOLS**

How much Cu+ in the surrounding environment can a cell withstand? Gram-negative bacteria, such as *P. aeruginosa* and *E. coli,* replicate at normal rates when grown at high Cu+ concentrations (1–2 mM). Such high Cu+ tolerance might be explained by the fact that bioavailable Cu+ (or Cu+ available in the media) might be several orders of magnitude below that range. However, cells could take up chelated-Cu+ as a silent piggyback rider while bound to nutrients or, more likely, by specialized Cu+ import systems after being stripped off the ligand molecule by specific exchange reactions. Both mechanisms would secure the Cu+ quota. However, the second mode of Cu+ entry would require the proper storage and sorting once Cu+ reaches the cytoplasm. In this regard, there is a lack of information on the constituents and sizes of Cu+ pools in the cell. Interestingly, it has been shown that oxidative and nitrosative stress, conditions where Cu plays a central role as part of the stress-tolerance machinery, triggers synthesis of cytosolic metallothioneins (Gold et al., 2008) and other extracellular cuproproteins (Raimunda et al., 2013). Thus, these proteins might have important functions not only in Cu+ storage, but also in sensing mechanisms. Recent work in *H. salinarum* suggests an interplay between Cu pools and Cu+-chaperones with relevant implications in Cu+ homeostasis (Pang et al., 2013).

#### **TOWARD INTEGRATION**

Previous sections have described the different elements participating in Cu homeostasis, including not only those that move the ion within and across compartments, but the various cuproproteins that require the metal for function. The requirement to maintain the cell free of Cu+ determines that the metal is transferred and delivered to final targets via chelator/protein-protein interactions. The molecular details of some of these events are not well understood and are just started to be uncovered. Similarly, although numerous examples are recorded in the literature, the presence of alternative arrangements of transporters and chelators has not been considered in an integrated fashion. These aspects are discussed in the following sections.

#### **ACHIEVING SELECTIVITY: Cu+-ATPASES AND CHAPERONES**

As pointed out, it is accepted that Cu+ ions are not free in cellular systems because of the high affinity binding of chaperone, sensing, and transport proteins. It is clear that these "high affinities" are for Cu+ binding/release into the aqueous media and they do not represent the molecular affinity for Cu+ when the ion is located at the interacting interphase of two partner proteins "exchanging" Cu+. Thus, Cu+ dependent protein-protein recognition is key for selectivity. An example of this phenomenon is the Cu+ transfer from the cytoplasmic chaperones to the TM-MBS of transport ATPases. In this case, the specific electrostatic complementation of the Cu+-bound chaperone with ATPase appears to determine the interaction (Padilla-Benavides et al., 2013b). As a consequence, structurally similar MBDs carrying a different electrostatic surface cannot deliver Cu+ to the ATPase nor can the apo-form of the chaperone "compete" with its holo-form (González-Guerrero and Argüello, 2008).

This parsimonious model might, however, be challenged in organisms with more than one Cu+-ATPase gene (Padilla-Benavides et al., 2013b). Thus, it could be hypothesized that in the genomes of organisms with more than one Cu+-ATPase gene might also contain genes for different Cu+-chaperones. Additionally, there may be alternative small proteins or other molecules that deliver Cu+ for transport through a particular ATPase.

#### **ACHIEVING SELECTIVITY: TRANSPORTERS AS TARGETING MECHANISMS**

While protein-protein interaction is a likely determinant for compartmental Cu+ distribution to various target molecules, directing Cu+ through distinct transporters also appears to play a central role (**Figure 1**). An example is again provided by alternative roles described for Cu+-ATPases. Bacterial genomes contain at least one gene encoding for a Cu+-ATPase, which is essential in conferring Cu+ tolerance (Rensing and Grass, 2003; Osman and Cavet, 2008; Argüello et al., 2011). However, the presence of multiple Cu+-ATPase coding genes in some genomes suggests their participation in different cellular processes. The idea of redundancy conferring reliability has been considered to understand the presence of multiple Cu+-ATPases. However, this increases complexity and energetic cost. In some cases, such as *Salmonella*, phenotypical and functional differences amongst two Cu+-ATPases, CopA and GolT, are a matter of debate (Checa et al., 2007; Osman et al., 2010). However, new subgroups of functionally distinct, non-redundant Cu+-ATPases have been described (Raimunda et al., 2011).

For instance, the *P. aeruginosa* genome encodes for two nonredundant Cu+-ATPases: CopA1 and CopA2 (**Figure 1**). CopA1 is the classical Cu+-ATPase, and its deletion leads to Cu+ accumulation and sensitivity for this metal. CopA2 presents slow kinetics of transport and higher affinity for Cu+ than that the observed in classic Cu+ detoxifying enzymes (González-Guerrero et al., 2010). This slow rate of transport is incompatible with a role in Cu+-detoxification but seems adequate to contribute to the assembly of cuproproteins. CopA2/FixI-like ATPases are co-transcribed with COX subunits. As such, it has been demonstrated that in *P. aeruginosa* CopA2 is required for the activity of this cuproprotein (González-Guerrero et al., 2010). One question immediately emerges, beyond these frequently observed functions (Cu+ detoxification and COX metallation) should we expect the distinct participation of Cu+-ATPases in other processes? Considering the presence of up to five Cu+-ATPase genes in certain bacterial genomes, it is tempting to hypothesize that unique/specific roles determined by their kinetic characteristics and structural determinants will emerge for each. As a corollary, if each Cu+-ATPase has a distinct function the simultaneous presence of multiple periplasmic chaperones serving different subgroups of cuproproteins is also necessary. In this hypothetical model, the presence of multiple cytoplasmic Cu+-chaperones might also be expected.

#### **ALTERNATIVE ARCHITECTURES FOR Cu+ EFFLUX SYSTEMS: COPPER HOMEOSTASIS IN γ-PROTEOBACTERIA**

Analysis of Cu transport and distribution in various bacteria has shown the frequent presence of polycistronic "transport systems" (See Maintaining the Cu+ quota: Cu+-sensing and transcriptional regulation of homeostatic systems). Recent bioinformatics analyses of systems involved in periplasmic homeostasis have shown that only 3% of the available γ-proteobacteria genomes present the full set of Cu homeostasis systems previously described (Rensing and Grass, 2003; Osman and Cavet, 2008; Hernández-Montes et al., 2012). That is, the vast majority of the organisms analyzed lack one or more components in one of these systems. While the Cu+-ATPases appear universal, the presence of other components seems to cluster in independent groups or clades: CueP, PcoC-YebZ-CutF-CusF-CueO, PcoE-PcoD, PcoA-PcoB, and CusC-CusA-CusB. This suggests that, depending on the presence or absence of some components, bacteria achieve Cu homeostasis through different strategies (Hernández-Montes et al., 2012). Since the selective pressure produced by the host immune system favored and preserved these detoxification mechanisms, it is not surprising that some pathogenic bacteria, such as *Klebsiella pneumonia, Enterobacter cloacae, E. coli, Cronobacter sakazakii*, and *Cronobacter turicensis*, carry the largest amount of proteins required for Cu homeostasis in their genomes (Hernández-Montes et al., 2012). Importantly, the linkage among components seems evolutionarily driven by protein-protein interactions rather than by function. For instance, CusC is distributed independently of CusB and CusA, as it is the most encountered protein after CopA in the clades. Alternatively, CueP was found in organisms also containing CusCFBA, arguing against the hypothesis that the former compensates for the latter in *Salmonella*. This presents challenges to any integrative parsimonious description of Cu homeostasis.

## **FUTURE DIRECTIONS**

Significant insights into the metal selectivity, regulation, transport mechanism, and interaction with metallochaperones have been gained. The high-resolution crystal structure of the Cu+- ATPase, various cytoplasmic and periplasmic chaperones, Cu+ sensors, and several components of the Cus system, as well as their biochemical characterization, has contributed to the elucidation of structure-function relationships in these molecules. The interaction between cytosolic Cu+ chaperones with the ATPases, as well as the access of Cu+ to the TM-MBSs, is relatively well understood. However, little information is available on the interaction of chaperones and sensors, or how the chaperones obtain Cu+ from their transmembrane transporters and subsequently deliver it to cuproenzymes. Despite the fact that biochemical studies will soon target these questions, a higher order of integration appears necessary to understand the homeostasis of this micronutrient. While global transcriptomics studies have provided some progress toward this goal, it appears that establishing the total composition of the cuproproteomes is an important requirement. Subsequently, a systems biology analysis, taking into account Cu+ pools sizes, their kinetics and thermodynamics, seems to be the next logical step.

#### **ACKNOWLEDGMENTS**

This work was supported by National Science Foundation Grant MCB-0743901 (to José M. Argüello).

#### **REFERENCES**


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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 24 August 2013; accepted: 17 October 2013; published online: 05 November 2013.*

*Citation: Argüello JM, Raimunda D and Padilla-Benavides T (2013) Mechanisms of copper homeostasis in bacteria. Front. Cell. Infect. Microbiol. 3:73. doi: 10.3389/fcimb. 2013.00073*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2013 Argüello, Raimunda and Padilla-Benavides. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Managing iron supply during the infection cycle of a flea borne pathogen, *Bartonella henselae*

#### *MaFeng Liu1 and Francis Biville2 \**

*<sup>1</sup> Key Laboratory of Animal Disease and Human Health of Sichuan Province, Avian Disease Research Center, Institute of Preventive Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu/Ya'an, Sichuan, China*

*<sup>2</sup> Unité des Infections Bactériennes Invasives, Département Infection et Epidémiologie, Institut Pasteur, Paris, France*

#### *Edited by:*

*Mathieu F. Cellier, Institut national de la recherche scientifique, Canada*

#### *Reviewed by:*

*Michael F. Minnick, The University of Montana, USA D. Scott Merrell, Uniformed Services University, USA*

#### *\*Correspondence:*

*Francis Biville, Unité des Infections Bactériennes Invasives, Département Infection et Epidémiologie, Institut Pasteur, 28, rue du Dr. Roux, 75015 Paris, France e-mail: fbiville@pasteur.fr*

*Bartonella* are hemotropic bacteria responsible for emerging zoonoses. Most *Bartonella* species appear to share a natural cycle that involves an arthropod transmission, followed by exploitation of a mammalian host in which they cause long-lasting intra-erythrocytic bacteremia. Persistence in erythrocytes is considered an adaptation to transmission by bloodsucking arthropod vectors and a strategy to obtain heme required for *Bartonella* growth. *Bartonella* genomes do not encode for siderophore biosynthesis or a complete iron Fe3<sup>+</sup> transport system. Only genes, sharing strong homology with all components of a Fe2<sup>+</sup> transport system, are present in *Bartonella* genomes. Also, *Bartonella* genomes encode for a complete heme transport system. *Bartonella* must face various environments in their hosts and vectors. In mammals, free heme and iron are rare and oxygen concentration is low. In arthropod vectors, toxic heme levels are found in the gut where oxygen concentration is high. *Bartonella* genomes encode for 3–5 heme-binding proteins. In *Bartonella henselae* heme-binding proteins were shown to be involved in heme uptake process, oxidative stress response, and survival inside endothelial cells and in the flea. In this report, we discuss the use of the heme uptake and storage system of *B. henselae* during its infection cycle. Also, we establish a comparison with the iron and heme uptake systems of *Yersinia pestis* used during its infection cycle.

**Keywords:** *Bartonella***, heme utilization, flea transmission, heme-binding protein,** *Yersinia*

## **INTRODUCTION**

*Bartonella* are α-proteobacteria that employ arthropods for transmission and erythrocyte parasitism as a common parasitism strategy (Dehio and Sander, 1999; Schulein et al., 2001). Currently, 26 distinct *Bartonella* species have been described (Kaiser et al., 2011). *Bartonella bacilliformis, Bartonella quintana,* and *B. henselae* are the three most important human pathogens (Dehio, 2005; Florin et al., 2008; Guptill, 2010). Humans are the reservoir host for *B. bacilliformis* and *B. quintana*, in which they cause various clinical manifestations associated with both intraerythrocytic bacteremia and endothelial cell infection (Hill et al., 1992; Maurin and Raoult, 1996). The cat is the reservoir host for *B. henselae* (Chomel et al., 2009). *B. bacilliformis* causes Oroya fever and verruga peruana (Herrer, 1953a,b). *B. quintana* causes trench fever (Vinson et al., 1969). *B. henselae* causes cat scratch disease (CSD) and bacillary peliosis (Jones, 1993). Both *B. quintana* and *B. henselae* can cause bacillary angiomatosis usually in immunodeficient patients (Spach et al., 1995; Sander et al., 1996).

### *Bartonella* **AND THEIR INFECTION CYCLE**

Each *Bartonella* species appears to be transmitted by specific bloodsucking arthropod vectors, and is highly adapted to one or several mammalian reservoir hosts, in which it causes longlasting intra-erythrocytic bacteremia (Schroder and Dehio, 2005). Intra-erythrocytic bacteremia caused by *Bartonella* in the host has been studied in different rodent models (*B. birtlesii*/mouse, *B. tribocorum*/rat) (Boulouis et al., 2001; Schulein et al., 2001; Marignac et al., 2010). After intravenous inoculation with *invitro*-grown *B. tribocorum*, the bacteria rapidly disappeared in the circulating blood system within a few hours, and blood remained sterile for 3–4 days (Schulein et al., 2001). Endothelial cells (Dehio, 2005) or CD34+ progenitor cells (Mandle et al., 2005) were proposed as the primary niche. About 4–5 days post-infection, bacteria seeded from the primary niche to the bloodstream are able to invade mature erythrocytes (Schulein et al., 2001). Inside erythrocytes, bacteria multiply until reaching a steady number (eight bacteria per infected erythrocyte for *Bartonella tribocorum* in rat erythrocytes), which is maintained for the remaining lifespan of the infected cell (Schulein et al., 2001). Intra-erythrocytic bacteremia in the *B. tribocorum*rat model is persistent for 8–10 weeks (Schulein et al., 2001). During this period, bloodsucking arthropods mediate the transmission to other susceptible hosts. This infectious procedure was also observed in a mouse model for *B. grahamii* and *B. birtlesii* (Koesling et al., 2001; Marignac et al., 2010).

## **IRON/HEME UPTAKE IN** *Bartonella*

Analysis of *Bartonella* genomes indicated that they neither encode for a siderophore biosynthesis pathway, nor for a complete Fe3<sup>+</sup> transport system. *Bartonella* genomes encode for homologs of YfeABCD from *Yersinia pestis* (Perry et al., 2007) and SitABCD from avian pathogenic *E. coli,* both characterized as Fe2<sup>+</sup> and Mn2<sup>+</sup> inner membrane transporters (Sabri et al., 2006; Anjem et al., 2009). *Bartonella* genomes encode for a complete heme uptake system. This heme uptake system is comprised of HutA, an outer membrane heme transporter, HutB, HutC, and HmuV the three components of an inner membrane ABC transporter and a cytoplasmic heme degrading enzyme (HemS) (Parrow et al., 2009; Liu et al., 2012a). HutA from *B. quintana* contains the FRAP and NPNL domains conserved in heme transporters like HemR of *Yersinia enterocolitica* and HumR of *Yersinia pestis* (Parrow et al., 2009). Also, it was shown that HutA from *B. quintana* can apparently transport heme when expressed in *E. coli hemA* mutant strain (Parrow et al., 2009). This activity is TonB-dependent (Parrow et al., 2009). *B. tribocorum* and *B. birtlessii hutA* mutants are unable to establish bacteremia in their reservoir hosts. This result suggests that HutA is required for *Bartonella* heme uptake in mammals (Saenz et al., 2007; Vayssier-Taussat et al., 2010). After its transport into the cytoplasm, heme must be degraded to release iron. *Ex vivo,* HemS of *B. henselae* promotes the release of iron from heme when expressed in *E. coli* (Liu et al., 2012a). *In vitro*, HemS of *B. henselae* binds heme and degrades it in the presence of suitable electron donors, such as ascorbate or NADPH-cytochrome P450 reductase (Liu et al., 2012a). Interestingly, HemS activity was shown to be involved in the oxidative stress response of *B. henselae* (Liu et al., 2012a). All these above data corroborate previous *ex vivo* results demonstrating that *Bartonella* can use heme as an iron source (Sander et al., 2000). In *B. quintana*, expression of the *hutA*, *hems*, *hutB*, *hutC,* and *hmuV* is repressed by heme in an Irrdependent manner (Parrow et al., 2009). Over expression of *fur* in the presence of heme repress *tonB* expression (Parrow et al., 2009).

#### **HEME-BINDING PROTEIN IN** *Bartonella*

*Bartonella* express 3–5 outer membrane heme-binding proteins (Battisti et al., 2006; Minnick and Battisti, 2009). Heme-binding proteins of *Bartonella* are a group of 30–40 kDa porin-like outer membrane proteins that lack similarity with known heme receptors (Minnick et al., 2003). HbpA of *B. quintana* was shown to bind heme *in vitro* (Carroll et al., 2000). However, it did not confer a heme-binding phenotype when expressed in *E. coli* (Carroll et al., 2000). Zimmermann et al., identified a prominent heme-binding protein Pap31 (HbpA), through a heme-binding blot performed with membrane proteins from *B. henselae*. They showed that expressing Pap31 in an *E. coli K12 hemA* mutant strain restored its growth when heme was added at 30 μM and above (Zimmermann et al., 2003). The activity of HbpA as a heme transporter was questioned by other authors. Recently, HbpA of *B. quintana* was shown to be unable to complement the *E. coli hemA* mutant in the presence of heme (Parrow, 2010). Complementation assays using the *E. coli hemA* mutant strain on solid medium in the presence of different heme concentrations also showed that HbpA of *B. birtlessii* cannot transport heme (Biville, unpublished results).

The heme uptake activity of four Hbps of *B. henselae*, expressed in an *E. coli* model strain, was investigated. All Hbps of *B. henselae* can bind heme *in vitro*. No heme transport activity was associated with expression of Hbps in *E. coli* (Liu et al., 2012b). In contrast, Hbps increase heme uptake efficiency when co-expressed with a heterologous heme transporter in an *E. coli* model strain (Liu et al., 2012b). Binding of heme by Hbps was proposed to increase its concentration around the bacteria and, as a consequence, facilitate its uptake. Another potential role for heme binding by Hbps was to provide an antioxidant barrier via heme's intrinsic peroxidase activity (Minnick and Battisti, 2009). Decreasing each Hbp amount using knockdown increases *B. henselae* sensitivity to hydrogen peroxide (Liu et al., 2012b). This antioxidant role of heme-binding proteins was evidenced to play an important role for survival of *B. henselae* in human endothelial cells and in the flea *Ctenocephalides felis*, where reactive oxygen species are produced. The expression levels of genes encoding for heme-binding proteins vary with oxygen, temperature and heme concentration (Battisti et al., 2006). One regulator, Irr, was shown to bind an "H-box" located in the promoter region of *hbp* genes (Battisti et al., 2007). Also, overexpression of RirA increased expression level of *hbpA*, *hbpD,* and *hbpE* (Battisti et al., 2007). Based on their regulatory pattern in *B. quintana*, *hbp* genes were divided into two groups. The first contained *hbpB* and *hbpC*, over expressed under conditions that mimic the gut arthropod environment [high heme concentration and low temperature (30◦C), high O2 concentration] (Battisti et al., 2006). The transcription of *hbpA, hbpD,* and *hbpE* was increased at low heme concentrations at 37◦C. HbpA, HbpD, and HbpE were suggested as being required when the free heme concentration is low, such as in blood circulation in the mammalian host. Transcription of *B. henselae hbpA* is also significantly increased at 28◦C, suggesting that HbpA could protect *B. henselae* from heme toxicity in the arthropod gut (Roden et al., 2012).

### **IRON SOURCES AND OXIDATIVE STRESS ENCOUNTERED DURING** *Bartonella* **INFECTION CYCLE**

Inside the arthropod gut, *Bartonella* encounter high heme and oxygen levels. Such conditions can generate a massive oxidative stress. Thus, inside the arthropod gut, bacteria confront oxidative stress after a blood meal (Graca-Souza et al., 2006). Inside mammals, getting iron required for bacterial growth is a challenge since 99.9% of total body iron is sequestered inside the cells (Wandersman and Stojiljkovic, 2000). Outside the cells, iron is bound to transferrin in the serum or to lactoferrin in mucosal secretions (Cassat and Skaar, 2013). Another iron source in mammals is heme that is mainly contained in hemoproteins like hemoglobin. After erythrocyte lysis, most hemoglobin is bound by haptoglobin. Hemoglobin degradation allows the release of heme that is sequestered by hemopexin to prevent its toxicity (Wandersman and Stojiljkovic, 2000). Thus, obtaining iron from mammals requires transport systems allowing uptake of heme or iron bound to proteins. After inoculation in mammals, in the primary niche proposed to be endothelial cells, the intracellular iron source is iron Fe2+. When bacteria reach the blood stream the iron sources encountered are iron loaded transferrin, heme loaded hemopexin and hemoglobin bound to haptoglobin. Free heme is present at a low concentration (0.5μM). *Bartonella* does not encode for a complete iron uptake system and cannot transport heme bound to hemopexin. Thus, the bacteria must use heme, stored on their surface, as an iron source. Inside erythrocytes the hemoglobin concentration is high and heme can be stored in heme-binding proteins. During their life cycle, *Bartonella* encounter heme rich environments and heme poor environments. To face this alternation, heme-binding proteins are hypothesized to play a crucial role in storing heme in the heme rich environment, and delivering it in iron/heme poor environment. The complex regulatory expression pathway of heme-binding proteins supports this hypothesis. *Bartonella* express one heme transport system HutA and control heme entry and toxicity using heme storage proteins, differentially expressed according to the bacterial infection cycle. The *Bartonella* heme uptake and storage pathways are regulated by Irr, RirA, and Fur similar to α-proteobacteria (Johnston et al., 2007). This iron/heme uptake pathway contrasts with that of another flea-borne pathogen *Yersinia pestis*.

#### **IRON/HEME UPTAKE AND HEME-BINDING PROTEINS IN** *Yersinia pestis*

*Yersinia pestis*, the causative agent of plague, is transmitted mainly by infected flea bites (Chouikha and Hinnebusch, 2012) (**Figure 1**). Like many highly-virulent pathogenic bacteria, *Y. pestis* possess siderophore-mediated iron acquisition systems. Yersiniabactin (Ybt), the siderophore synthesized and secreted by *Y. pestis*, can remove iron from various host iron-binding proteins, such as transferrin and lactoferrin (Fetherston et al., 2010). Fe-Ybt assimilation occurs through the TonB-dependent outer membrane receptor Psn (Lucier et al., 1996). After passage through the outer membrane, Ybt-bound iron is transported into the bacterial cell by the YbtP-YbtQ ABC transporter (Fetherston et al., 1999). In *Y. pestis*, iron Fe2<sup>+</sup> is transported by the YfeABCD and FeoABC systems (Perry et al., 2007). Also, *Y. pestis* encode for two heme transport systems. The *hmu* locus (hmuRSTUV) allows *Y. pestis* to use heme as well as host hemoproteins including hemoglobin, myoglobin, hemealbumin, heme-hemopexin, and hemoglobin-haptoglobin as an iron source (Hornung et al., 1996) (**Figure 2**). The second hemeprotein acquisition system consisted of a heme receptor HasR, HasA hemophore, and HasA-dedicated ABC transporter factor HasDE, as well as a TonB homolog, HasB. However, the Has system appears not to allow the bacteria to use hemoglobin as an iron source under laboratory conditions (Rossi et al., 2001). In *Y. pestis*, the iron and heme uptake systems are Fur regulated (Gao et al., 2008). *Y. pestis*'s main transmission mode depends on the flea foregut blockage (Hinnebusch et al., 1996; Chouikha and Hinnebusch, 2012) resulting in continuous attempts to feed (**Figure 1**). Thus, the contaminated blood is regurgitated back into the mammalian host, where the bacteria rapidly establish an infection (Hinnebusch et al., 1996). Blockage of the flea foregut requires the activity of *hmsHFRS* gene products which synthesize and export the polysaccharide extracellular matrix required for formation of biofilm (Bobrov et al., 2008). Biofilm formation at the surface of proventricular spines is required for infection of the proventriculus (Jarrett et al., 2004). The Hms system is responsible for absorption of exogenous heme or Congo red when the bacteria are grown at 26◦C (Burrows and Jackson, 1956; Surgalla and Beesley, 1969). However, the Hms system never encodes for a heme uptake system. Moreover, the stockpiled heme is not used under irondeficient conditions (Lillard et al., 1999). Consistently, a deletion of the Hms system has no effect on *Y. pestis* virulence in mice infected by a subcutaneous route. This indicated that the Hms system is not required for the virulence of bubonic plague in a mouse model (Lillard et al., 1999). The Hms heme storage system is essential for establishing an infection within the flea midgut and to block the proventriculus (Hinnebusch et al., 1996). Heme absorbed by the Hms system protects *Y. pestis* from the nitric oxide and superoxide anion toxicity, but not from the H2O2 killing effect (Lillard et al., 1999). HmsH and HmsF were

characterized as outer membrane proteins (Pendrak and Perry, 1991) while HmsR, HmsS, and HmsT are inner membrane proteins (Perry et al., 2004). HmsF and HmsR possess domains found in polysaccharide-modifying enzymes and glycosyltransferases, respectively, and HmsT belongs to the family of GGDEF proteins (Lillard et al., 1997; Ausmees et al., 2001; Perry et al., 2004). Transcription of heme acquisition operon *hmsHFRS* and *hmsT* is not regulated by the iron status inside the cells and the availability of exogenous heme (Perry et al., 2004). The temperature-dependent regulation of HmsH, HmsF, HmsR, and HmsT amount is post transcriptional and is related to a decreased stability of these proteins at 37◦C (Perry et al., 2004).

### **CONCLUSION**

The two flea-borne pathogens *B. henselae* and *Y. pestis* can invade the flea gut and mammals. As a consequence, they must adapt their physiology to face the change related to these two environments. Inside the flea gut, bacteria can use heme as an iron source and must face its toxicity. Flea bite is responsible for *Y. pestis* transmission and *B. henselae* can also be transmitted by flea feces contamination (**Figure 1**). This transmission mode is possible since *B. henselae* is able to survive several days within feces. Inside the flea gut, *Y. pestis* forms a biofilm in the proventriculus responsible for blocking. Blocking and biofilm formation have not been described for *B. henselae*. Inside mammals, *Y. pestis* can use different iron and heme sources (**Figure 2**). In contrast, the sole iron/heme source for *B. henselae*, outside the cells, is heme contained in hemoglobin (**Figure 2**). Inside the cells, *B. henselae* could use the reduced form of iron. For *B*. *henselae*, the ability to invade erythrocytes is thus a good opportunity to obtain heme. In *Y. pestis*, no heme transport activity was associated with HmsH, HmsF, HmsR, and HmsT. Moreover, heme absorbed by the Hms system is not used as an iron source (Lillard et al., 1999). In contrast, heme-binding proteins from *B. henselae* were shown to increase the heme uptake efficiency. Inside the flea, the presence of all heme-binding proteins is required for *B. henselae* survival. The pathogenesis of *Y. pestis* is not affected by the absence of a heme storage phenotype. In contrast, disruption of one heme-binding protein in *Bartonella* impairs bacteremia establishment. In regard to the cell invasion *B. henselae* and *Y. pestis* exhibit a similar phenotype since HpbA knockdown in *B. henselae* and Hms− phenotype in *Y. pestis* decrease the ability to invade cells (Lillard et al., 1999; Liu et al., 2012b). Both in *Y. pestis* and *B. henselae* the heme storage phenotype was evidenced to be involved in protection against oxidative stress. In *B. henselae*, all heme-binding proteins were associated with protection against oxidative stress generated by exposure to hydrogen peroxide. In *Y. pestis*, Hms− phenotype decreases the ability to face exposure to paraquat.

For *Bartonella*, the ability to store heme looks like it is more crucial than that for *Y. pestis*. This could explain why small *Bartonella* genomes (Alsmark et al., 2004) encode for 3–5 highly homologous heme-binding proteins whose expression level is submitted to a complex regulatory pathway (Battisti et al., 2007). Since heme storage is a very important process for *Bartonella*, this can explain why decreasing the amount of only one heme-binding protein generates some defects in spite of the presence of others heme-binding proteins. In addition to the regulatory events managing expression of the various *hbp* genes, it cannot be excluded that heme-binding proteins coordinate their activity. A precise biochemical characterization of heme-binding proteins will give fruitful information about a possible cooperative activity of these outer membrane proteins.

## **REFERENCES**


*Microbiology* 145(Pt 1), 197–209. doi: 10.1099/13500872-145-1-197


pathogen Bartonella. *J. Exp. Med.* 193, 1077–1086. doi: 10.1084/jem. 193.9.1077


Sander, A. (2003). Hemin binding, functional expression, and complementation analysis of Pap 31 from *Bartonella henselae*. *J. Bacteriol.* 185, 1739–1744. doi: 10.1128/JB.185.5.1739-1744.2003

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 17 July 2013; paper pending published: 01 August 2013; accepted: 19 September 2013; published online: October 2013. 18*

*Citation: Liu M and Biville F (2013) Managing iron supply during the infection cycle of a flea borne pathogen, Bartonella henselae. Front. Cell. Infect. Microbiol. 3:60. doi: 10.3389/fcimb. 2013.00060*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2013 Liu and Biville. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Role and regulation of heme iron acquisition in gram-negative pathogens

## *Laura J. Runyen-Janecky\**

*Department of Biology, University of Richmond, Richmond, VA, USA*

#### *Edited by:*

*Frédéric J. Veyrier, Institut Pasteur, France*

#### *Reviewed by:*

*Erin R. Murphy, Ohio University Heritage College of Osteopathic Medicine, USA Zehava Eichenbaum, Georgia State University, USA*

#### *\*Correspondence:*

*Laura J. Runyen-Janecky, Department of Biology, Gottwald Science Center, University of Richmond, Richmond, VA 23173, USA e-mail: lrunyenj@richmond.edu*

Bacteria that reside in animal tissues and/or cells must acquire iron from their host. However, almost all of the host iron is sequestered in iron-containing compounds and proteins, the majority of which is found within heme molecules. Thus, likely iron sources for bacterial pathogens (and non-pathogenic symbionts) are free heme and heme-containing proteins. Furthermore, the cellular location of the bacterial within the host (intra or extracellular) influences the amount and nature of the iron containing compounds available for transport. The low level of free iron in the host, coupled with the presence of numerous different heme sources, has resulted in a wide range of high-affinity iron acquisition strategies within bacteria. However, since excess iron and heme are toxic to bacteria, expression of these acquisition systems is highly regulated. Precise expression in the correct host environment at the appropriate times enables heme iron acquisitions systems to contribute to the growth of bacterial pathogens within the host. This mini-review will highlight some of the recent findings in these areas for gram-negative pathogens.

**Keywords: heme, hemin, hem, hemoglobin, iron, pathogens, regulation, Fur**

## **INTRODUCTION**

Almost all living organisms require iron for growth. One notable exception is the Lyme disease pathogen, *Borrelia burgdorferi*, which uses manganese in place of iron (Posey and Gherardini, 2000). Iron is critical for a wide range of cellular functions; however, high levels of iron are toxic because iron catalyzes the formation of reactive oxygen species, and iron acquisition by cells is highly regulated as a result. In the complex interaction between human host and bacterium, iron plays a critical role. Free ferric (Fe3+) iron is poorly soluble in aerobic conditions at neutral pHs; however, ferrous (Fe2+) iron is much more soluble. Additionally, the host sequesters free iron in iron binding proteins (such as ferritin, transferrin, lactoferrin) and in heme and hemoproteins to prevent iron toxicity and to withhold nutrients from pathogens, thereby limiting pathogen growth. Thus, free iron is not readily available to the bacterial pathogen inside the host. Pathogens have evolved numerous mechanisms to capture this limited supply of free iron and iron from host iron proteins. Since the type of iron available varies depending on the location of the pathogen within the human host and since pathogens occupy a wide variety of host niches, there is a diversity of iron acquisition mechanisms employed by both intracellular and extracellular pathogens. This mini-review focuses on acquisition of iron in gram-negative pathogens from one of the most abundant sources—host heme.

### **AVAILABILITY OF HEME AND HEME-CONTAINING MOLECULES IN THE HUMAN HOST**

Approximately 70% of the iron in the human body is within heme, a heterocyclic organic ring called porphryin covalently bound to one ferrous iron atom (Bridges and Seligman, 1995). Heme is critical for functions including oxygen transport, enzymatic reactions, and cellular respiration. Heme is synthesized in almost all human cell types (the majority in erythroid cells, and to a lesser extent in hepatocytes) and can be obtained from the diet (reviewed in Hamza and Dailey, 2012).

Heme is an essential biomolecule; however, excess free heme is toxic to cells due to its lipophilic nature, lipid peroxidation capacity, and ability to catalyze the production of reactive oxygen species (reviewed in Anzaldi and Skaar, 2010). Thus, over 95% of the heme is bound to proteins (hemoproteins), the majority of which are intracellular (Bridges and Seligman, 1995). The intracellular free heme pool is approximately 0.1 μM, which is less than 0.1% of total cellular heme (Granick et al., 1975). The majority of heme in the human body (∼67%) is in hemoglobin, which is primarily found in erythrocytes (Bridges and Seligman, 1995). Other major hemoproteins include myoglobin and cytochromes. Recently, additional hemoproteins have been described, including cytoglobin and neuroglobin, which appear to play a role in oxygen homeostasis/oxygen stress (Liu et al., 2012b; Watanabe et al., 2012; Storz et al., 2013). Additional heme binding proteins exist that are most likely important in scaffolds for synthesis and scavenging heme. The existence of heme chaperones for incorporating heme into apo-hemoproteins has been proposed, but such proteins have yet to been identified in humans (Severance and Hamza, 2009). All of these proteins represent potential heme sources for intracellular pathogens.

Although the majority is intracellular, limited amounts of heme can be found extracellular and thus available to extracellular pathogens. One of the major locations for extracellular heme is in blood hemoglobin (estimated to be 80–800 nM in serum) (Schryvers and Stojiljkovic, 1999). Hemoglobin from lysed erythrocytes is bound by haptoglobin for eventual recycling by macrophage and hepatocytes (Tolosano et al., 2010). Free heme, from damaged hemoglobin, is bound by serum hemopexin and, to a lesser extent, serum albumin. In the gut, dietary heme may be bioavailable to bacteria, either free or complexed with hemopexin. Heme levels are thought to be low in the respiratory track; however, since the heme auxotroph *Haemophilus influenzae* can live in this environment, there must be enough heme to support bacterial growth (Fournier et al., 2011). The urogenital track has varying amounts of heme: the bladder, urethra, and male genital track likely have low heme levels; however, there may be high heme levels in the female urogenital track during menses (Schryvers and Stojiljkovic, 1999). Finally, even in environments where heme is typically low, heme and hemoproteins are released by cells damaged during infection.

#### **BACTERIAL HEME TRANSPORTERS AND LIBERATION OF IRON FROM HEME**

Host microenvironments that have potential heme sources have selected for bacteria with high-affinity heme transport systems which locate and transport heme into the bacterial cell. Heme auxotrophs can use the intact heme for insertion into bacterial hemoproteins. Additionally for both heme prototrophs and autotrophs alike, the iron can be extracted from the heme for other uses (e.g., building Fe-S cluster proteins). Most commonly, there is direct uptake of heme by a cell surface receptor which binds heme or host hemoproteins. A variation of this method includes bipartite systems in which a lipoprotein facilitates heme or hemoproteins binding to the cell surface receptor (Lewis et al., 1998, 2006). Alternatively, some pathogens produce hemophores, small secreted proteins that capture free heme or heme bound to host hemoproteins and then deliver this heme to bacterial surface receptors (Cescau et al., 2007).

There are over 30 well-characterized outer membrane heme receptors that transport heme in gram-negative pathogens, although there are many more putative receptors in genomic databases (**Table 1**). The overall structure of these proteins includes a membrane spanning beta-barrel with extracellular loops that bind to free heme, host hemoproteins, or bacterial hemophores (reviewed in Wilks and Burkhard, 2007). Most are characterized by the presence of FRAP/NPNL domains with a conserved histidine residue that coordinates that heme (Stojiljkovic et al., 1995), although there are reports of heme transporters lacking some of these elements (e.g., PhuR from *Pseudomonas aeruginosa*) suggesting that there are other motifs for heme coordination in outer membrane heme transporters (Tong and Guo, 2009). The energy for heme transport is transduced from the inner to the outer membrane using the TonB/ExbB/ExbD system (reviewed in Krewulak and Vogel, 2011). Thus, all heme outer membrane transporters have a characteristic "TonB box" motif, through which the receptor interacts with TonB. Given the presence of multiple hemoproteins as potential iron sources, there are at least two strategies for bacteria to optimize access to heme iron (**Figure 1**). Some species have multiple receptors, presumably for different hemoproteins or for expression

in different host environments (e.g., *Haemophilus influenza*). Other species have one outer membrane receptor capable of binding to multiple hemoproteins (e.g., *Yersinia enterocolitica* HemR), suggesting the recognition is at the level of the heme molecule (Stojiljkovic and Hantke, 1992; Bracken et al., 1999).

Once the heme molecule has been transported through the outer membrane receptor, ABC transport systems then transport heme though the periplasm, across the inner membrane, and into the cytoplasm (**Table 1** and **Figure 1**). Each ABC transport system consists of a high-affinity periplasmic ligand-binding protein which shuttles heme through the periplasm, two subunits of a cytoplasmic membrane permease, and a peripheral membrane ATPase that supplies the energy for transport. Although there is low sequence homology among the approximately 50 identified periplasmic heme binding proteins, all but one has a conserved tyrosine which is believed to coordinate heme (Tong and Guo, 2009). Frequently, these ABC transporter genes are located in the same operon as or near outer membrane receptor genes; however, orphan ABC transporters that can transport heme exist (e.g., the *E. coli* DppABCD system, which also transports dipeptides) (Letoffe et al., 2006).

Upon entry into the bacterial cell, heme storage, transfer and degradation proteins sequester heme and facilitate extraction of the iron from heme (**Table 1** and **Figure 1**). Bacterial proteins that sequester heme likely prevent heme from catalyzing the formation of reactive oxygen species [e.g., *Shigella dysenteriae* ShuS Wyckoff et al. (2005)]. Other cytoplasmic hemebinding proteins transfer heme to heme degradation proteins [e.g., *Pseudomonas aeruginosa* PhuS Lansky et al. (2006)]. Many pathogens contain homologues of mammalian heme oxygenases (HO), enzymes that cleave the heme to release the iron, generating biliverdin and CO as end products (e.g., *Pseudomonas aeruginosa* HO and *Neisseria meningitidis* HemO). Recently, new structural classes of HOs have been identified such as the "splitbarrel fold class" in *Helicobacter pylori*(HugZ) and *Campylobacter jejuni* (ChuZ) (Guo et al., 2008; Zhang et al., 2011). Additional bacterial enzymes that degrade heme to liberate iron, but release different end products than those released by classical HOs, have been identified. For example, MhuD in *Mycobacterium tuberculosis* cleaves heme to release the iron, generating a novel tetrapyrrole product of called mycobilin, but not CO (Nambu et al., 2013).

For pathogens that can transport heme, the ability to increase the local concentration of heme and/or hemoproteins would be advantageous for growth in the host. Production of cytolysins/hemolysins that lyse cells releasing hemoproteins is common in almost all extracellular and facultative intracellular pathogens that use heme as an iron source. Additionally, some pathogens secrete proteases that degrade hemoproteins to release heme. For example, *Porphyromonas gingivalis* produces hemolysins to lyse cells and proteases called gingipans that have hemaglutin domains and degrade hemoproteins (Chu et al., 1991; Sroka et al., 2001). Alternatively, some bacteria secrete hemophores, small, secreted proteins that capture free heme or heme bound to host hemoproteins and that deliver the heme to bacterial cells. There are several distinct families


*(Continued)*

**Table 1 |** 

**Characteristics**

 **of heme iron acquisition**

 **in some major pathogens.**


*(Continued)*

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**Table 1 | Continued**

**gram-negative bacteria.** Bacteria factors damage host cells releasing heme, Hb, and other hemoproteins. Additionally, secreted bacterial hemophores capture host heme. Extracellular host Hb and heme may be bound by host Hp and Hpx, respectively. A bacterium could acquire iron from these host heme sources using one or more TonB-dependent outer membrane (OM) receptors for these heme compounds, which transport the heme through the outer membrane into the periplasm. Some OM receptors are specific for one

Transport though the periplasmic and across the inner membrane is facilitated by ABC transport systems (green). Inside the bacterium, the heme is degraded using heme oxidases or stored in heme storage protein. Intracellular pathogens would have access to host heme and hemoproteins via similar mechanisms. cyto, bacterial cytoplasm; IM, bacterial inner/cytoplasmic membrane; OM, bacterial outer membrane. Although all the OMR are TonB-dependent, TonB is not shown in the figure.

of hemophores, which share little to no sequence similarity, suggesting convergent evolution of this strategy for increasing local heme concentration (**Table 1**; **Figure 1**). The first class of hemophores identified was the HasA group, initially characterized in *Serratia marcescens* (Letoffe et al., 1994). HasA captures heme, using conserved His32 and Tyr75 residues, and relays it to the outer membrane receptor HasR for transport. Homologues of the HasA/HasR system have only been found in gram-negative bacteria including *Yersinia pestis*(Rossi et al., 2001) and *Pseudomonas aeruginosa* (Ochsner et al., 2000). A second type of hemophore, only found in *Haemophilus influenza*, is HxuA, which captures heme from hemopexin, and the released heme is transported into the cell by outer membrane heme transporters (Fournier et al., 2011).

## **REGULATION OF EXPRESSION OF HEME IRON ACQUISITION GENES BY IRON, HEME AND OTHER STIMULI**

Most genes encoding components of heme iron acquisition systems are not transcribed in iron-replete condition because (1) high-affinity heme iron acquisition is generally not needed and, thus, would be energetically wasteful and (2) excess iron is cytotoxic. One of the most common mechanisms of regulation of heme iron acquisition system expression by iron levels utilizes iron-responsive transcriptional regulators that repress transcription of high-affinity iron acquisition systems when iron is plentiful. The prototypical example is Fur (ferric uptake regulation). In the classical model of iron-repression, Fe-Fur binds to a DNA sequence called the Fur-box in promoters of many highaffinity iron acquisition genes. Fe-Fur occupation of the promoter prevents RNA polymerase binding, thereby repressing transcription. When iron levels decrease, the Fe-Fur equilibrium shifts, Apo-Fur cannot bind to the Fur-box, and transcription occurs [for review Carpenter et al. (2009)]. DtxR and IdeR are iron responsive regulators with similar functions to Fur, and most heme acquisition genes are regulated by repressor proteins from the Fur or DtxR families (**Table 1**).

Not only is excess iron toxic to bacteria, but heme can also be cytotoxic due to its ability to catalyze the formation of reactive oxygen species, its peroxidase activity, and its lipophilic nature which disrupts cell membranes. Thus, for these reasons and for energetic reasons similar to those for iron regulation, expression of a subset of heme iron acquisition systems is regulated by heme levels in some pathogens. In *Bartonella quintana*, transcription of the *hut* operon increases when heme concentrations are lower than required for optimal growth, but decreases at very high heme concentrations. The decrease in expression is predicted to be mediated by the heme-responsive Irr transcriptional regulator, which is only found in some alpha-proteobacteria (Parrow et al., 2009). *Bordetella pertussis* employs an extracytoplasmic function σ factor (ECF) called HurI and its cognate anti-sigma factor HurR to modulate transcription of the *bhuRSTUV* heme uptake operon by heme though a mechanisms in which iron regulation and heme regulation converge. In low iron, Fur repression of *hurIR* is relieved; however, HurI is inactive because it is bound by HurR when heme is absent. Heme binding by BhuB alleviates HurR repression of HurI activity, and HurI can activate transcription of the *bhuRSTUV* operon. (Vanderpool and Armstrong, 2003, 2004). In the presence of heme, the *Vibrio vulnificus* LysR-family transcriptional regulator HupR increases transcription of the Furregulated outer membrane heme receptor gene *hupA* (Litwin and Quackenbush, 2001). In *Pseudomonas aeruginosa*, transcription of the *phu* operon is up-regulated via an uncharacterized, but Furindependent, mechanism (Kaur et al., 2009). Regulatory patterns like these enable expression of heme iron acquisition systems when some heme is available for transport and/or prevent expression of the systems when heme levels are too high. It is unclear why more heme iron acquisition systems are not under such control; however, most expression studies have not formally tested this possibility and, thus, this mode of regulation may be more widespread than reported.

In addition to heme/iron levels, other host-related environmental stimuli may fine-tune expression of heme iron acquisition genes, allowing integration of the iron/heme conditions with other physiological and environmental signals. The cyclic AMP receptor protein, which actives transcription when glucose levels are low, activates expression of *Vibrio vulnificus hupA* (Oh et al., 2009). In *Shigella dysenteriae* and pathogenic *E. coli*, expression of the Fur-regulated outer membrane heme receptor genes *shuA* and *chuA* increases at 37◦C due to post-transcriptional regulation by the 5' untranslated region of these genes (Kouse et al., 2013). The Fur-regulated *Yersinia pestis hasRADEB* and *Vibrio vulnificus hupA* genes have increased expression at 37◦C and 40◦C, respectively, as compared to lower temperatures (Rossi et al., 2001; Oh et al., 2009). *phuR* and *hasA* expression in *Pseudomonas aeruginosa* and *hmuRY* expression in *Porphyromonas gingivalis* are quorum/cell density-regulated (Arevalo-Ferro et al., 2003; Wu et al., 2009). *Haemophilus influenzae* and *Neisseria meningitidis* overlay phase variation on expression of heme acquisition systems, perhaps to counteract the host response to immunogenic OMPs (Ren et al., 1999; Richardson and Stojiljkovic, 1999). Finally, the pathogen's niche may change during the course of infection due to the interaction between host and pathogen and the movement of the pathogen through the host, and available iron sources may change as a result. Tissue specific expression of heme receptors has been show in several pathogens including *Yersinia enterocolitica*, where *hemR* expression is higher in spleen and peritoneum, as compared to liver and intestinal lumen. Furthermore, peritoneum expression of *hemR* is higher than in *in vitro* iron-limited media suggesting there are additional host specific signals besides low iron that allow for maximal *hemR* expression (Jacobi et al., 2001). Finally, there are examples of transcriptional regulation by other regulators suggesting there are more regulatory signals and integration with other regulatory pathways to be discovered.

In summary, each pathogen fine-tunes expression of heme iron acquisition genes to generate the appropriate physiological response for each environmental niche. This response is characterized by particular host heme iron sources/levels, total iron levels, other environmental inputs, and the phylogenetic history of the pathogen. Thus, there are varying patterns of regulation of heme iron acquisition system and regulation of the expression of these systems sometimes overlaps with other global regulatory circuits, creating intricate regulatory pathways in some pathogens. Alternatively, regulation of heme acquisition systems in other pathogens may be relatively simple (e.g., only regulated by an iron-responsive transcriptional regulator) because the pathogen is in a stable environment with low free iron and access to heme.

#### **CONCLUSIONS AND FUTURE OUTLOOK**

Although much is known about heme transport mechanisms and their regulation in many of well-studied pathogens, these topics have not been investigated as extensively in less-common and emerging pathogens, leaving the potential for novel discoveries. Furthermore, the possible fates of the transported heme molecule within the bacterial cell are just beginning to be clarified fully. Additional families of heme iron acquisition and utilization proteins may be waiting to be identified using biochemical (e.g., heme binding assays), genetic (e.g., complementation of *E. coli* heme mutants), and bioinformatic (e.g., mining expression databases for Fur- or iron-regulated genes and searching for heme binding motifs in proteins databases) approaches. Defining the role of each particular heme iron acquisition system in virulence is ongoing for many pathogens, but has been complicated by the presence of redundant systems in some pathogens and/or the use of certain systems in just one niche in the host. Thus, deletions of particular heme iron acquisition genes do not always show an effect in all animal models. It is clear, however that in many pathogens there is a role for some heme iron acquisition proteins, demonstrating the importance of heme for pathogenesis (Henderson and Payne, 1994; Morton et al., 2004, 2007, 2009a; Palyada et al., 2004; Domenech et al., 2005; Brickman et al., 2006; Hagan and Mobley, 2009). A more complete description of heme acquisition and utilization in human pathogens may serve as a reference point for understanding iron acquisition in nonpathogenic symbiotic bacteria that reside in humans and other animals, an area that is currently under-investigated. With respect to gene regulation, expression of the genes encoding most heme iron acquisition systems increases when iron is low due to alleviation of transcriptional repression by ironresponsive transcriptional repressors. However, whether heme levels and/or other regulatory RNAs or proteins modulate this expression further has not been examined for many of these genes.

Pathogens and their human hosts have evolved together, and as a consequence, there is a complex interplay between sequestration of iron from the pathogen by the host and elaboration of mechanism to capture that iron by the pathogen. From the host side, human hemoglobin is quite variable in amino acid sequence; thus, individuals may have differing susceptibility to

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pathogens due to differences in the ability of the pathogen to bind hemoglobin to access the heme (Pishchany and Skaar, 2012). Thus, bacteria pathogen acquisition of heme iron could have been a driving force for hemoglobin evolution. From the pathogen side, the fact that most heme is intracellular and bound to hemoproteins may have been a selective pressure for intracellular growth and protease/hemolysin production in pathogen evolution. Furthermore, heme acquisition genes have been found associated with mobile genetic elements in some pathogens (e.g., *Neisseria meningitidis* and *Shigella dysenteriae*), suggesting potential for rapid spread of these genes via horizontal gene transfer (Wyckoff et al., 1998; Kahler et al., 2001).

#### **AUTHOR CONTRIBUTIONS**

Laura Runyen-Janecky conceived and wrote the entire manuscript.

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**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 02 August 2013; accepted: 10 September 2013; published online: 08 October 2013.*

*Citation: Runyen-Janecky LJ (2013) Role and regulation of heme iron acquisition in gram-negative pathogens. Front. Cell. Infect. Microbiol. 3:55. doi: 10.3389/ fcimb.2013.00055*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2013 Runyen-Janecky. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## *Pseudomonas aeruginosa* adapts its iron uptake strategies in function of the type of infections

## *Pierre Cornelis 1,2\* and Jozef Dingemans 1,2*

*<sup>1</sup> Research Group Microbiology, Department of Bioengineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium*

*<sup>2</sup> Department Structural Biology, VIB, Vrije Universiteit Brussel, Brussels, Belgium*

#### *Edited by:*

*Mathieu F. Cellier, Institut National de la Recherche Scientifique, Canada*

#### *Reviewed by:*

*Angela Wilks, University of Maryland, USA Isabelle J. Schalk, Centre National de Recherche Scientifique, France*

#### *\*Correspondence:*

*Pierre Cornelis, Research Group Microbiology, Department of Bioengineering Sciences, Department Structural Biology Brussels, VIB, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium e-mail: pcornel@vub.ac.be*

*Pseudomonas aeruginosa* is a Gram-negative *γ* -Proteobacterium which is known for its capacity to colonize various niches, including some invertebrate and vertebrate hosts, making it one of the most frequent bacteria causing opportunistic infections. *P. aeruginosa* is able to cause acute as well as chronic infections and it uses different colonization and virulence factors to do so. Infections range from septicemia, urinary infections, burn wound colonization, and chronic colonization of the lungs of cystic fibrosis patients. Like the vast majority of organisms, *P. aeruginosa* needs iron to sustain growth. *P. aeruginosa* utilizes different strategies to take up iron, depending on the type of infection it causes. Two siderophores are produced by this bacterium, pyoverdine and pyochelin, characterized by high and low affinities for iron respectively. *P. aeruginosa* is also able to utilize different siderophores from other microorganisms (siderophore piracy). It can also take up heme from hemoproteins via two different systems. Under microaerobic or anaerobic conditions, *P. aeruginosa* is also able to take up ferrous iron via its Feo system using redox-cycling phenazines. Depending on the type of infection, *P. aeruginosa* can therefore adapt by switching from one iron uptake system to another as we will describe in this short review.

**Keywords:** *Pseudomonas aeruginosa***, iron, siderophores, pyoverdine, pyochelin, heme uptake, Feo, phenazines**

#### *Pseudomonas aeruginosa*

*Pseudomonas aeruginosa* is a ubiquitous *γ* -proteobacterium found in many diverse environments, such as water or the rhizosphere and it produces a large array of colonization and virulence factors allowing it to establish itself in plants, nematodes, insects, and in mammals, including humans where it can cause different types of infections (Rahme et al., 1997; Mahajan-Miklos et al., 1999; Goldberg, 2000; Lyczak et al., 2000, 2002; Pukatzki et al., 2002). *P. aeruginosa* produces different virulence factors, including the extracellular exotoxin A, extracellular proteases, lipase, phospholipases, and toxins injected via the type III secretion system (Coggan and Wolfgang, 2012; Jimenez et al., 2012; Balasubramanian et al., 2013). The production of many virulence factors is coordinately regulated by small diffusing molecules, via a mechanism termed quorum sensing (Juhas et al., 2005; Schuster and Greenberg, 2006; Venturi, 2006; Williams et al., 2007; Girard and Bloemberg, 2008; Winstanley and Fothergill, 2009). The other important factor allowing colonization of the host is the efficient uptake of iron by the bacterium. In the mammalian host iron is not freely available since it is either present in the heme molecule found in hemoproteins (hemoglobin, cytochromes*...*) or strongly chelated by extracellular proteins (transferrin and lactoferrin) (Cornelissen and Sparling, 1994). *P. aeruginosa* is able to switch its lifestyle from planktonic unicellular to a sessile form in biofilms (Goodman et al., 2004; Mikkelsen et al., 2011). Two conflicting sensor systems control the switch from planktonic to sessile lifestyle: RetS for the switch to acute virulence with the production of toxins and expression of type III secretion system, and LadS/GacS for the conversion to the sessile mode with the expression of genes involved in type VI secretion system, production of exopolysaccharides, resulting in biofilm formation and chronic infections (Goodman et al., 2004; Mikkelsen et al., 2011; Coggan and Wolfgang, 2012; Balasubramanian et al., 2013). It is not the purpose of this opinion article to go into the details of the mechanisms at the basis of the switch, but rather to emphasize the importance of this duality in relation to iron uptake systems.

#### **THE TWO OXIDATION STATES OF IRON AND THEIR IMPORTANCE FOR BIOLOGICAL SYSTEMS**

Iron is the fourth most abundant element on earth and it is widely used by organisms because it can exist in two oxidation states, Fe2<sup>+</sup> and Fe3+, which is why it is involved in numerous oxido-reduction reactions (Andrews et al., 2003). Fe3<sup>+</sup> dominates in oxygenated environments, which presents a problem for microorganisms with an aerobic lifestyle because of the extremely low solubility of this form of the metal (Andrews et al., 2003). Conversely, the soluble Fe2<sup>+</sup> is the most abundant form in anaerobic environments or in microaerobic conditions at low pH (Andrews et al., 2003).

### *Pseudomonas aeruginosa* **USES MULTIPLE IRON UPTAKE SYSTEMS**

As already mentioned, bacterial pathogens are confronted with a problem of iron availability in the host since it is sequestered in the heme molecule or by circulating proteins such as transferrin or lactoferrin (Finkelstein et al., 1983; Cornelissen and Sparling, 1994). Although some pathogens, like *Neisseria*, are able to take-up iron directly from transferrin, this is not an option for *P. aeruginosa* (Cornelissen, 2003; Noinaj et al., 2012).

*P. aeruginosa* can use different strategies to acquire iron:


Depending on the type of infection it causes (acute vs. chronic), *P. aeruginosa* can adapt its iron uptake strategy to best fulfill its needs for the metal without spending too much energy.

## **THE** *P. aeruginosa* **PYOVERDINE AND PYOCHELIN-MEDIATED Fe3<sup>+</sup> UPTAKE SYSTEMS**

Siderophores are low-molecular weight excreted molecules that specifically chelate Fe3<sup>+</sup> with a high affinity and are taken up by specific receptors dependent on the energy provided by the TonB cytoplasmic membrane protein (Braun and Killmann, 1999; Boukhalfa and Crumbliss, 2002; Hider and Kong, 2011; Schalk et al., 2012; Schalk and Guillon, 2013). Siderophores are of different types, based on the way the iron is complexed: phenolate-, catecholate-, hydroxamate-, carboxylate-, or mixed type of siderophores have been described.

#### **PYOVERDINES: THE HIGH AFFINITY SIDEROPHORES ARE NEEDED TO CAUSE ACUTE INFECTIONS**

*P. aeruginosa* pyoverdine is a composite (mixed) siderophore comprising a peptide chain and a chromophore (Meyer, 2000; Ravel and Cornelis, 2003; Visca et al., 2007) and the structure of one *P. aeruginosa* pyoverdine (type I) is shown in **Figure 1**. Pyoverdines are the hallmark of fluorescent *Pseudomonas* species (*P. fluorescens*, *P. putida*, *P. syringae*, *P. aeruginosa*) and are produced when the bacteria are grown in low iron conditions (Meyer, 2000; Ravel and Cornelis, 2003; Visca et al., 2007). The conserved chromophore part of the molecule provides the catecholate function participating in the binding of Fe3<sup>+</sup> while the peptide chain is highly variable among representatives of the different *Pseudomonas* species or even within a species (Meyer et al., 2008). Pyoverdines peptide chains contain between 6 and 12 amino-acids (Meyer, 2000; Ravel and Cornelis, 2003; Visca et al., 2007). Within the species *P. aeruginosa*, three different types of pyoverdines have been recognized, which differ by the composition of their respective peptide chain (Cornelis et al., 1989; Meyer et al., 1997). Pyoverdines bind iron with a very high affinity, are able to displace iron from transferrin and pyoverdine production is absolutely needed to cause infection in a burned mouse model or in case of mouse pulmonary infections (Albrecht-Gary et al., 1994; Meyer et al., 1996; Meyer, 2000; Takase et al., 2000a,b; Imperi et al., 2013). Likewise, a mutant in the TonB protein is

avirulent in a mouse model (Takase et al., 2000b). In their recent study, Imperi et al. screened different FDA-approved compounds for their inhibitory activity toward pyoverdine production by *P. aeruginosa*. One such compound, the antimycotic drug flucytosine, caused a strong decrease in pyoverdine production in several strains of *P. aeruginosa* producing the different pyoverdine types and the inhibitory activity was found to target the extracytoplasmic sigma factor (ECF σ) PvdS, which is needed for the transcription of pyoverdine biosynthesis genes (Imperi et al., 2013). Pyoverdine is not only a siderophore, but also a signal molecule since it triggers the production of two extracellular virulence factors, the protease PrpL and the potent toxin exotoxin A (**Figure 2**) (Lamont et al., 2002; Redly and Poole, 2003, 2005; Visca et al., 2007; Cornelis, 2010). Although pyoverdine seems to be essential to *P. aeruginosa* to cause acute infections, it has also been shown to be involved in the establishment of thick mature biofilms (Banin et al., 2005; Patriquin et al., 2008; Glick et al., 2010).

#### **PYOCHELIN: THE LOW-AFFINITY SIDEROPHORE**

Pyochelin (**Figure 1**), the second siderophore of *P. aeruginosa*, is produced by all *P. aeruginosa* isolates, but its affinity for iron is much lower compared to pyoverdine (Cox et al., 1981; Ankenbauer et al., 1988; Brandel et al., 2012). Pyochelin biosynthesis involves a lower number of genes compared to pyoverdine (Serino et al., 1997) and it has been recently demonstrated that *P. aeruginosa* first produces pyochelin and switches to pyoverdine production only when the concentration of iron becomes really low (Dumas et al., 2013). Pyochelin-iron can redox-cycle and has been shown to cause oxidative damage and inflammation, especially in the presence of another *P. aeruginosa* extracellular compound, pyocyanin (Coffman et al., 1990; Britigan et al., 1992, 1997). In chronic infections, such as in CF lungs, the production of pyochelin could play a role in the sustained inflammatory response which is known to occur and cause damage to tissues (Lyczak et al., 2002). It has been shown that pyochelin production is increased in a synthetic CF sputum medium (Hare et al., 2012).

## **HEME UPTAKE SYSTEMS**

*P. aeruginosa* has the capacity to take up heme from hemoproteins via the two systems Has and Phu (Ochsner et al., 2000). Heme is not found in its free form because it is highly hydrophobic causing it to associate with membranes where it promotes non-enzymatic redox reactions (Wyckoff et al., 2005). Heme must therefore be extracted from hemoproteins such as hemoglobin or hemopexin. In the *P. aeruginosa* Phu system heme is directly extracted by an outer membrane TBDR while in the Has system, heme is first extracted by a secreted protein, the hemophore. The hemophoreheme complex is recognized by another TBDR, HasR (Letoffe et al., 1998; Wandersman and Delepelaire, 2004, 2012). Once in the periplasm, heme is bound by a periplasmic binding protein and transported to the cytoplasm by an ABC transporter where

shows the FpvA receptor empty since only apo-pyoverdine is present. FpvA is a TonB-dependent receptor which is also associated with the FpvR anti-σ factor. FpvR sequesters two ECF σ, PvdS and FpvI via its cytoplasmic domain. In this condition there is little possibility for the respective σ factors to associate with the core RNA polymerase and the pyoverdine biosynthesis genes are not transcribed (PvdS) neither the *fpvA* gene (FpvI). The right panel shows what happens when ferripyoverdine binds to the FpvA receptor. This binding triggers a conformational change resulting in the proteolysis of FpvR, which liberates the two σ factors which now can associate with the RNA polymerase. PvdS is not only involved in the transcription of pyoverdine genes, but also in the expression of two virulence genes, *prpL* encoding an extracellular protease and *exoA* encoding the potent exotoxin A.

it is first bound to a heme chaperone, PhuS, before being delivered to the heme oxygenase HemO where the heme molecule will be degraded to form biliverdin, CO, and Fe2<sup>+</sup> (Bhakta and Wilks, 2006; Lansky et al., 2006; Barker et al., 2012; O'Neill et al., 2012). A single mutant in the *phu* system or in the *has* system is still able to take up heme while a double *phu has* mutant is virtually unable to use heme as a source of iron (Ochsner et al., 2000). The Phu and Has systems are schematically presented in **Figure 3**.

## **UPTAKE OF XENOSIDEROPHORES**

*P. aeruginosa* strains have generally more than 30 genes encoding TBDRs, the majority of them involved in the uptake of ferrisiderophores (Bodilis et al., 2009; Cornelis and Bodilis, 2009; Cornelis et al., 2009). The different TBDRs can be classified into two categories, the simple TBDR and the TonB-dependent transducers (TBDT) (Hartney et al., 2011). The TBDT, of which the ferripyoverdine receptor FpvA is an example, can sense the presence of the cognate ferrisiderophore by interacting with a membrane protein which acts as an anti-sigma factor (Hartney et al., 2011). Upon recognition of the ferrisiderophore by the cognate receptor, the anti-sigma factor undergoes a proteolytic degradation liberating the extracytoplasmic sigma factor (ECF σ), which associates with the RNA polymerase to transcribe the receptor gene, causing an auto-induction reaction (Cornelis et al., 2009; Mettrick and Lamont, 2009; Cornelis, 2010; Hartney et al., 2011). The majority of *P. aeruginosa* strains (98%) have a second receptor for type I ferripyoverdine, FpvB, which means that almost all strains have the capacity to use this pyoverdine as source of iron (Ghysels et al., 2004; Bodilis et al., 2009). *P. aeruginosa* is also able to utilize the *E. coli* siderophore enterobactin via two

**FIGURE 3 |** *P. aeruginosa* **has two heme uptake systems, Phu, and Has.** The PhuR TonB-dependent receptor binds directly hemoproteins extracting heme while the HasR receptor binds heme complexed to a secreted hemophore protein HasA. In the periplasm heme is bound to a periplasmic protein which delivers it to an ABC transporter. In the cytoplasm heme is directed to the heme oxygenase HemO by the PhuS chaperone. HemO cleaves the tetrapyrrole ring, leaving biliverdin, CO, and Fe2+.

different receptors, PfeA and PirA since only a double *pfeA pirA* mutant is unable to take up ferrienterobactin (Dean and Poole, 1993; Ghysels et al., 2005; Cornelis and Bodilis, 2009). Other characterized receptors are FoxB and FiuA for the uptake of ferrioxamine and ferrichrome (Llamas et al., 2006; Cuiv et al., 2007; Banin et al., 2008; Hannauer et al., 2010), FemA for the utilization of mycobactin and carboxymycobactin (Llamas et al., 2008), FecA for Fe-citrate uptake (Marshall et al., 2009), ChtA for rhizobactin, aerobactin, and schizokinen (Cuiv et al., 2006), and FvbA for the uptake of vibriobactin (Elias et al., 2011). However, the importance of these xenosiderophore uptake systems in infections has not, to the best of our knowledge, been established. They could however be of importance in case of polymicrobial infections where *P. aeruginosa* could be at advantage because of its capacity to steal siderophores produced by other microorganisms (siderophore piracy) (Traxler et al., 2012) while depriving the competitors from iron because they would be unable to recognize the complex pyoverdine siderophore.

## **UPTAKE OF Fe2<sup>+</sup> VIA THE Feo SYSTEM: INVOLVEMENT OF PHENAZINES**

Unlike Fe3+, Fe2<sup>+</sup> is soluble and is present in anaerobic conditions or in microaerobic environments at lower pH (Andrews et al., 2003). Fe2+probably diffuses through the outer membrane and is further transported inside the cytoplasm by the FeOABC system, which is present in many Gram-negative bacteria (Cartron et al., 2006). The soluble Fe2<sup>+</sup> is transported inside the cells via a transport system composed of the permease FeoB, and the proteins FeoA and FeoC (Cartron et al., 2006). The uptake of Fe2<sup>+</sup> by *P. aeruginosa* is probably relevant when the bacterium finds itself in a microaerobic or anaerobic environment, a situation known to exist in the CF lung mucus where *P. aeruginosa* forms biofilms (Worlitzsch et al., 2002; Yoon et al., 2002). Phenazines are secondary metabolites produced by *P. aeruginosa* (**Figure 4**). Phenazine-1-carboxylic acid (PCA) is the precursor of pyocyanin, a blue-green compound typical of *P. aeruginosa*, and both phenazine compounds can redoxcycle (Wang and Newman, 2008). PCA, and to a lesser extent pyocyanin, is able to reduce Fe3<sup>+</sup> bound to host proteins to Fe2+, allowing the uptake of iron in biofilms via the Feo system (**Figure 4**) (Wang et al., 2011). Recently, it was demonstrated that both phenazines and Fe2<sup>+</sup> accumulate in the lungs of CF patients when their condition deteriorates (Hunter et al., 2012, 2013). In their last article, Hunter et al. (2013) also show that maximal *P. aeruginosa* biofilm disruption is achieved using a combination of both Fe3<sup>+</sup> and Fe2<sup>+</sup> chelators.

## **ADAPTATION OF** *P. aeruginosa* **IRON UPTAKE STRATEGIES: THE EXAMPLE OF CF LUNG INFECTIONS**

As already mentioned, *P. aeruginosa* can switch from the production of pyochelin, the low affinity siderophore, to the more energy demanding high affinity pyoverdine in function of the availability of Fe3<sup>+</sup> (Dumas et al., 2013). One typical example is the adaptation of *P. aeruginosa* to the CF lung environment (Lyczak et al., 2002). When *P. aeruginosa* invades the lungs, it is probably able to produce pyoverdine, but with longer colonization times the bacterium induces a strong inflammatory response, due to the production of pyochelin among other causes, resulting in tissue damage and release of cellular contents, including hemoproteins and other iron-containing proteins (Britigan et al., 1997). Although siderophores, including pyoverdine, have been detected in the sputum samples of CF patients (Martin et al., 2011), pyoverdine-negative mutants accumulate with longer times of colonization (De Vos et al., 2001; Lamont et al., 2009), suggesting

that alternative systems are used by *P. aeruginosa* to fulfill its needs for iron. This is the case since *P. aeruginosa* can take up heme from hemoproteins released by the inflammatory process and Fe2<sup>+</sup> generated by the redox activity of phenazines, in particular PCA (Lamont et al., 2009; Wang et al., 2011; Hunter et al., 2013; Konings et al., 2013).

#### **CONCLUSIONS**

From the analysis of the abundant literature on *P. aeruginosa* iron uptake systems, it is clear that this bacterium can exquisitely adapt its iron capture strategy in function of the type of infection it causes. When causing acute infections, it uses its high-affinity pyoverdine siderophore, which at the same time acts as a signal molecule for the production of acute virulence factors. On the other hand, when establishing itself in a niche where it can persist and cause inflammation, it tends to lose its capacity to produce pyoverdine and to rely on alternative iron uptake strategies, including the uptake of heme from hemoproteins and the uptake of Fe2<sup>+</sup> generated via the redox activity of phenazines. This has certainly implications in terms of finding treatments based on iron chelation since not only Fe3<sup>+</sup> scavenging should be considered (Ballouche et al., 2009; Hunter et al., 2013).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 02 October 2013; paper pending published: 11 October 2013; accepted: 22 October 2013; published online: 14 November 2013.*

*Citation: Cornelis P and Dingemans J (2013) Pseudomonas aeruginosa adapts its iron uptake strategies in function of the type of infections. Front. Cell. Infect. Microbiol. 3:75. doi: 10.3389/fcimb.2013.00075*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2013 Cornelis and Dingemans. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Shared and distinct mechanisms of iron acquisition by bacterial and fungal pathogens of humans

## *Mélissa Caza and James W. Kronstad\**

*The Michael Smith Laboratories, Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada*

#### *Edited by:*

*Mathieu F. Cellier, Institut national de la recherche scientifique, Canada*

#### *Reviewed by:*

*Philippe Delepelaire, Institut de Biologie Physico-Chimique, France Roland Strong, Fred Hutchinson Cancer Research Center, USA Simon C. Andrews, University of Reading, UK*

#### *\*Correspondence:*

*James W. Kronstad, The Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC V6T 1Z4, Canada e-mail: kronstad@msl.ubc.ca*

Iron is the most abundant transition metal in the human body and its bioavailability is stringently controlled. In particular, iron is tightly bound to host proteins such as transferrin to maintain homeostasis, to limit potential damage caused by iron toxicity under physiological conditions and to restrict access by pathogens. Therefore, iron acquisition during infection of a human host is a challenge that must be surmounted by every successful pathogenic microorganism. Iron is essential for bacterial and fungal physiological processes such as DNA replication, transcription, metabolism, and energy generation via respiration. Hence, pathogenic bacteria and fungi have developed sophisticated strategies to gain access to iron from host sources. Indeed, siderophore production and transport, iron acquisition from heme and host iron-containing proteins such as hemoglobin and transferrin, and reduction of ferric to ferrous iron with subsequent transport are all strategies found in bacterial and fungal pathogens of humans. This review focuses on a comparison of these strategies between bacterial and fungal pathogens in the context of virulence and the iron limitation that occurs in the human body as a mechanism of innate nutritional defense.

**Keywords: heme, hemoglobin, transferrin, siderophores, iron, microbial pathogenesis**

### **INTRODUCTION**

Iron is an extremely versatile cofactor that is essential for many biochemical reactions in both mammalian hosts and pathogenic microbes. Ferrous (Fe2+) and ferric (Fe3+) iron, the biologically relevant forms, are found in prosthetic groups, such as iron-sulfur clusters and heme, that are incorporated into many metalloproteins (e.g., aconitase and cytochromes), where the iron serves as a biocatalyst or as an electron carrier. Iron is also found in many mono- and di-nuclear non–heme iron proteins like ferritin and ribonucleotide reductase. The redox potential of iron makes it especially useful for biological processes, in particular for oxidative phosphorylation where iron reduction/oxidation facilitates electron transfer in the respiratory chain. Moreover, iron is present in multiple proteins with diverse functions that include replication and repair of DNA, transport of oxygen, metabolism of carbon [e.g., via the tricarboxylic acid (TCA) cycle] and regulation of gene expression. Several reviews on the importance of iron in biological processes have appeared recently (Evstatiev and Gasche, 2012; Tandara and Salamunic, 2012; Dlouhy and Outten, 2013; Ilbert and Bonnefoy, 2013).

Because of its utility, iron is an essential element and an object of extreme competition between pathogens and their hosts. However, upon oxygenation of the Earth's atmosphere, the predominant form of iron switched from the relatively soluble ferrous state to the extremely insoluble ferric form at neutral pH. In fact, ferric iron is oxidized and polymerized into insoluble polymers of ferric (oxy)hydroxide at pH 7.0, thus further limiting its biological accessibility (Griffiths, 1999; Ilbert and Bonnefoy, 2013). On the other hand, ferrous iron is quite toxic due to its propensity to react with oxygen to generate reactive oxygen species (ROS) via the Fenton and Haber-Weiss reactions. ROS can damage membrane lipids, proteins and DNA (Imlay, 2003). Therefore, iron acquisition, storage, and incorporation into proteins must be carefully managed by mechanisms that promote solubility, control the redox state, and avoid toxicity.

In this review, we discuss and compare selected examples of how pathogenic bacteria and fungi perform iron uptake in the context of competitive sequestration by host proteins. Detailed studies have been performed in a large number of bacterial species and we will focus on illustrative examples. For the fungi, we will describe iron acquisition systems in the three best-studied opportunistic pathogens. These are the mold *Aspergillus fumigatus* (a saprotroph that is also responsible for invasive pulmonary aspergillosis), the polymorphic fungus *Candida albicans* (the cause of skin or mucosal infections and invasive candidiasis), and the yeast *Cryptococcus neoformans* (the agent of cryptococcosis, a disease involving life-threatening meningoencephalitis). We have mainly focused our discussion on iron sources and uptake mechanisms in the context of virulence, with limited coverage of regulation. This is because many excellent reviews have summarized regulatory aspects of iron acquisition and homeostasis in bacteria, fungi and mammals (Andrews et al., 2003; Hentze et al., 2010; Cornelis et al., 2011; Schrettl and Haas, 2011; Wang and Pantopoulos, 2011; Pantopoulos et al., 2012; Philpott et al., 2012; Salvail and Masse, 2012; Kronstad et al., 2013).

## **IRON DISTRIBUTION IN THE MAMMALIAN HOST: OPPORTUNITIES FOR MICROBIAL EXPLOITATION**

A large quantity of iron is potentially available to microbes upon infection of vertebrate hosts, although pathogens must extract this iron from a variety of proteins in blood, different cell types, and tissue locations. In fact, the average human adult contains 3–5 g of iron, the majority of which (65–75%) is found in heme associated with hemoglobin within erythrocytes (red blood cells or RBCs) (McCance and Widdowson, 1937; Andrews, 2000). Each day, 20–25 mg of iron is required to support the synthesis of hemoglobin. The daily intake of iron is very low (1–2 mg per day); therefore a considerable amount of iron is recycled each day mainly by macrophages. Macrophages recognize and phagocytose damaged or senescent RBCs, with the spleen playing a major role in recycling. Phagocytized RBCs are first degraded to extract heme and iron is subsequently released by heme oxygenase (HO-1) for reutilization in erythropoiesis. Hence, approximately 1 g of iron is stored in hepatocytes and macrophages of the liver (Kupffer cells) and spleen. A number of recent reviews have appeared that summarize iron homeostasis in humans (Bleackley et al., 2009; Evstatiev and Gasche, 2012; Ganz, 2012; Tandara and Salamunic, 2012).

Dietary iron is taken up in the intestine (duodenum and upper jejunum) either as ferrous iron [after reduction of ferric iron by the intestinal ferric reductase, duodenal cytochrome B (DcytB)], or as heme (McKie et al., 2001; Latunde-Dada et al., 2008; Evstatiev and Gasche, 2012). The ferrous iron is transported by divalent metal transporter 1 (DMT1), located at the apical membrane of enterocytes (Fleming et al., 1997; Gunshin et al., 1997). The mechanism of dietary heme uptake remains to be clarified. The heme carrier protein 1 (HCP1) was proposed as a heme receptor in duodenal enterocytes (Shayeghi et al., 2005); however, its primary role may be to transport folic acid rather than heme (Qiu et al., 2006). HRG-1, the heme responsive gene-1, was first identified in *Caenorhabditis elegans* as a heme importer (Rajagopal et al., 2008). The human homologue of HRG-1 appears to transport heme as well, but rather from the lysosome into the cytosol (Yanatori et al., 2010; Delaby et al., 2012). FLVCR2 (feline leukemia virus, subgroup C receptor 2) was also recently reported to mediate the endocytosis of heme by mammalian cells (Duffy et al., 2010). The availability of dietary iron to pathogens and the microbiota in the intestine is relevant to colonization, commensalism, and invasion, as demonstrated by recent studies with both bacterial and fungal pathogens (Chen et al., 2011; Kortman et al., 2012; Deriu et al., 2013).

Iron can also be found in blood upon the release of hemoglobin and heme from ruptured erythrocytes and enucleated erythroblasts. However, free hemoglobin is trapped by haptoglobin and taken up by hepatocytes or macrophages via the CD163 receptor (Kristiansen et al., 2001). Heme that is released into the bloodstream can also be bound by hemopexin, albumin, and high and low density lipoproteins (HDL and LDL) (Ascenzi et al., 2005). The hemopexin-heme complex is cleared by hepatocytes and macrophages via the CD91 receptor (Hvidberg et al., 2005). Plasma heme can also originate from the degradation of myoglobin and heme-containing enzymes such as catalases, peroxidases and cytochromes, and from myeloperoxidase secreted from neutrophils (Ascenzi et al., 2005). All these mechanisms promote iron recycling and also protect the host from iron toxicity.

Transferrin in the circulatory system can also potentially be exploited by microbes during bloodstream infections. Approximately 2–3 mg of iron is bound to partly saturated transferrin in plasma (Tandara and Salamunic, 2012). However, transferrin in serum is partially saturated (about 30–40%) to limit the availability of free iron (Williams and Moreton, 1980; Aisen et al., 2001). The transferrin polypeptide has two homologous globular lobes that each binds one iron atom, and ferric iron is tightly bound at physiological pH (*Ka* about 1020 M−1) (Aisen and Brown, 1977). Consequently, the plasma concentration for free ferric iron is <sup>∼</sup>10−<sup>24</sup> M (Otto et al., 1992). Transferrin delivers ferric iron to cells via the transferrin receptor (TfR1) expressed on almost every cell, and also by another receptor, TfR2, expressed in hepatocytes (Hu and Aisen, 1978; Kawabata et al., 1999; Fleming et al., 2000). Iron-loaded transferrin bound to its receptor is endocytosed through a clathrin-dependent pathway, and acidification during endosome maturation dissociates ferric iron from transferrin; the iron-depleted complex is then recycled (Dautry-Varsat et al., 1983; Aisen, 2004; Steere et al., 2012). Subsequent reduction of iron to the ferrous form is achieved in endosomes by the Steap 3 (six-transmembrane epithelial antigen of the prostate 3) protein in erythrocytes and other Steap proteins in non-erythroid cells (Ohgami et al., 2005, 2006). Iron is exported from endosomes to the cytosol by DMT1 (Fleming et al., 1998).

Lactoferrin is a member of the transferrin family that is predominantly found in milk, but can also be present in mucosal secretions like tears and saliva, and in neutrophil granules (Evans et al., 1999). Like transferrin, lactoferrin can bind two atoms of iron, but it retains iron at a much lower pH (∼3.0) than transferrin (∼5.5) (Mazurier and Spik, 1980; Baker and Baker, 2004). Lactoferrin contributes to immunity by iron sequestration at sites of infection. Similarly, the host protein siderocalin (also called NGAL and lipocalin2) plays a role in the innate immune response against microbial pathogens by iron sequestration (Flo et al., 2004). It has been proposed that the small molecule 2,5 dihydroxybenzoic acid, also known as gentisic acid, functions as a mammalian siderophore (a low molecular weight iron chelator) (Devireddy et al., 2010). This molecule is able to bind iron and it was proposed that it delivers the metal to cells via interaction with the siderocalin and the cell surface receptor 24p3R (Devireddy et al., 2005). However, binding studies contradict this hypothesis, since gentisic acid does not form high-affinity complexes with siderocalin and iron (Correnti et al., 2012). The interesting role of siderocalin and it physiological importance in mammalian iron homeostasis are yet to be defined; however, its function in the competition for iron with bacterial pathogens is better understood and described in more detail below in the discussion of siderophores.

Once iron is taken into a cell, it is stored in ferritins for later use or incorporated into metalloproteins in complexes with heme (e.g., catalase, cytochromes, hemoglobin and myoglobin), as mono and dinuclear iron (e.g., ribonucleotide reductase), or as Fe-S clusters (e.g., aconitase, succinate dehydrogenase) (Rouault and Tong, 2008; Dlouhy and Outten, 2013). Ferritins are ironstorage proteins composed of 24 subunits and are able to accumulate up to 4500 iron atoms (Fischbach and Anderegg, 1965; Hoare et al., 1975). These proteins are present in the cytoplasm, nucleus, and mitochondria of cells and also in plasma, and they release iron during iron deficiency via a mechanism involving lysosome acidification and autophagy (De Domenico et al., 2006; Asano et al., 2011). Iron can be exported from cells by ferroportin, a ferrous iron transporter (Donovan et al., 2000). Ferrous iron can be oxidized by hephaestin in intestinal enterocytes and by ceruloplasmin in macrophages, immune cells and other cell types, and loaded onto transferrin for subsequent distribution via the bloodstream (Curzon and O'reilly, 1960; Osaki et al., 1966; Yang et al., 1986; Vulpe et al., 1999). Iron homeostasis in humans is maintained by the major regulator hepcidin that binds to ferroportin and promotes its degradation. This triggers a series of event resulting in a loss of intestinal iron absorption and cellular iron efflux (Anderson et al., 2002; Nemeth et al., 2004). The regulation of iron homeostasis has been reviewed recently (Evstatiev and Gasche, 2012; Tandara and Salamunic, 2012; Finberg, 2013).

The trafficking of iron in mammalian host cells is summarized in **Figure 1**. This figure and the information outlined above define the range of target iron sources that microbes can potentially exploit to proliferate in a variety of host tissues. It is clear that iron homeostasis and availability are tightly controlled by binding proteins and that the competition for iron is therefore a key aspect of infectious diseases. The microbial strategies to compete for iron are outlined in the following sections.

### **MICROBIAL STRATEGIES FOR IRON ACQUISITION FROM MAMMALIAN SOURCES**

Pioneering work by Schade and Caroline in 1944 revealed that high affinity iron binding proteins present in blood and egg whites are able to inhibit the growth of several bacterial species including *Escherichia coli,* as well as the yeast *Saccharomyces cerevisiae* (Schade and Caroline, 1944). They deduced that iron was too tightly bound to these proteins to be available to bacteria and yeast cells, thus inhibiting their growth. Importantly, growth could be restored by addition of iron, and this study was the first to establish a link between iron-related natural host resistance and microbial growth. Subsequently, Bullen et al. demonstrated that iron injection into guinea pigs considerably decreased the lethal dose of *E. coli*, thus suggesting an important role of iron in bacterial infection (Bullen et al., 1968). These and other studies led Kochan to propose the concept of "nutritional immunity," the phenomenon that host control of access to essential nutrients, including iron, could impact the survival and proliferation of microbial pathogens (Kochan, 1973). In response, successful pathogens can overcome nutritional immunity by efficiently acquiring iron within the host via four strategies that target specific iron sources: (1) iron acquisition from heme and hemecontaining proteins; (2) iron acquisition from transferrin, lactoferrin, and ferritin; (3) ferric iron acquisition by siderophores and; (4) uptake of ferrous iron. These strategies are described in the following sections.

## **IRON ACQUISITION FROM HEME AND HEME-CONTAINING PROTEINS**

One strategy for microbes to obtain iron during infection of mammals is to target heme, hemoglobin, or complexes containing these molecules (e.g., haptoglobin-hemoglobin, hemopexinheme). This strategy requires access to host heme sources, and several pathogenic bacteria and fungi therefore secrete hemolysins to lyse red blood cells and release hemoglobin, and/or produce hemoglobin proteases to degrade the protein. Hemolysins have been characterized in Gram-negative bacteria, such as pathogenic *E. coli* (α-hemolysin HlyA, ClyA, Hpb, and EspC) (Felmlee et al., 1985; Otto et al., 1998; Ludwig et al., 2004; Drago-Serrano et al., 2006), *Vibrio cholerae* El-Tor (HlyA) (Stoebner and Payne, 1988) and *Bordetella pertussis* (CyaA) (Glaser et al., 1988), as well as in Gram-positive bacteria including *Staphylococcus epidermis* (δ-hemolysin Hdl) (Verdon et al., 2009) and *Bacillus cereus* (hemolysin BL) (Senesi and Ghelardi,

**FIGURE 1 | Iron transport and homeostasis in human cells. (A)** Iron recycling in macrophages via phagocytosis of senescent red blood cells, uptake of heme-hemopexin and hemoglobin-haptoglobin complexes, and iron-loaded transferrin. **(B)** Dietary iron and heme absorption by intestinal endocytes via DMT1 and the heme receptor HCP1/FLVCR2, respectively. Iron-loaded siderocalin can also be absorbed via the receptor 24p3R. Iron is

extracted from these carriers by heme oxygenase in lysosomes or by reductases in endosomes and is used for metabolic processes (mitochondria, storage, or export). Export is performed by ferroportin in partnership with ceruloplasmin in macrophages and with hephaestin in intestinal cells. Iron is loaded on transferrin for distribution. The descriptions of the specific proteins are given in the text.

2010). Fungi also produce hemolysins or have been reported to possess hemolytic activity. For example, the mold *A. fumigatus* secretes the hemolysin Asp which has hemolytic activity on chicken erythrocytes (Yokota et al., 1977). The polymorphic fungus *C. albicans* also possesses hemolytic activity but the yeast *C. neoformans* reportedly does not (Manns et al., 1994). Moreover, microbial pathogens have evolved two mechanisms to acquired iron from heme and heme-containing proteins: 1) direct uptake of heme and 2) use of hemophores (heme-binding proteins). These strategies have been extensively studied in numerous Gram-negative bacteria, while only a few examples are known in Gram-positive bacteria and in fungi. Iron acquisition strategies from heme and heme-containing proteins are illustrated in **Figure 2** and described below.

## **HEME UPTAKE IN BACTERIA**

## *Gram-negative bacteria*

The direct uptake of heme by Gram-negative bacteria is a wellcharacterized strategy for iron acquisition. In general, heme uptake is achieved by recognition and binding to a specific receptor in the outer membrane (OM). These surface receptors can directly bind heme and process its transport, but they are usually also able to bind heme-containing proteins like hemoglobin, haptoglobin-hemoglobin, hemopexin-heme, and myoglobin (Wandersman and Delepelaire, 2004). In that case, heme is extracted from these complexes and transported into the periplasm in a TonB-dependent manner (in Gram-negative bacteria). TonB-ExbB-ExbD is an energy-transducing complex that energizes outer membrane receptors to facilitate translocation of specific cargo (Braun and Braun, 2002). The majority of the outer membrane heme uptake receptors, as well as siderophore transporters and some transferrin/lactoferrin receptors (see below),

**FIGURE 2 | Diagrams of hemoglobin and heme uptake and utilization.** Mechanisms are depicted for the Gram-negative bacterium *Pseudomonas aeruginosa* via the Phu system, and for the Gram-positive bacterium *Staphylococcus aureus* via the Isd system. For comparison, the scheme in the fungal pathogen *Candida albicans* is also illustrated and components include the receptors Rbt5, Rbt51, and Pga7. A schematic is also included to depict endocytosis (via ESCRT functions) and processing (with the heme oxygenase Hmx1). Additional details about the specific proteins are given in the text.

are members of the TonB-dependent outer transporter (TBDT) family. Two unique domains are found in the TBDT fold: (1) a β-barrel with 22 anti-parallel strands and an internal diameter of 35–47 Å and (2) an N-terminal cork domain that blocks the internal space and prevents passive diffusion through the barrel (Ferguson and Deisenhofer, 2002). Furthermore, two His residues, or in some cases one Tyr residue, are required for heme transport across the barrel. Two conserved amino acid motifs (FRAP and NPNL) have also been identified on the extracellular loop containing one of the His residues (Bracken et al., 1999; Hagan and Mobley, 2009). The NPNL motif plays a role in binding heme due to its surface exposed location, whereas the FRAP motif is likely involved in heme transport across the cell membrane because it is buried inside the barrel (Liu et al., 2006). Once in the periplasm, heme is bound to a heme transport protein (HTP) and delivered to an ABC transporter in the inner membrane. Heme is then transported into the cytoplasm in an ATP-dependent fashion with subsequent degradation and iron release by bacterial heme oxygenases (Anzaldi and Skaar, 2010). The expression of the majority of these systems is controlled by the bacterial "ferric uptake regulator" protein Fur. Fur is a dimeric DNA-binding repressor that uses ferrous iron as a co-factor. Fur plays a central role in the bacterial response to iron starvation as it binds to promoter regions of iron-regulated genes at a so-called "fur box" and represses their expression under iron-replete conditions. Upon iron limitation, the Fur-Fe(II) complex dissociates from the DNA, thereby allowing transcription of iron-regulated genes (Hantke, 1981; Bagg and Neilands, 1987; De Lorenzo et al., 1987). Other proteins can also participate in the regulation of these systems under different conditions.

One of the two heme uptake system in *Pseudomonas aeruginosa* is illustrated in **Figure 2** as an example. This system is encoded by the *phuR-phuSTUVW* genes, and PhuR is the outer membrane receptor, PhuT is the HTP, PhuUVW is the inner membrane ABC transporter and PhuS is an intracellular heme trafficking protein that delivers heme to the heme oxygenase (*pa*-HO, PigA, or HemO) (Ochsner et al., 2000; Ratliff et al., 2001; Lansky et al., 2006). This system facilitates the uptake of heme and the use of heme from hemoglobin since mutation of any component reduces growth on these iron sources (Ochsner et al., 2000). However, the specific mechanism of heme extraction from hemoglobin at the cell surface by PhuR or another protein is currently unknown. The amino acid sequence of PhuR shares similarity with several heme and hemoglobin receptors, such as HutA from *V. cholerae* (Henderson and Payne, 1994), ChuA *from E. coli* O157:H7 (Torres and Payne, 1997) and HmuR from *Y. pestis* (Hornung et al., 1996). Furthermore, three conserved motifs were identified in the amino acid sequence of PhuR, including a "TonB box," and this strongly suggests a TonB-dependent translocation mechanism (Ochsner et al., 2000). Once heme is translocated into the periplasmic space, it is bound by PhuT. This heme transport protein binds heme and protoporphyrin IX at a ratio of 1:1 with high affinity (Kd ∼ 1*.*2 and 14nM, respectively) (Tong and Guo, 2007). It is believe that PhuT delivers heme to the inner membrane transporter PhuUVW, although direct transfer of heme has not been demonstrated. Once in the cytoplasm, heme is bound by PhuS and delivered to the heme oxygenase HemO (Lansky et al., 2006). Protein-protein interaction studies identified a mechanism in which a heme-dependent conformational switch in PhuS drives heme release to HemO in a unidirectional fashion (Bhakta and Wilks, 2006; O'Neill et al., 2012). HemO is a δ-regioselective heme oxygenase that cleaves heme and produces biliverdin IX-β and -δ (Ratliff et al., 2001). Interestingly, the metabolic flux of heme uptake is driven by HemO, since mutation of the heme oxygenase results in loss of heme uptake and no production of biliverdin (Barker et al., 2012; O'Neill and Wilks, 2013). Expression of *phuR* and the *phuSTUVW* operon is controlled by the Fur regulator and two "Fur boxes" were identified by DNase footprinting (Ochsner et al., 2000).

Other similar heme and hemoglobin uptake systems have been characterized in several pathogenic Gram-negative bacteria including *Yersinia pestis* (HmuRSTUV) (Hornung et al., 1996; Thompson et al., 1999), *Yersinia enterocolitica* (HemRSTUV) (Stojiljkovic and Hantke, 1992, 1994), *Vibrio cholerae* (HutABCD) (Occhino et al., 1998) and the uropathogenic *E. coli* strain CFT073 (ChuA-Hma-DppABCDF) (Torres and Payne, 1997; Torres et al., 2001; Letoffe et al., 2006; Hagan and Mobley, 2009). Expression of the outer membrane receptors of these systems is regulated by Fur, they are all members of the TBDT family and they possess conserved FRAP and NPNL motifs. Site-direct mutagenesis of the TBDT HemR from *Y. enterocolitica* identified two conserved His residues as being required for heme transport through the receptor pore, while binding activity of heme was not affected (Bracken et al., 1999). The Hma receptor of *E. coli* requires a cell-surface exposed Tyr residue for heme use rather than the conserved His residues (Hagan and Mobley, 2009). The contributions of these systems to virulence have been evaluated for some of these bacteria. For example, virulence was tested for mutants lacking the Hmu and ChuA-Hma systems of *Y. pestis* and *E. coli,* respectively. Heme acquisition via the receptors ChuA and Hma in uropathogenic *E. coli* contributes to disease in mice, while the Hmu system in *Y. pestis* does not (e.g., when inoculated by subcutaneous or retro-orbital injection), presumably due to redundancy in iron acquisition systems for this species (Thompson et al., 1999; Torres et al., 2001; Hagan and Mobley, 2009).

Other systems are present in gram-negative bacteria for the use of hemoglobin as a sole source of iron. For example, *Haemophilus influenzae* type B (Hib) is able to use hemoglobin via three TonB-dependent cell surface receptors, HgpA, HgpB, and HgpC, that bind hemogloblin and hemoglobin-haptoglobin (Jin et al., 1996; Morton et al., 1999). Deletion of the *hgp* genes abolishes growth on hemoglobin-haptoglobin as a sole heme/iron source, although only a partial reduction occurred in the ability to use hemoglobin (Morton et al., 1999). The heme utilization protein Hup is responsible for this residual heme uptake activity from hemoglobin, since mutation of all of the *hgp* and *hup* genes resulted in a severe growth defect in the presence of low concentrations of hemoglobin or heme as the only iron source (Morton et al., 2004). It is believe that heme is extracted from hemoglobin at the cell surface by these receptors, although this activity has not yet been demonstrated. Nevertheless, once heme is translocated into the periplasm, it is taken up by the lipoprotein HbpA (Hanson and Hansen, 1991; Hanson et al., 1992b). Deletion of the *hpbA* gene in Hib resulted in growth diminution in the presence of low concentrations of heme, heme-hemopexin, and heme-albumin, but not in the presence of hemoglobin or hemoglobin-haptoglobin. These data indicate that *H. influenzae* may possess other periplasmic heme transporters in addition to HbpA (Morton et al., 2007b, 2009a). It has been proposed that HbpA delivers heme to the DppBCDF membrane transporter (Morton et al., 2009b), although several homologues of heme ABC transporters (SapACBDF and OppABCDF) have been discovered and may participate in heme transport in different strains of *H. influenzae* (Fleischmann et al., 1995; Mason et al., 2011). Nothing is known about how iron is extracted from heme once it enters the cytoplasm. Mutations in the *hgp*, *hup, hpbA* and *hel* (encoding lipoprotein *e* (P4), another periplasmic heme binding protein) genes had no impact on virulence in a bacteremia model with 5-day old rats (Morton et al., 2004, 2007a). However, mutation of the *hgp*, *hbpA*, and *hel* genes in Hib caused a significantly lower rate of bacteremia relative to the wild-type strain in a 30 day old rat model of infection (Seale et al., 2006; Morton et al., 2007b, 2009a). The level of plasma hemopexin and haptoglobin increases with age in rats, which may explain the requirement of different heme and hemoprotein acquisition systems for the virulence of *H. influenza* in older rats (Seale et al., 2006). This system of heme acquisition from hemoglobin is similar to the heme acquisition system from *P. aeruginosa* in that heme is transported into the cytoplasm by specific TonB-dependent outer membrane receptors, periplasmic proteins, and inner membrane ABC transporters.

Outer membrane receptors have also been identified that facilitate the use of hemoglobin. For example, *Neisseria meningitidis* is able to bind hemoglobin but not heme through the outer membrane receptor HmbR. This protein, like many outer membrane receptors for heme and iron acquisition systems, requires a functional TonB system and is regulated by Fur. HmbR functions by binding to hemoglobin and removing heme for subsequent translocation into the periplasm, and an NPNL motif has a possible role in heme removal. The cork domain of HmbR is also involved in heme passage to the periplasm (Perkins-Balding et al., 2003). Furthermore, an *hmbR* mutant is attenuated in an infant rat model for meningococcal infection, indicating that the use of hemoglobin as an iron source is important for *N. meningitidis* virulence (Stojiljkovic et al., 1995). *N. meningitidis* and *Neisseria gonorrhoeae* also possess a distinct bipartite TonBdependent receptor for hemoglobin designated HpuAB*.* HpuB is an outer membrane receptor and HpuA encodes a lipoprotein, and together they transport heme from hemoglobin and the hemoglobin-haptoglobin complex. Expression of the *hpuAB* operon is regulated by iron and Fur (Lewis and Dyer, 1995; Lewis et al., 1997; Turner et al., 1998; Rohde et al., 2002). Both receptors (HmbR and HpuAB) are also subject to phase variation (Lewis et al., 1999) and the presence of either HmbR or HpuAB was found to be highly correlated with clinical isolates causing disease, suggesting a role in virulence for iron acquisition from hemoglobin (Tauseef et al., 2011). It is believe that these bacteria employ phase variation to more effectively adapt to the hostile environment of the host. So far, nothing is known about the intracellular transport of heme into the cytoplasm of these bacteria, although the process likely involves an ABC transporter. Similarly to the heme uptake system of *P. aeruginosa*, a heme oxygenase, HemO has been identified in *Neisseria* species and is required for the degradation of heme into ferric iron, biliverdin, and CO (Zhu et al., 2000a,b).

### *Gram-positive bacteria*

Heme acquisition systems in Gram-positive bacteria share properties with those in Gram-negative bacteria in that they consist of cell surface receptors for heme, cell wall chaperone proteins that facilitate internalization of heme, ABC transporters that perform membrane translocation and heme oxygenase activities to release iron from heme. The HtaAB-HmuOTUV heme acquisition system identified in *Corynebacterium diphtheriae* illustrates the organization of one such system. Cell surface exposed HtaA binds hemoglobin and transfers heme to HtaB (Allen and Schmitt, 2009). Heme is believed to be transported inside the cell by the activities of the cell wall protein HmuT, the ATP transporter HmuUV and the cytoplasmic heme oxygenase HmuO that extracts the iron (Wilks and Schmitt, 1998; Drazek et al., 2000; Allen and Schmitt, 2009, 2011). Recently, another heme/hemoglobin system was identified in *C. diphtheriae* (Allen et al., 2013). Specifically, three proteins that are exposed on the cell surface, ChtA, ChtB, and ChtC, are able to bind heme and hemoglobin, with ChtA showing the highest affinity. A mutant lacking both *chtB* and *htaB* had significantly impaired iron use from heme, indicating a contribution of both systems for heme iron acquisition. No evaluations of virulence have been reported for these systems. As in Gram-negative bacteria, it appears that multiple heme acquisition systems are generally present in the Gram-positive bacteria characterized to date (i.e., with several surface receptors and ABC transporters).

## **USE OF HEMOPHORES BY BACTERIA** *Gram-negative bacteria*

Hemophores are secreted proteins with the ability to bind heme and/or heme-containing proteins in the extracellular environment. This definition has recently been expanded to include any surface-exposed (or secreted) protein involved in the transfer of heme to a transporter for import (Wandersman and Delepelaire, 2012). A hemophore system was first discovered in 1994 in *Serratia marcescens* and others have been identified subsequently in Gram-negative and Gram-positive bacteria (Letoffe et al., 1994a; Wandersman and Delepelaire, 2012). As described below, a candidate hemophore has also recently been described in the fungal pathogen *C. neoformans* (Letoffe et al., 1994a; Cadieux et al., 2013). The hemophore system in *S. marcescens* (Has) includes the secreted HasA protein that is able to extract heme from hemoglobin, hemopexin and myoglobin (Letoffe et al., 1994a; Wandersman and Delepelaire, 2012). HasA is secreted by the export complex HasDEF, where HasD is an ATPase, HasE is a membrane fusion protein and HasF is an outer membrane protein (Letoffe et al., 1994b). Heme is transferred from hemoproteins to HasA by a passive mechanism due to higher affinity of HasA for heme, without protein-protein complex formation (Letoffe et al., 1999). HasA interacts with and delivers heme to the specific outer membrane receptor HasR (Izadi-Pruneyre et al., 2006). HasR can perform the uptake of heme from hemoglobin alone, but the process is 100 times more efficient with the participation of HasA (Arnoux et al., 2000). The determination of the structure of the HasR receptor revealed a cork and a β-barrel organization like other heme receptors, with two conserved His residues being important for heme binding (Izadi-Pruneyre et al., 2006; Krieg et al., 2009). This receptor actively transports heme with the help of HasB, a TonB orthologue that functions specifically with HasR (Benevides-Matos et al., 2008). After heme transfer from HasA to HasR, apo-HasA remains bound to HasR. The release of apo-HasA from the receptor is performed in an energy-driven process by HasB (Paquelin et al., 2001). This recycling process for HasA is only observed in the presence of heme, which is also required for the induction of *hasB* expression (Rossi et al., 2003; Wandersman and Delepelaire, 2012). The Has system is negatively regulated by iron and Fur, and positively regulated by a sigma and anti-sigma (HasI and HasS) signaling cascade triggered by hemeloaded hemophore binding to HasR (Rossi et al., 2003; Cwerman et al., 2006). Systems with similarity to Has have been reported in *P. aeruginosa* (Letoffe et al., 1998), *P. fluorescens* (Idei et al., 1999), and *Y. pestis* (Rossi et al., 2001). The contribution of the Has system to the virulence of *Y. pestis* has been assessed in a mouse model of bubonic plague and no role was found, even in the absence of the Hmu system for heme uptake (Rossi et al., 2001).

*H. influenza* type b (Hib) also produces a hemophore system (Hxu) that is synthesized from the *hxuCBA* gene cluster. The hemophore HxuA is able to bind the human heme-hemopexin complex and to release heme into the medium. HxuA is either anchored to the cell surface or partially released into culture medium depending on the strain (Wong et al., 1995). Unlike HasA, HxuA does not directly bind heme, but rather it interacts with hemopexin and interferes with its ability to sequester heme (Hanson et al., 1992a; Fournier et al., 2011). Free heme is then internalized by the TonB-dependent outer membrane receptor HxuC, while HxuB is involved in secretion of HxuA (Cope et al., 1995). It was also reported that HxuC is involved in residual use of heme from hemoglobin, as seen in an *hgp* triple knockout mutant, and in the direct use of heme from heme-albumin complexes. (Cope et al., 2001; Morton et al., 2007a). Moreover, deletion of the *hxuABC* genes significantly impaired the virulence of the strain in a 5-day-old rat model of bacteremia, but not in a 30-day old rat model, suggesting that these age related differences may be related to changes in levels of host heme-binding proteins during the development of the rat (Morton et al., 2007a). Subsequent heme transport across the inner membrane is likely to be performed by various ABC transporters as previously discussed.

Similar to HpuAB from *Neisseria* species, a bipartite receptor for heme has been described for *Porphyromonas gingivalis*. In this bacterium, the TonB-dependent heme receptor HmuR mediates heme uptake with the help of a heme-binding lipoprotein HmuY (Simpson et al., 2000; Olczak et al., 2008; Wojtowicz et al., 2009). HmuY has low affinity for heme but the proteolytic activity of secreted proteases (gingipains) on host heme-containing proteins facilitates heme release. For example, it has been demonstrated that HmuY can extract heme from hemoglobin after pre-treatment with gingipains (Olczak et al., 2001; Smalley et al., 2007, 2011). In fact, R-gingipains cleave hemoglobin to allow oxidation from ferrous to ferric iron thus facilitating release of heme and subsequent degradation of globin by K-gingipain. Free heme is then bound by HmuY. HmuY was proposed to be a hemophorelike protein because it was found either attached to the outer membrane or release in the supernatant. This release is dependent on proteolytic cleavage by gingipains (Wojtowicz et al., 2009). Once heme is bound to HmuY, it is transferred to HmuR for uptake. As with other outer membrane receptors, HmuR has two conserved His residues and the NPDL motif for heme binding and utilization (Liu et al., 2006). The *hmuY* and *hmuR* genes are regulated by the transcriptional activator PG1237 and are part of a larger locus (*hmuYRSTUV*) (Wu et al., 2009). The *hmuSTUV* genes may be responsible for heme transport to the cytoplasm. HmuS has sequence similarity to the cobN/Mg chelatase, HmuT and HmuU are similar to permeases and HmuW is annotated as an ATP-binding protein involved in hemin import (Lewis et al., 2006). Further studies are required to investigate these roles.

#### *Gram-positive bacteria*

The Isd (iron regulated surface determinant) system found in *Staphylococcus aureus* is one of the best-characterized mechanisms of iron acquisition from heme in Gram-positive bacteria. As illustrated in **Figure 2**, the Fur-regulated Isd machinery is composed of four cell wall-anchored proteins (IsdABCH), two cell wall sortases (SrtA and SrtB), a membrane transporter (IsdDEF) and two cytoplasmic heme oxygenases (IsdG and IsdI) (Mazmanian et al., 2003). Cell surface exposed IsdA binds heme, IsdB binds hemoglobin and heme, and IsdH binds heme, hemoglobin, haptoglobin and the complex of hemoglobinhaptoglobin (Dryla et al., 2003, 2007). Once heme is extracted by IsdH or IsdB, it is transferred unidirectionally to either IsdA or IsdC. Transfer can also occur from IsdA to IsdC, and bidirectionally between IsdH and IsdB. As well, IsdC transfers heme unidirectionally to the lipoprotein IsdE (Liu et al., 2008; Muryoi et al., 2008; Zhu et al., 2008). The IsdABCH proteins in *S. aureus* have been structurally characterized and found to all possess one or more NEAT domains. The NEAT domain is a poorly conserved 120 amino acid region that is encoded in variable numbers in genes located in the vicinity of putative siderophore transporter genes; NEAT therefore stands for near transporter (Andrade et al., 2002). NEAT domains can bind heme, hemoglobin, or hemoglobin-haptoglobin. As an example, IsdH possesses three NEAT domains (N1, N2, and N3) and it has been demonstrated that N1 and N2 bind hemoglobin and hemoglobin-haptoglobin, whereas N3 binds heme (Pilpa et al., 2009). It is thought that the transfer of heme across the cell wall of *S. aureus* occurs by protein-protein interactions that shuttle heme from one NEAT domain to another until the membrane is reached (Wandersman and Delepelaire, 2012). The heme molecule is believed to be transported across the inner membrane via the action of the ABC transporter IsdDEF. However, it has been shown that an *isdDEF* mutant does not significantly reduce heme use, suggesting that another ABC transporter might be present in *S. aureus* (Mazmanian et al., 2003). Nonetheless, iron is then released in the cytoplasm by degradation via the action of IsdG and IsdI, which have similarity to monooxygenases (Wu et al., 2005). Virulence assays revealed that an *isdB* mutant, but not an *isdH* mutant, showed reduced virulence in a murine abscess model of disease (Torres et al., 2006).

The Isd system has also been identified in several Grampositive bacteria including *Streptococcus pyogenes* and *Bacillus anthracis* (Maresso et al., 2006; Nygaard et al., 2006). In the latter species, the Isd system is composed of three genes (*isdX1, isdX2,* and *isdC*) that encode proteins with one or more NEAT domains. It was shown that IsdX1 and IsdX2 are secreted proteins that extract heme from hemoglobin and deliver it to cell wallbound IsdC (Fabian et al., 2009). The IsdX1 and IsdX2 proteins do not possess a cell-wall anchoring motif, and they are therefore thought to be secreted hemophores (Maresso et al., 2008). So far, it is unclear how heme is transported into the cell for *B. anthracis*. A new hemophore, Hal, has also been discovered recently in this bacterium (Balderas et al., 2012). Hal contains one NEAT domain that binds heme, the protein has several leucine-rich repeats and is proposed to be covalently coupled by a sortase to the cell wall via its C-terminal Gram-positive bacterium anchor (GPA). Deletion of *hal* resulted in a growth defect on heme or hemoglobin as the sole iron source (Balderas et al., 2012). Recently, another iron regulated leucine-rich surface protein (IlsA) was identified in *Bacillus cereus*. This protein has a conserved NEAT domain and directly binds heme. Inactivation of *ilsA* decreases the ability of the bacterium to grow in the presence of hemoglobin, heme, and ferritin, indicating a role in iron acquisition for IlsA. Moreover, the *ilsA* mutant showed a reduction in growth and virulence in an insect model, suggesting an important role for iron acquisition in disease caused by *B. cereus* (Daou et al., 2009).

A similar heme/hemoglobin uptake system (Shp-Shr-HtsABC) was found in *Streptococcus pyogenes*, where HtsABC encodes an ABC transporter, Shp binds heme on the cell surface and Shr binds hemoglobin and the hemoglobin/haptoglobin complex (Lei et al., 2002, 2003; Bates et al., 2003). Furthermore, the direct transfer of heme from hemoglobin by Shr to Shp has been demonstrated (Lu et al., 2012), and further characterization of Shp revealed two NEAT domains, a series of leucine-rich repeats and the absence of a cell wall-anchoring motif. It was also demonstrated that Shr spans the cell wall and is exposed to the extracellular environment, reminiscent of the Hal protein of *B. anthracis* (Fisher et al., 2008; Ouattara et al., 2010).

#### *Mycobacterium tuberculosis*

*M. tuberculosis* is known to acquire iron from transferrin and lactoferrin through secretion of the siderophores mycobactin and exochelins (Gobin and Horwitz, 1996). However, a pathway of heme utilization involving a secreted hemophore (Rv0203) and two trans-membrane proteins, MmpL11 and MmpL13, has been discovered recently. Mutation of either *rv0203* or *mmpL11* significantly reduces growth on heme or hemoglobin as a sole iron source, while mutation of *mmlp13* was unsuccessful and the gene may be essential (Tullius et al., 2011). It also has been shown that Rv0203 binds heme with a similar affinity constant to the heme binding proteins PhuS and HmuT from *P. aeruginosa* and *Y. pestis*, respectively (Owens et al., 2012). Upon binding, Rv0203 rapidly transfers heme to either of the inner membrane transporters MmpL11 and MmpL13 (Owens et al., 2013).

#### **HEME UPTAKE BY FUNGI**

Much less is known about heme use by pathogenic fungi compared with bacterial pathogens. The ability to utilize heme and hemoglobin as an iron source by *C. albicans* was first described in 1992 (Moors et al., 1992). It was initially demonstrated that *C. albicans* binds erythrocytes via complement-receptor-like molecules (Moors et al., 1992). Subsequently, it was found that *C. albicans* possesses a hemolytic factor described as a secreted mannoprotein, although further characterization is needed for this factor (Watanabe et al., 1999). Nevertheless, the uptake of hemoglobin is mediated by specific receptors exposed on the surface of *C. albicans,* as illustrated in **Figure 2**. The first two heme/hemoglobin receptors to be identified were Rbt5 and Rbt51. Both of these are extracellular, glycosylphophatidylinositol (GPI)-anchored proteins and they harbor a conserved CFEM domain that may be involved in heme binding (Weissman and Kornitzer, 2004). CFEM domains are composed of eight cysteine residues of conserved spacing and they are found in a number of fungal membrane proteins (Kulkarni et al., 2003). Three other members of the hemoglobin-receptor family (Csa1, Csa2, and Pga7) have been identified based on the presence of the CFEM domain (Almeida et al., 2009). Rbt51 is sufficient by itself to confer the ability to use hemoglobin on *S. cerevisiae*, while a mutant of *RBT5* also showed a strong reduction of heme and hemoglobin use by *C. albicans* (Weissman and Kornitzer, 2004). Furthermore, Rbt5 facilitates the rapid endocytosis of hemoglobin into vacuoles in *C. albicans* cells. This endocytic process requires Myo5, a type I myosin that may be involved in endocytic vesicle scission, CaSla2, which is an actin-binding protein also required for endocytosis, an active vacuolar ATPase, and a member of the HOPS complex (CaVps41) (Weissman et al., 2008). Components of the ESCRT (endosomal sorting complex required for transport) system are also involved in the utilization of hemoglobin. ESCRT complex proteins are generally involved in transporting membrane proteins to the multivesicular body compartment and from there to the vacuole, where proteins are degraded (Hurley and Emr, 2006). Therefore, it was interesting that the ESCRT components are involved in heme/hemoglobin utilization, and that individual mutants of *C. albicans* (i.e., *vps2, vps23, vps24, vps38, vps36* and *snf7*) show a growth delay in the presence of hemoglobin (Weissman et al., 2008). It is not clear how heme and hemoglobin are processed upon Rbt5 binding and endocytosis, but it has been proposed that acidification of the vacuole might be sufficient to extract heme from hemoglobin. Heme degradation by the heme oxygenase CaHmx1 may occur in the vacuole or in the cytosol via transport of the heme molecule by a vacuolar transporter (Pendrak et al., 2004; Weissman et al., 2008). Importantly, CaHmx1 is required for full virulence in a mouse model of disseminated candidiasis (Navarathna and Roberts, 2010).

The pathogenic yeast *C. neoformans* is also able to grow on hemoglobin and heme as sole iron sources (Jung and Kronstad, 2008). *C. neoformans* secretes a 43 KDa serine proteinase that degrades hemoglobin and other substrates, although further characterization of this proteinase is needed (Yoo Ji et al., 2004). Information is starting to accumulate about heme use by *C. neoformans*. For example, an *Agrobacterium-*mediated T-DNA insertion screen for mutants with reduced growth on heme identified the ESCRT-I protein Vps23 as being important for iron acquisition from heme. Deletion of *vps23* resulted in growth defect on heme presumably due to a defect in endocytosis and proper sorting of the heme cargo (Hu et al., 2013). Recently, the first candidate hemophore in fungi was described in *C. neoformans*. This mannoprotein, Cig1, was shown to support iron acquisition from heme and to make a contribution to virulence in a mouse model of cryptococcal disease (Cadieux et al., 2013). However, the contribution of Cig1 to virulence was only evident in a mutant that also lacked a reductive, high affinity uptake system (described further below).

It is likely that other pathogenic fungi are able to use heme and hemoproteins. For example, the dimorphic pathogen *Histoplasma capsulatum* uses heme as a sole source of iron via a putative cellsurface receptor, although further studies are needed to elucidate the mechanism of heme uptake (Foster, 2002). It is also known that some important fungal pathogens, such as *A. fumigatus,* lack the ability to use heme as an iron source (Eisendle et al., 2003; Schrettl et al., 2004; Haas, 2012).

#### **IRON ACQUISITION FROM TRANSFERRIN, LACTOFERRIN AND FERRITIN**

#### **DIRECT ACQUISITION OF IRON FROM TRANSFERRIN AND LACTOFERRIN IN BACTERIA**

Several bacterial pathogens can utilize non-heme, ironcontaining proteins like transferrin, lactoferrin, and ferritins as sources of iron. As illustrated in **Figure 3**, the Gram-negative bacteria *N. meningitidis* and *N. gonorrhoeae* possess the receptors TbpAB and LbpAB that mediate the uptake of ferric iron from transferrin and lactoferrin, respectively (Cornelissen et al., 1992; Biswas and Sparling, 1995). The TbpAB system consists of two transferrin-binding proteins expressed from a biscistronic operon regulated by Fur and encoding the TonB-dependent protein TbpA and the lipoprotein TbpB that acts as a co-receptor (Ronpirin et al., 2001). TbpA binds apo and holo-transferrin with similar affinities, whereas TbpB only binds preferentially to iron-containing transferrin (Cornelissen and Sparling, 1996; Boulton et al., 1998). TbpA is able to extract iron from transferrin in the absence of its co-receptor, but the process is considerably more efficient in the presence of TbpB. In fact, it has been estimated that TbpB helps to internalize about half of the iron obtained from transferrin and also participates in the dissociation of apo-transferrin from the cell surface (Anderson et al., 1994; Derocco et al., 2009). The affinities for transferrin are distinct for TbpA and TbpB, and for the combined receptor (TbpAB), which suggests that formation of the dual receptor results in unique characteristics in the interaction with transferrin (Cornelissen and Sparling, 1996). Upon transferrin binding, TbpB forms a transient triple complex with TbpA. TbpA catalyzes a conformational change that leads to iron release and dissociation of apo-transferrin with the help of the TonB complex. TbpA is a TBDT protein and the conformational change moves the cork domain allowing the formation of a transient docking site for iron inside the β-barrel and transfer to the periplasmic ferric binding protein FbpA (Noinaj et al., 2012a,b). FbpA then initiates transport into the cytosol (Siburt et al., 2009).

descriptions of the specific proteins are given in the text.

FbpA is also known as the bacterial transferrin due to its similarities in structure and function to human transferrin (Parker Siburt et al., 2012). The *fbpABC* operon encodes an ABC transport system, where FbpB is a permease and FbpC is a nucleotidebinding protein that provides energy to transport iron across the cytoplasmic membrane (Adhikari et al., 1996; Strange et al., 2011). The FbpABC system is also involved in transport of iron from lactoferrin but is not required for the acquisition of iron from heme and hemoglobin (Khun et al., 1998). The FbpABC transporter is also required for the transport of xenosiderophores (i.e., siderophores such as enterobactin and salmochelin S2 from other microbes) in a TonB-independent fashion (see below) (Strange et al., 2011). Virulence was assessed in a murine model of *N. meningitidis* bacteremia, and both a *tbpA tbpB* mutant and a *tbpA* mutant are avirulent in mice suggesting a role for iron acquisition through transferrin in disease (Renauld-Mongenie et al., 2004). A *tbpB* mutant was as virulent as the wild-type strain. Importantly, a transferrin receptor mutant (*tbpA tbpB*) for *N. gonorrhoeae* was unable to initiate urethritis in human volunteers, demonstrating that a bacterial iron acquisition system is an essential virulence factor for human infection (Cornelissen et al., 1998). This bipartite receptor mechanism of iron acquisition from transferrin and lactoferrin is reminiscent of the heme bipartite receptor HupAB in *Neisseria* spp. and the hemophore Has system in *S. marcescens*. In addition, the use of an inner membrane ABC transporter is a recurrent mechanism shared by many pathogenic bacteria for iron transport.

The lactoferrin uptake system LbpAB in the *Neisseria* species is very similar to TbpAB in that LbpA is a TonB-dependent outer membrane protein and LbpB is a lipoprotein that serves as a coreceptor for LbpA (Biswas and Sparling, 1995). In contrast to the situation with TbpB and transferrin, LbpB is not required for uptake of iron from lactoferrin (Biswas et al., 1999). The specific mechanism of iron extraction from lactoferrin remains to be elucidated. Lactoferrin receptors are only found in about 50% of clinical isolates, whereas all isolates of *N. gonorrhoeae* express receptors that bind human transferrin. However, *in vivo* experiments demonstrated that the expression of the lactoferrin receptor in the absence of the transferrin receptor is sufficient for establishment of infection. Furthermore, in a mixed infection of male volunteers, expression of both lactoferrin and transferrin receptors gave a competitive advantage over a strain expressing only the transferrin receptor, thereby further indicating a role in virulence for iron acquisition from lactoferrin (Anderson et al., 2003).

#### **INVOLVEMENT OF CATECHOLAMINES IN IRON ACQUISITION FROM TRANSFERRIN AND LACTOFERRIN**

The availability of iron from transferrin and lactoferrin for bacterial use is also influenced by catecholamine stress hormones (epinephrine, norepinephrine and dopamine) and inotropes (isoprenaline and dobutamine) (Freestone et al., 2000, 2002; Neal et al., 2001; O'Donnell et al., 2006). Catecholamine stress hormones are able to bind transferrin and lactoferrin, to form direct complexes with ferric iron, and to reduce ferric to ferrous iron with subsequent liberation from transferrin (Sandrini et al., 2010). Free iron can then be used for bacterial growth via other specific iron uptake systems. This ability of stress hormones to mediate bacterial iron acquisition from transferrin and lactoferrin has been proposed to function in biofilm formation in intravenous lines by the Gram-positive bacterium *S. epidermidis* (Lyte et al., 2003). It may also play a role in the development of intra-abdominal sepsis by *E. coli* (Freestone et al., 2002) and be a contributing factor in biofilm formation on endotracheal tubing during ventilator-associated pneumonia caused by *P. aeruginosa* (Freestone et al., 2012).

For bacterial pathogens, iron acquisition involving catecholamines is mediated by siderophores or by mechanisms that are partially or completely independent of siderophore function. For example, enterohemorrhagic *E. coli* O157:H7 and *Salmonella enterica* can grow on transferrin in the presence of norepinephrine. The growth of both species in the presence of transferrin and norepinephrine also requires the synthesis, transport, and degradation of the siderophore enterobactin, suggesting that once iron is release from transferrin by a catecholamine, it is transported inside the bacteria by enterobactin. (Freestone et al., 2003; Methner et al., 2008). The transport of iron by enterobactin is discussed in more detail below. *Bordetella bronchiseptica* also uses catecholamines (norepinephrine, epinephrine, and dopamine) to obtain iron from both transferrin and lactoferrin (Anderson and Armstrong, 2008; Armstrong et al., 2012). The efficiency of iron acquisition from transferrin in the presence of catecholamine is increased by addition of enterobactin, but the siderophore is not essential, since norepinephrine alone can stimulate growth in presence of transferrin. This growth stimulation is dependent on TonB because a mutation in *tonB* abolishes growth in presence of transferrin and NE (Anderson and Armstrong, 2008). A genetic screen identified three TonB-dependent outer membrane receptors (BfrA, BfrD, and BfrE) for catecholamines that are required for growth in the presence of catecholamines and transferrin. These receptors can also mediate the uptake of enterobactin and 2,3-dihydroxybenzoic acid. The characterization of catecholamine-mediated iron uptake for *B. bronchiseptica* revealed a siderophore-independent pathway. However, its features imply that siderophores may act to shuttle iron between transferrin and outer membrane receptors (Armstrong et al., 2012).

Growth stimulation by norepinephrine in the presence of transferrin has been also shown to be independent of siderophore production for *E. coli* and *Bacillus subtilis* (Miethke and Skerra, 2010). Mutants with defects in siderophore biosynthesis in both bacteria are still able to grow in the presence of norepinephrine and transferrin, indicating that iron-complexed norepinephrine can directly serve as an iron source. However, the FeuABC uptake system for bacillibactin was also identified in *B. subtilis* to be involved in the use of iron-complexed norepinephrine, since deletion of this locus abolished growth stimulation by NE and transferrin (Miethke et al., 2006). Furthermore, this iron acquisition could be abolished by the addition of siderocalin, the host innate immune protein that binds enterobactin and inhibits its use by the bacteria (Miethke and Skerra, 2010). A similar system may operate in other Gram-positive bacteria because a siderophoredeficient strain of *S. aureus* can grow in human serum in the presence of catecholamines (epinephrine, norepinephrine, and dopamine). In this case, iron uptake via catecholamine sequestration is mediated by the transporter SstABCD, as shown in **Figure 3**. Based on sequence similarities, the *sst* genes encode two putative cytoplasmic membrane proteins (SstA and SstB), an ATPase (SstC), and a membrane-bound lipoprotein (SstD) (Morrissey et al., 2000). Moreover, *S. aureus* can use its endogenous siderophores, staphyloferrin A and staphyloferrin B, to access the transferrin iron pool (Beasley et al., 2011). The collective activities of the siderophore transporters (Hts and Sir) and the Sst transport system are required for full virulence of *S. aureus* in intravenously challenged mice. However, *sst* inactivation was sufficient to significantly decrease colonization of the mouse heart (Beasley et al., 2011).

### **FUNGAL ACQUISITION OF IRON FROM TRANSFERRIN AND LACTOFERRIN**

Transferrin and lactoferrin are known to have an inhibitory effect on the growth of the pathogenic fungi *A. fumigatus*, *C. albicans* and *C. neoformans* (Sridhar et al., 2000; Ahluwalia et al., 2001; Lahoz et al., 2008; Almeida et al., 2009; Kornitzer, 2009; Okazaki et al., 2009). The mechanism of inhibition is probably due to iron sequestration by partially iron-loaded protein because additional studies have shown that these fungi can acquire iron from fully iron-loaded transferrin under specific conditions. For example, iron-loaded transferrin, but not apo-transferrin, restores growth to iron-starved cells of *C. albicans* (Knight et al., 2005). In this fungus, the use of transferrin iron is dependent on fungal contact with the transferrin and on a reductive, high affinity uptake system that includes the iron permease Ftr1 and a reductase Fre10 (**Figure 3**). Importantly, Ftr1 is required for virulence thus suggesting iron acquisition from transferrin during infection (Ramanan and Wang, 2000). Siderophore and heme uptake systems did not play a role in iron acquisition from transferrin by *C. albicans*. In contrast, *A. fumigatus* uses secreted siderophores to obtain iron from transferrin and this may be important during disease (Hissen et al., 2004; Hissen and Moore, 2005; Haas, 2012). The situation for *C. neoformans* resembles that of *C. albicans* where an iron permease, Cft1, of the reductive, high affinity system is required for iron use from transferrin and for full virulence (Jung et al., 2008).

## **IRON ACQUISITION FROM FERRITINS**

Ferritins represent a potentially rich source of iron for bacteria and fungi. For example, *N. meningitides* is able to use iron from ferritin after a rapid redistribution and degradation of cytosolic ferritin in infected epithelial cells (Larson et al., 2004). Ferritin is in fact aggregated and recruited by intracellular meningococci and degradation of ferritin provides an excellent source of iron (Larson et al., 2004). For the fungi, ferritin use as a sole iron source has been best characterized for *C. albicans*. This pathogen uses the adhesin Als3 as a ferritin receptor, as demonstrated by the findings that deletion of *als3* blocks ferritin binding and that heterologous expression of Als3 in *S. cerevisiae* confers the ability to bind ferritin (Almeida et al., 2008).

## **FERRIC IRON ACQUISITION BY SIDEROPHORES**

Many bacteria and fungi (and perhaps mammals) produce siderophores (low molecular weight, high affinity ferric chelators) to acquire and transport iron, as detailed in several reviews (Andrews et al., 2003; Miethke and Marahiel, 2007; Winkelmann, 2007; Haas et al., 2008). The first three siderophores were isolated and identified from bacteria (mycobactin and coprogen) and fungi (ferrichrome). Snow and collaborators first reported in 1949 that supplementation with purified mycobactin enhanced the growth of *Mycobacterium johnei* (also known as *M. paratuberculosis*) (Francis et al., 1949). Mycobactin was considered to be a growth factor, although a high affinity for ferric chloride was also noted (Francis et al., 1953; Snow, 1954). Early experiments identified other growth factors with apparently dissimilar structures but strong chelating activity for ferric iron, including the Terregens Factor (later identified as arthrobactin), coprogen, and ferrichrome (Hesseltine et al., 1952; Lochead et al., 1952; Neilands, 1957). Garibaldi and Neilands reported the key finding that the production of ferrichrome A was enhanced when the fungus *Ustilago sphaerogena* was grown in iron-depleted medium, and that several other microbes (e.g., the bacteria *B. subtilis* and *Bacillus megaterium,* and the fungus *Aspergillus niger*) produced iron-binding compounds under similar conditions (Garibaldi and Neilands, 1956). This work led to the suggestion that the growth factors might be involved in a system for sequestering and transferring iron that is induced during iron deficiency. This key observation led to a refined view of the function of siderophores and their biological significance. In fact, siderophores enhance growth by coordinating ferric iron for uptake by microorganisms using facilitative transport machinery.

Numerous reviews have appeared describing the types of siderophores produced by microbes (Crosa and Walsh, 2002; Winkelmann, 2002, 2007; Andrews et al., 2003; Miethke and Marahiel, 2007; Haas et al., 2008). Therefore, we will focus on selected principles and examples for bacterial and fungal pathogens to illustrate general properties. Importantly, in addition to a role in iron acquisition in the context of infection, some siderophores are secreted by microorganisms to deprive competing organisms of iron (Emery, 1982). Conversely, many microorganisms have evolved the transport machinery to use heterologous siderophores produced by other microbes (xenosiderophores) (Winkelmann, 2007). This is the case for opportunistic pathogen *P. aeruginosa* which produces two different siderophores, pyoverdine and pyochelin (Cox, 1980; Cox and Adams, 1985), but can utilize a variety of heterologous siderophores from other bacteria and fungi, including ferrioxamine B, ferrichrome and enterobactin (Poole et al., 1990; Cuiv et al., 2007). A focus on the use of xenosiderophores is also the case for the fungal pathogens *C. albicans* and *C. neoformans,* as described below. Of course, many pathogenic microorganisms produce siderophores that are directly implicated in their virulence (Miethke and Marahiel, 2007; Garenaux et al., 2011). In this case, siderophores of bacterial and fungal pathogens can directly remove iron from host proteins such as transferrin to support proliferation in vertebrates (Konopka et al., 1982; Brock et al., 1983).

#### **ENTEROBACTIN, THE ARCHETYPICAL SIDEROPHORE**

The archetypical bacterial siderophore is the catecholate enterobactin, also known as enterochelin. This siderophore was identified simultaneously by O'Brien and Gibson (1970), who isolated enterochelin from *E. coli*, and Pollack and Neilands (1970), who characterized enterobactin from *S. enterica* Typhimurium. Enterobactin has been extensively studied over the past 40 years and it is the siderophore with the strongest known affinity for ferric iron (*K*<sup>d</sup> of 10−52M) (Harris et al., 1979). Enterobactin participates in the retrieval of iron from transferrin, as discussed earlier, and the siderophore is produced by *E. coli, Salmonella spp.*, *Klebsiella spp*, and by some strains of *Shigella* (Wyckoff et al., 2009)*.* Enterobactin can, however, be sequestered by the host innate immune protein siderocalin (also known as lipocalin 2) as a defense mechanism to prevent bacteria from accessing iron (Goetz et al., 2002; Flo et al., 2004). In response, the pathogenic enterobacteria don't rely solely on enterobactin to gain access to iron within the host and they possess multiple siderophore systems. In particular, enterobactin can be modified into salmochelins by the addition of up to three glucose molecules on its catechol moieties (Hantke et al., 2003; Bister et al., 2004). This glycosylation blocks binding by siderocalin without altering iron binding by the siderophore (Fischbach et al., 2006). Hence, the production of salmochelins contributes to virulence of pathogenic *E. coli, S.* Typhimurium, and *K. pneumoniae* (Caza et al., 2008, 2011; Crouch et al., 2008; Bachman et al., 2012). Two other types of siderophores can be produced by these bacteria, aerobactin and yersiniabactin, and these can also escape siderocalin sequestration and contribute to the virulence of pathogenic *E. coli* and *K. pneumoniae* (Dozois et al., 2003; Fischbach et al., 2006; Bachman et al., 2011; Correnti and Strong, 2012).

A common observation is that pathogens often deploy multiple iron acquisition systems or siderophores to support proliferation in the host (Dozois et al., 2003; Garenaux et al., 2011; Kronstad et al., 2013). In particular, redundancy in siderophore iron acquisition systems can mask the contribution of each individual system to virulence. A good example comes from the production of pyochelin and pyoverdine by *P. aeruginosa*. In an intramuscular infection model with immuno-compromised mice, only the strain mutated for the production of both pyochelin and pyoverdine showed attenuation of virulence. However, in an intranasal murine model of infection, only pyoverdine is required for pathogenesis, although loss of both molecules more severely attenuated virulence (Takase et al., 2000).

#### **SIDEROPHORE TRANSPORT IN GRAM-NEGATIVE BACTERIA**

Typically, the internalization of siderophores in bacteria is facilitated by ABC type transporters. Although in some cases, inner membrane permeases driven by energy proton motrive force can also translocate iron-loaded siderophores. The iron-loaded siderophore is first recognized and internalized by specific cellsurface receptors, which are all members of the TBDT family and are usually regulated by Fur. The ferri-siderophore is then processed through the different membranes and the cell wall by chaperone proteins and facilitators. Once the molecule reaches the intracellular space, the iron atom can be released by physical degradation of the siderophore or by a redox-mediated process, the affinity of siderophores for ferrous iron being much less than that for ferric iron. In some cases, such as with pyoverdine uptake by *P. aeruginosa*, iron can be released in the periplasmic space with subsequent transport of siderophore-free iron into the cytoplasm and recycling of the empty siderophore to the extracellular medium (Faraldo-Gomez and Sansom, 2003; Wandersman and Delepelaire, 2004; Schalk and Guillon, 2013).

For Gram-negative bacteria, iron-loaded siderophores need to pass two membranes and a peptidoglycan cell wall to reach the intracellular space (**Figure 4**). Recognition and internalization requires specific receptors on the cell surface and examples include FepA, IroN, and PfeA from *E. coli*, *S. enterica* and *P. aeruginosa,* respectively (Lundrigan and Kadner, 1986; Dean and Poole, 1993; Hantke et al., 2003). A single transport system can also internalize different siderophores. For example, the internalization of the siderophore aerobactin in *E. coli* is supported by the receptor IutA and the ABC transporter FhuBCD (De Lorenzo et al., 1986; Wooldridge et al., 1992). This transporter also mediates the uptake of ferrichrome, coprogen and rhodotorulic acid with the help of the specific receptors FhuA, FhuE, and Fiu (Fecker and Braun, 1983; Hantke, 1983). This illustrates the versatility of receptor-substrate recognition and also the piracy for iron acquisition that exists among competitive pathogens. The detailed processes of siderophore internalization are illustrated in **Figure 4** for the well-characterized mechanism of the fur-regulated catecholate siderophores system, enterobactin, and salmochelins. Iron-loaded catecholate siderophores are translocated upon recognition by the outer membrane receptor FepA (for enterobactin only) or IroN coupled to the energy transducing TonB-ExbD-ExbB complex (Pierce et al., 1983). After internalization, ferri-siderophore moves through the inner membrane. This passage requires proteins located in the periplasmic space and an inner membrane transporter. Cyclic molecules can be linearized in the periplasm by the esterase IroE (Lin et al., 2005; proteins are given in the text.

Zhu et al., 2005). The periplasmic protein FepB and the ABC transporter FepCEG translocate iron-loaded siderophores into the bacterial cytoplasm (Shea and McIntosh, 1991; Sprencel et al., 2000; Crouch et al., 2008). Once in the cytoplasm, the release of iron requires degradation of the molecule. The esterases Fes and IroD cleave iron-loaded enterobactin and salmochelins at ester bonds creating monomers, dimers, and trimers of DHBS and their glycosylated versions (Langman et al., 1972; Lin et al., 2005). These molecules can then be resecreted outside the bacteria, via their specific efflux pump EntS and IroC and reutilized as siderophores (Caza et al., 2011). This recycling characteristic of siderophore molecules is similar to the recycling of transferrin receptors and hemophores.

#### **SIDEROPHORE TRANSPORT IN GRAM-POSITIVE BACTERIA**

Siderophore transport in gram-positive bacteria is similar to the process in Gram-negative bacteria in that ABC transporters mediate translocation into the cytoplasm. The system in *S. aureus* provides a good illustration of the process. This bacterium produces for two siderophores, staphyloferrin A and staphyloferrin B, which are transported into the cytoplasm through the ABC transporters HtsABC and SirABC, respectively (**Figure 4**) (Meiwes et al., 1990; Beasley et al., 2009). HtsA and SirA are receptors exposed on cell surface while HtsBC and SirBC are components in the membrane responsible for the transport into the cell (Beasley et al., 2011). HtsBC also participates in the uptake of heme, suggesting a dual role for the HtsABC transporter (Skaar et al., 2004). The *sfa* and *sbn* loci encode the enzymes for staphyloferrin A and staphyloferrin B biosynthesis, respectively, and are regulated negatively by Fur and iron (Beasley et al., 2009, 2011). As discussed earlier, these siderophores are able to acquire iron from transferrin and lactoferrin with the help of catecholamine, although they are also able to mediate the uptake of ferric iron directly. In addition, *S. aureus* can utilize exogenous hydroxamate siderophores like aerobactin, ferrichrome, ferrioxamine B and coprogen through the Fhu (FhuCBG, FhuD1and FhuD2) uptake system (Sebulsky et al., 2000; Sebulsky and Heinrichs, 2001). FhuB and FhuG are membrane components and FhuC is the ATP-binding protein. FhuD1 and FhuD2 are lipoproteins thought to function as binding proteins for hydroxamate siderophores and staphylobactin (Sebulsky et al., 2003). Assays with a *fhuCBG* mutant revealed a significant contribution to virulence in a murine kidney abscess model (Speziali et al., 2006).

*Listeria monocytogenes* provides a useful additional example because this facultative intracellular pathogen uses several iron uptake systems. It can acquire iron from host proteins such as transferrin, lactoferrin, ferritin, and hemoglobin, but it does not secrete any siderophores. Rather it can use several hydroxamate (ferrichrome, ferrichrome A and ferrioxamine B) and catecholate (enterobactin and corynebactin) siderophores from other organisms and it can use additional iron-binding compounds, including catecholamines (Simon et al., 1995; Jin et al., 2006). As in *S. aureus,* the ABC transporter FhuCDBG system in *L. monocytogenes* is responsible for uptake of the hydroxamate siderophore ferrichrome and the HupDGC transporter mediates the uptake of iron from hemoglobin (Jin et al., 2006).

#### **SIDEROPHORE PRODUCTION AND TRANSPORT IN PATHOGENIC FUNGI**

As a group, fungi produce a number of structurally different siderophores and, as mentioned, some of the earliest studies of siderophores involved ferrichrome and ferrichrome A (Burnham and Neilands, 1961; Zalkin et al., 1964). The ferrichrome siderophore family illustrates the potential for complexity because it consists of 20 structurally different hexapeptides where modifications can occur on a common ferrichrome backbone molecule. These modifications include the addition, for example, of a hydroxymethyl group, a methyl group, or a lateral side chain. These alterations can generate derivatives such as ferricrocin, ferrichrysin, asperchrome D1 and B1, ferrirubin, ferrirhodin, ferrichrome A and other molecules (Winkelmann, 2007). At the other end of the spectrum, there are also fungi that do not produce any known siderophores (as with *L. monocytogenes* discussed above), but readily make use of xenosiderophores through the deployment of specific transporters. In general, fungi use transporters of the major facilitator protein superfamily, rather than ABC transporters, for siderophore internalization (Haas et al., 2003, 2008).

The importance of siderophores in fungal virulence in humans is nicely illustrated by detailed studies with the airborne pathogen *A. fumigatus* (and parallel comparative studies with the related saprotrophic species *Aspergillus nidulans*) (Eisendle et al., 2003; Schrettl et al., 2004, 2007; Haas, 2012)*. A. fumigatus* produces the siderophores fusarinine C (FsC)/triacetylfusarinine C (TAFC) and ferricrocin, and much is known about the regulation, biosynthesis, uptake and role in virulence for these molecules (Hissen et al., 2004; Schrettl et al., 2004, 2007; Kragl et al., 2007; Wallner et al., 2009; Haas, 2012). FsC and TAFC are excreted in response to iron deprivation and they function in extracellular iron binding with subsequent uptake by siderophore iron transporters (SITs) (**Figure 4**). *A. fumigatus* is predicted to encode 10 SITs (Haas, 2012) and two from *A. nidulans* have been functionally characterized: MirA was found to transport enterobactin and MirB was shown to take up TAFC in both *A. nidulans* and *A. fumigatus* (Haas et al., 2003; Raymond-Bouchard et al., 2012). After internalization, the intracellular release of iron from TAFC is achieved by hydrolysis of the siderophore backbone by the esterase EstB (Kragl et al., 2007). Interestingly, *A. fumigatus* possesses ferricrocin intracellular siderophores, and their production is coordinated with the morphology of the fungus. That is, ferricrocin (FC) is produced during filamentous hyphal growth, while hydroxyferricrocin (HFC) is produced within the conidial spores that are the infectious particles (Schrettl et al., 2007; Wallner et al., 2009). The intracellular siderophores are believed to function in iron storage (Schrettl et al., 2007; Wallner et al., 2009). Both intracellular and extracellular siderophores contribute to the virulence of *A. fumigatus* because the deletion of key genes for production results in avirulence in a murine model of invasive pulmonary aspergillosis (Schrettl et al., 2007). A recent study demonstrated that topical treatment with the human tear lipocalin (TL, also known as Lcn1) or lactoferrin reduced *A*. *fumigatus* growth in the cornea of mice, suggesting that therapeutic inhibition of fungal iron acquisition can be used to treat infections (Leal et al., 2013). TL is a secretory protein that interferes with microbial growth by scavenging microbial siderophores. In contrast to siderocalin, TL binds to a broader array of siderophores, including fungal siderophores such as coprogen, TAFC and rhodotorulic acid (Fluckinger et al., 2004).

The pathogenic yeasts *C. albicans* and *C. neoformans* don't produce siderophores but can scavenge xenosiderophores from other microbes. This iron parasitism depends on specific siderophore transporters in the plasma membrane. For example, the transporter Sit1 (also designated Sit1p/Arn1p) from *C. albicans* mediates the uptake of ferrichrome-type siderophores including ferricrocin, ferrichrysin, ferrirubin, coprogen and TAFC (Heymann et al., 2002). A mutant lacking Sit1 had a reduced ability to damage cells in a reconstituted human epithelium model of infection (Heymann et al., 2002). In *C. neoformans,* the transporter Sit1 is required for the uptake of ferrioxamine B, but does not make a contribution to virulence in a mouse model of cryptococcosis (Tangen et al., 2007).

## **UPTAKE OF FERROUS IRON**

Activities for the reduction of ferric iron and subsequent uptake of ferrous iron are present in bacteria and fungi. The ferrous form can exist in acidic environments and under anoxic conditions, and it can be generated by cell-associated or exported reductase activities. Ferrous iron ions are believed to diffuse freely through the outer membrane of Gram-negative bacteria, with subsequent transport through the inner membrane by the ABC transporter FeoABC. This system is conserved in many species, and it was first discovered in the non-pathogenic *E. coli* strain K-12 (Kammler et al., 1993). FeoB is the main transmembrane transporter that acts as a permease, while FeoC has been proposed to regulate FeoB. The role of FeoA is not well-understood, but it interacts with the highly conserved core region of FeoB (Lau et al., 2013). This system is under control of *fnr* and *fur* regulatory elements, where Fnr is an anaerobically-induced transcriptional activator and Fur inhibits transcription of *feo* genes in iron-replete conditions (Spiro and Guest, 1990; Kammler et al., 1993). The Feo system also contributes to intracellular replication for facultative intracellular pathogens like *Legionella pneumophila* (Robey and Cianciotto, 2002), *Shigella flexneri* (Runyen-Janecky et al., 2003) and *Francisella tularensis* (Thomas-Charles et al., 2013).

Other ferrous iron transport systems are present in various bacterial species. For example, the SitABCD system exists in *S. enterica* and *E. coli*, and a similar system has been described in *Y. pestis* (YfeABCD) (Bearden and Perry, 1999). The Yfe system is a typical ABC transporter for ferrous iron and manganese. YfeA is a periplasmic binding protein, YfeC and YfeD are the two inner membrane permeases, and YfeB is the ATPase required for energy transduction transport. No outer membrane protein has been identified and transport is independent of TonB (**Figure 5**) (Bearden et al., 1998; Perry et al., 2003; Fetherston et al., 2010). The Fur-regulated EfeUOB system is found in enterohaemorrhagic *E. coli* O157:H7 and *E. coli* Nissle 1917, and is also thought to transport ferrous iron (Grosse et al., 2006; Cao et al., 2007). This system promotes growth under aerobic, low-pH and lowiron conditions in response to ferrous iron (Cao et al., 2007). Interestingly, the EfeUOB system is analogous to the reductive, high-affinity iron uptake system of *S. cerevisiae* (Ftr1-Fet3-Fre1). Frt1 is an iron permease (with homology to EfeU), Fet3 is a multicopper oxidase (MCO) that will oxidize ferrous iron to its ferric state during the translocation and Fre1 is one of seven reductases that reduce ferric iron (Kosman, 2003). The EfeUOB system also exists in the Gram-positive bacterium *B. subtilis* where it permits acquisition of both ferrous and ferric iron species depending on extracellular conditions (Miethke et al., 2013). The ferrous iron peroxidase EfeB and the ferric iron binding protein EfeO act in succession during ferrous oxidation and ferric iron delivery to the membrane permease EfeU. However, EfeB is dispensable for direct ferric uptake via EfeUO, but instead promotes growth under microaerobic conditions where ferrous iron is more abundant. EfeUOB thus has a dual mechanism for acquisition of iron (**Figure 5**) (Miethke et al., 2013). More recently, the FtrABCD system for ferrous iron transport has been characterized in *Bordetella pertussis* and *Bordetella bronchiseptica,* and in *Brucella abortus* (Brickman and Armstrong, 2012; Elhassanny et al., 2013). Importantly, a *B. abortus* mutant lacking FtrA is attenuated in murine macrophages and mice thereby indicating the importance of ferrous iron in the mammalian host.

As mentioned above for the yeast *S. cerevisiae*, fungi have a high affinity system consisting of reductases, an iron permease and a MCO to generate ferrous iron for uptake, and this is the case for *A. fumigatus, C. albicans,* and *C. neoformans*. The components of this system and its contribution to iron acquisition in a vertebrate host were first characterized for *C. albicans.* Two *C. albicans* reductases, Cfl1 and Cfl95, were identified that promote reduction of ferric iron upon heterologous expression in a *S. cerevisiae* reductase-deficient strain. A large number of additional reductases are predicted from the *C. albicans* genome sequence (Hammacott et al., 2000; Knight et al., 2002). As in *S. cerevisiae*, reduced iron is transported into the cell by a complex consisting of an MCO and a permease. Five MCO candidates are predicted

**FIGURE 5 | Schemes for ferrous iron uptake.** The Gram-negative bacterium *Yersinia pestis* uses the YfeABCD proteins and the Gram-positive bacterium *Bacillus subtilis* uses the EfeUOB complex to accomplish ferrous iron uptake. A comparable process is shown for the fungal pathogen *Cryptococcus neoformans*. This pathogen used the Cfo1-Cft1 multicopper oxidase-iron permease complex, the cell wall pigment melanin, and the secreted reductant 3-hydroxyanthranilic acid to perform reduction and ferrous iron uptake. Note that ferrous iron is oxidized by Cfo1 prior to transport into the cell by Cft1. Physiological evidence for an additional low affinity transport system for ferrous iron has been presented for *C. neoformans* and this is indicated by a question mark (Jacobson et al., 1998). Additional details for each system are provided in the text.

for *C. albicans,* and *CaFET3*, *CaFET31* and *CaFET43*, can rescue the growth of a *fet3-* (MCO) mutant of *S. cerevisiae* in response to iron limited conditions (Ziegler et al., 2011; Cheng et al., 2013). Moreover, deletion of *CaFET33* and *CaFET34* decreased cellular iron content and iron acquisition during iron starvation, and *CaFET3* can compensate for the loss of *CaFET33* and *CaFET34* (Cheng et al., 2013). *C. albicans* has two iron permeases, *CaFTR1* and *CaFTR2*. The expression of *CaFRT1* is induced by iron starvation and this gene is required for iron acquisition from ferritin and transferrin (Ramanan and Wang, 2000; Almeida et al., 2009). A mutant that lacks the gene cannot cause damage to oral epithelial cells and, as mentioned earlier, is unable to cause systemic disease in a mouse model of candidiasis (Ramanan and Wang, 2000).

A screen of the *A. fumigatus* genome revealed 15 putative reductases, and FreB was shown to participate in adaptation to iron starvation and to function as a reductase (Blatzer et al., 2011). In this pathogen, the reduced ferrous iron is then reoxidized by the MCO FetC and imported by the iron permease FtrA; FetC and FtrA are 52% and 55% identical to the *C. albicans* Fet3 and Frt1 proteins, respectively (Schrettl et al., 2004). However, in *A. fumigatus,* the reductive iron uptake system does not play a role in virulence, and the siderophore system appears to be much more important for proliferation in the host (Schrettl et al., 2007; Haas, 2012).

The situation in *C. neoformans* is similar to that of *C. albicans.* Reductase activities have been characterized and the MCO Cfo1 as well as the iron permease Cft1 are required for reduction of iron from transferrin (Jung et al., 2009). Interestingly, in addition to enzymatic reductase activity, two other reduction systems exist at the cell surface for *C. neoformans*, the secreted reductant 3-hydrozyanthranilic acid (3HAA) and melanin which is responsible for a black cell wall pigmentation in presence of L-DOPA (**Figure 5**) (Nyhus et al., 1997). Cfo1 and Cft1 are both required for full virulence of *C. neoformans* in an inhalation murine model of cryptococcosis, however, mutants lacking these enzymes still cause disease (Jung et al., 2008, 2009). Therefore, additional iron acquisition functions are needed during disease. One of these functions includes the mannoprotein Cig1 that was recently shown to participate in heme uptake, as described earlier (Cadieux et al., 2013).

## **CONCLUSIONS AND PERSPECTIVES**

Pathogenic bacteria and fungi have evolved a number of mechanisms to acquire iron from different sources in the mammalian host. Although many of these mechanisms share functional similarities, it is clear that far more is known about bacterial systems. This is particularly evident for mechanisms that mediate iron acquisition from heme and heme-containing proteins. Considered as a group, key components have been identified in several bacterial pathogens and these include hemolysins, hemophores, receptors, ABC transporters for internalization and heme oxygenase activities. In contrast, the components that perform analogous functions in fungal pathogens are just now being identified and characterized. While there is some information about hemolysins and receptors, a candidate hemophore has only recently been described in a fungal pathogen and the details of uptake via endocytosis require considerable more investigation.

For other iron-containing host proteins, such as transferrin, lactoferrin and ferritin, there are clear differences between bacterial and fungal pathogens, although again the lack of information for fungi precludes detailed comparisons. It is clear that some bacteria, particularly *Neisseria* species, have sophisticated mechanisms for using transferrin, lactoferrin, and ferritin iron. In addition, there is a fascinating body of information on the participation of catecholamines in bacterial iron acquisition. This aspect of iron acquisition remains to be explored in fungal pathogens. For fungi, transferrin and lactoferrin tend to inhibit growth by an iron sequestration mechanism, although some fungi can overcome this limitation by reductive iron uptake or siderophore elaboration. Reductive iron uptake systems show similarities between bacteria and fungi in the uptake of ferrous iron, and this mechanism is clearly important for virulence in some but not all fungi. In addition, the recent discovery of a ferritin receptor in *C. albicans* has generated considerable interest in fungal exploitation of this iron source.

Siderophore-mediated acquisition of iron is one area where fungi as a group (i.e., not just pathogens) have provided significant structural and mechanistic information in parallel with studies in bacterial pathogens. In particular, the detailed studies in *A. fumigatus* (and the related species *A. nidulans*) on siderophore biosynthesis rival the sophisticated and advanced state of analysis for bacteria. However, the critical area of siderophore transport needs considerable attention for fungal pathogens. This is because little information is available for any of the species and because some of the most important species, *C. albicans* and *C. neoformans*, rely on transporters to steal siderophores. Considerable attention is now directed at siderophore-based drug development where siderophore transporters might be exploited as Trojan horse delivery systems. Therefore, an understanding of fungal siderophore transporters might facilitate the application of these drugs to fungal diseases. It is evident, however, that pathogenic bacteria and fungi generally possess more than one mechanism for exploiting the potential iron sources in vertebrate hosts. This is clear from virulence studies that often reveal only partial attenuation upon loss of a single uptake mechanism. Therefore, therapeutic approaches that target iron acquisition must inactivate the most critical of these mechanisms and/or exploit them for the delivery of antibacterial and antifungal drugs.

#### **ACKNOWLEDGMENTS**

Our research is supported by the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada and the National Institute of Allergy and Infectious Diseases (RO1 AI053721). James W. Kronstad is a Burroughs Wellcome Fund Scholar Award in Molecular Pathogenic Mycology. We apologize to those authors whose work could not be cited due to space limitations.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 11 August 2013; accepted: 30 October 2013; published online: 19 November 2013.*

*Citation: Caza M and Kronstad JW (2013) Shared and distinct mechanisms of iron acquisition by bacterial and fungal pathogens of humans. Front. Cell. Infect. Microbiol. 3:80. doi: 10.3389/fcimb.2013.00080*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2013 Caza and Kronstad. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## The roles of transition metals in the physiology and pathogenesis of *Streptococcus pneumoniae*

## *Erin S. Honsa†, Michael D. L. Johnson† and Jason W. Rosch\**

*Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, TN, USA*

#### *Edited by:*

*Mathieu F. Cellier, Institut National de la Recherche Scientifique, Canada*

#### *Reviewed by:*

*Zehava Eichenbaum, Georgia State University, USA James C. Paton, University of Adelaide, Australia*

#### *\*Correspondence:*

*Jason W. Rosch, Department of Infectious Diseasesm, St. Jude Children's Research Hospital, Mail Stop 320, 262 Danny Thomas Place, Memphis, TN 38105-3678, USA e-mail: jason.rosch@stjude.org*

*†These authors have contributed equally to this work.*

For bacterial pathogens whose sole environmental reservoir is the human host, the acquisition of essential nutrients, particularly transition metals, is a critical aspect of survival due to tight sequestration and limitation strategies deployed to curtail pathogen outgrowth. As such, these bacteria have developed diverse, specialized acquisition mechanisms to obtain these metals from the niches of the body in which they reside. To oppose the spread of infection, the human host has evolved multiple mechanisms to counter bacterial invasion, including sequestering essential metals away from bacteria and exposing bacteria to lethal concentrations of metals. Hence, to maintain homeostasis within the host, pathogens must be able to acquire necessary metals from host proteins and to export such metals when concentrations become detrimental. Furthermore, this acquisition and efflux equilibrium must occur in a tissue-specific manner because the concentration of metals varies greatly within the various microenvironments of the human body. In this review, we examine the functional roles of the metal import and export systems of the Gram-positive pathogen *Streptococcus pneumoniae* in both signaling and pathogenesis.

**Keywords:** *Streptococcus pneumoniae***, pathogenesis, metal transport, virulence factors, infection**

#### **METALS AND INFECTION**

*Streptococcus pneumoniae* can cause a variety of infections, including meningitis, otitis media, bacteremia, and pneumonia—the infection causing the most deaths worldwide from this pathogen (Wardlaw et al., 2006). During these various forms of bacterial infection, *S. pneumoniae* must acquire all necessary nutrients for survival and replication from within the host. Transition metals are an important subset of nutrients because they are required as cofactors and structural components of many proteins and play vital roles in metabolism and cellular defenses (Andreini et al., 2008). The bioavailability of metals in various host sites of pneumococcal colonization and infection vary significantly, which is reflected by the contribution to virulence of different pneumococcal metal import and export systems in these various host niches.

Although the efficient acquisition of metals is important, overaccumulation of intracellular metals can have deleterious effects on multiple cellular pathways, including antioxidant defense and central metabolic pathways. As such, bacteria have evolved highly efficient efflux mechanisms and precise regulatory systems to ensure appropriate modulation of intracellular metal accumulation. In addition to the concentration of a particular metal, the relative concentration of a particular metal in relation to that of other metals is a vital aspect of bacterial physiology because these metals can compete for intracellular binding sites within proteins (Dudev and Lim, 2008).

Baseline levels of metals vary greatly between various sites in the human body. As a reference, we have provided published concentrations of these metals in various body sites (McDevitt et al., 2011), as well as the pneumococcal transporters associated with these transition metals (**Figure 1**). In response to infection and inflammation, the bioavailability of metals can be rapidly altered, with the host sequestering nutrients from the bacterium to limit bacterial growth (Corbin et al., 2008; Weinberg, 2009; White et al., 2009). For example, the calprotectin protein chelates zinc and manganese during infection, rendering them unavailable to the pathogenic bacterium (Kehl-Fie and Skaar, 2010). In response to pneumococcal infection, the levels of metals in host tissues can vary dramatically. For example, zinc in the blood increases more than 10-fold during infection (McDevitt et al., 2011). Another example of the host modulating metal concentrations occurs within the phagolysosome of immune cells. Here, the innate immune cell actively pumps out necessary metals such as manganese and iron and pumps in toxic metals such as copper and zinc to eliminate the pathogen (Jabado et al., 2000; Forbes and Gros, 2003; White et al., 2009; Botella et al., 2011). This arms race for metals can determine whether or not an infection would be successful. In this review, we examine the role of the transition metals manganese, iron, copper, and zinc in *S. pneumoniae* physiology and pathogenesis.

#### **MANGANESE**

Although metals are toxic to bacteria, which require export systems for sustained viability, metal ion homeostasis must be maintained because metals are essential for bacterial viability and survival. For *S. pneumoniae*, one such metal that is critical for sustained colonization and invasive disease is manganese (Mn). Mn2<sup>+</sup> is found in various concentrations within the human host, depending on the body site, and is an essential cofactor for many pneumococcal proteins. In this section, we will discuss the specific

roles that this divalent metal plays in *S. pneumoniae* growth and virulence, as well as the antioxidant properties of Mn2+. **Table 1** lists all Mn2+-related proteins and genes of *S. pneumoniae* that will be discussed.

#### **MANGANESE IMPORTER: PsaBCA**

The pneumococcal Mn2+-importer is the PsaBCA ATP-binding cassette (ABC) transporter, which acquires and pumps Mn2<sup>+</sup> ions from the extracellular environment (i.e., mammalian host) into the cytosol. PsaBCA belongs to the Cluster A-I substrate binding protein transporters, which can transport Mn2+, Fe2+, and Zn2<sup>+</sup> (**Figure 1**). However, deletion of the transmembrane and ATP-binding components (PsaC and PsaB, respectively) results in an absolute requirement for Mn2<sup>+</sup> in the growth medium (Novak et al., 1998; Johnston et al., 2006; Berntsson et al., 2010), suggesting that Mn2<sup>+</sup> is the transporter's preferred substrate. Furthermore, cells lacking the PsaA component are deficient in growth in defined media lacking Mn2<sup>+</sup> (Dintilhac et al., 1997; Novak et al., 1998).

The PsaA component was the first member of the Cluster A-I family substrate binding transportation proteins to be crystallized, with structures showing a Zn2<sup>+</sup> ion within the metalcoordination site. However, free Zn2<sup>+</sup> is unable to downregulate PsaBCA expression, suggesting that Zn2<sup>+</sup> is not the natural ligand of PsaA (Kloosterman et al., 2008). Two published studies have shown that PsaA binds to Zn2<sup>+</sup> with a higher affinity than it does to Mn2<sup>+</sup> and that PsaA-Zn2<sup>+</sup> binding is responsible for an <sup>∼</sup>40% reduction in Mn2<sup>+</sup> accumulation in the cytosol (Jacobsen et al., 2011; McDevitt et al., 2011). Zn2<sup>+</sup> binding to PsaA, therefore, partially inhibits Mn2<sup>+</sup> binding and subsequent import. This phenomenon of inhibiting nutrient acquisition by the bacterium may partially explain why extracellular Zn2<sup>+</sup> is toxic to *S. pneumoniae*, as will be discussed later.

PsaA's mechanism of Mn2<sup>+</sup> import has recently been determined through a series of elegant structural and biochemical experiments (Counago et al., 2013). The metal-coordinating residues of PsaA were shown to interact with both Mn2<sup>+</sup> and Zn2+, similar to previously published observations, with the subsequent release and intracellular import of Mn2<sup>+</sup> found to be facilitated by the sub-optimal coordination of Mn2<sup>+</sup> in the metalbinding site. In contrast, optimal coordination of zinc resulted in the locking of the conformation of PsaA in a manner whereby the zinc ion was not released. This situation highlights the importance of ligand specificity and biological function, as many such import machineries must discriminate between metals of varying reactivity and abundance in the host.

#### **MANGANESE-DEPENDENT GENE REGULATION**

The *psaBCA* operon repressor PsaR negatively regulates the Psa import machinery. The recently elucidated crystal structure of the *S. pneumoniae* PsaR forms a stable homodimer and contains two metal-binding sites (Lisher et al., 2013). Site one coordinates Zn2<sup>+</sup> with a *KD* <sup>≥</sup> <sup>10</sup><sup>13</sup> <sup>M</sup>−1. Site two mediates metal selectivity and DNA activation of PsaR. Although Zn2<sup>+</sup> bound to this site with a higher affinity than did Mn2+, Mn2<sup>+</sup> was essential for the activation of DNA binding by PsaR. This requirement


**Table 1 | Summary of** *Streptococcus pneumoniae* **genes and their products involved in transition metal import/export, and metal-dependent proteins.**

**Table 1 | Continued**


*N/A, not available.*

for Mn2<sup>+</sup> is due to the higher allosteric coupling free energy released when Mn2<sup>+</sup> binds to site two than when Zn2<sup>+</sup> binds. Altogether, this study elucidated the first PsaR crystal structure and showed that PsaR requires Mn2<sup>+</sup> to be bound within site two for DNA binding activity, and most likely *psaBCA* gene repression.

Mn2<sup>+</sup> also plays an important role in the ability of the pneumococcus to regulate its response to stressors (Balogun et al., 2010). Global transcriptomic and proteomic analyses of WT and *psaA* pneumococci, grown under high- or low-Mn2+, revealed the overall effect of Mn2<sup>+</sup> on various biological processes of the pneumococcus. In the absence of supplemental Mn2+, the *psaBCA* gene expression was increased, consistent with the Mndependent binding of PsaR (repressor) to the promoter of the *psa* operon, when excess Mn2+ was present in the bacterial cytosol (Johnston et al., 2006). Additional genes were also controlled by the PsaR regulator, including the virulence factors *prtA* (*sp0641*) and *pcpA* (*sp2136*), which encode a cell wall-associated serine protease and CbpA, respectively (Hoskins et al., 2001).

Mn2<sup>+</sup> has also been implicated in the development of natural competence in the pneumococcus. In fact, the *psaA* strain absolutely requires Mn2<sup>+</sup> supplementation in the growth media for competence and transformation (Dintilhac et al., 1997). Transcriptome analysis results provided the first real evidence that PsaA was needed to induce competence. Because PsaA is also required for Mn2<sup>+</sup> import, this finding suggested that a certain level of Mn2<sup>+</sup> must be present in the cytosol to trigger competence. Indeed, the competence transcriptional regulator *comX1*, the competence operon, and the competence-stimulating peptide were all downregulated in strains lacking *psaA*.

#### **THE ROLE OF MANGANESE IN PROTECTION AGAINST OXIDATIVE STRESS**

When the expression of the ligand-binding lipoprotein *psaA* was knocked out, the resultant *psaA* pneumococcus was hypersensitive to oxidative stress and had lowered ability to neutralize the reactive oxygen species (ROS) superoxide anions (Tseng et al., 2002). For *S. pneumoniae*, ROS accumulation is a particularly volatile situation because this bacterium does not express a catalase, an enzyme essential for breaking down and neutralizing H2O2 (Hoskins et al., 2001). Furthermore, during the normal function of the pyruvate oxidase SpxB, which is required for the decarboxylation of pyruvate to acetyl phosphate and CO2, high levels of H2O2 are produced as a byproduct (Spellerberg et al., 1996). In fact, when SpxB is absent from a mutant strain of pneumococcus, the relative levels of cytosolic H2O2 drop 99%, suggesting that SpxB is the major H2O2-producing protein. Therefore, the pneumococcus and many other bacteria have evolved systems to break down ROS molecules to prevent oxidative stress. Because the *psaA* mutant is unable to acquire Mn2+, it is proposed that the loss of the antioxidant property of Mn2<sup>+</sup> is responsible of the accumulation of ROS, resulting in oxidative damage and cell death (Spellerberg et al., 1996). Although Mn2<sup>+</sup> is a cofactor for many proteins, the Mn-bicarbonate complex can also reduce ROS molecules directly, without the need for enzymes (Daly et al., 2004). However, this Mn-dependent ROS neutralization activity has not yet been found in the pneumococcus.

The superoxide anion (O− <sup>2</sup> ) is a major source of oxidative stress within the pneumococcus. Sod metalloenzymes neutralize superoxide anions by converting them to molecular water and H2O2, and are a major cellular defense against oxidative stress (McCord and Fridovich, 1969; Hassan, 1989). Although the production of H2O2 itself may be deleterious to the pneumococcus, previous results suggest that the Mn-dependent superoxide dismutase SodA is responsible for only 1% of pneumococcal H2O2 production. Furthermore, as we have discussed, SpxB was responsible for the remaining 99% (Spellerberg et al., 1996). Therefore, although the mechanism of H2O2 neutralization by the pneumococcus remains unknown, the role of SodA is highly critical for the survival and virulence of the pneumococcus when grown in a high oxidative stress environment.

Yesilkaya et al. reported the first detailed information on the pneumococcal Mn-dependent SodA, its role in protecting against oxidative stress, and its possible role in virulence (Yesilkaya et al., 2000). They showed that the pneumococcus produces two Sod proteins: a Mn-dependent Sod (SodA) and an iron-dependent Sod that is inhibited by H2O2. The pneumococcal genome clearly contained *sodA*, but no other discernible superoxide dismutase homologs have been identified to date. It should be noted that the presence of an iron-dependent SodA has not been addressed since the original study. Deleting *sodA* impairs pneumococcal growth under aerobic conditions, most likely due to the loss of the Mn-driven neutralization of O− <sup>2</sup> produced during growth in a high molecular oxygen environment (Yesilkaya et al., 2000). Furthermore, the mutation is lethal under anaerobic conditions. The authors suggest that this result may be due to the loss of superoxide scavenging function during anaerobic growth in this highly limiting growth medium and that the cells are highly susceptible to death by oxidative stress without the Mndependent SodA.

#### **MANGANESE EFFLUX**

As with all biological systems, pneumococci must achieve equilibrium between the amount of nutrient acquired and the amount of nutrient required. In the case of Mn2+, which serves a positive role in the growth and virulence of the pneumococcus, there may be negative side effects to its accumulation within the cytosol if levels exceed those needed. To help control this tight regulation, the pneumococcus encodes a Mn2+ efflux protein MntE, which selectively removes excess Mn2<sup>+</sup> from the cytosol to maintain metal homeostasis (**Figure 1**) (Rosch et al., 2009). MntE was originally predicted to be a CDF inorganic cation transporter, however, its substrate was unknown (Tettelin et al., 2001). Subsequent analysis of a *mntE* mutant showed its enhanced sensitivity to high levels of Mn2<sup>+</sup> in the growth medium, attributed to the inability of the mutant to export internal Mn2<sup>+</sup> ions (Rosch et al., 2009). Also, the levels of intracellular Mn2<sup>+</sup> in the mutant were significantly higher than those of the parental strain. These data indicate that MntE functions as a manganese efflux transporter in the pneumococcus.

#### **MANGANESE HOMEOSTASIS AND VIRULENCE**

Based on the importance of manganese to the pneumococcus, it was not unexpected that the *psaA* mutant was highly attenuated for multiple modes of pneumococcal infection, including otitis media, respiratory infection, and systemic invasion (Berry and Paton, 1996; Marra et al., 2002). One of the critical steps in pneumococcal pathogenesis is the initial adherence to and invasion of host cells, and a *psaA* strain of pneumococcus has decreased adherence to mammalian cells (Berry and Paton, 1996). Although it was originally hypothesized that PsaA was a surface-exposed adhesin, subsequent studies showed that PsaA plays a predominantly regulatory role in adherence, attributed to the important cellular role of the Mn-dependent repressor PsaR. Furthermore, perturbing manganese import affected the levels of the structural adhesin CbpA, an important virulence determinant for the pneumococcus, on the bacterial surface (Novak et al., 1998). It was, therefore, proposed that Mn-import by PsaBCA and the Mn-homeostasis role of PsaR are ultimately responsible for patterns of CbpA expression and pneumococcal adherence (Paton, 1998). Subsequent work showed that although PsaR is responsible for the negative expression of the Mn-transporter PsaBCA and several other surface proteins, PsaR inactivation does not alter pneumococcal adherence to cultured nasopharyngeal cells (Johnston et al., 2006). Also, though PsaR-mediated repression is essential for the pneumococcus to establish a systemic infection, Mn-dependent signaling through PsaR is not required for nasopharyngeal colonization (Johnston et al., 2006). This discrepancy may be due to the differences between the Mn levels of the lungs and those of the nasopharynx, which are shown in **Figure 1**. Regardless, these findings highlight the importance of Mn2<sup>+</sup> acquisition during pneumococcal infection.

The results of initial studies of intranasal models of infection suggested that SodA may also have a role in the initial colonization of the lung tissue and subsequent invasion into the bloodstream (Yesilkaya et al., 2000). To confirm this hypothesis, animals were intravenously infected with WT and *sodA* pneumococcal strains. No differences in median survival times were observed, suggesting that SodA is dispensable during a systemic pneumococcal infection (Yesilkaya et al., 2000). These findings may suggest that the microenvironment of the lungs, being rich in molecular oxygen, is the site of maximal SodA activity, protecting against the high oxidative stress levels. The delay in infection as the bacteria traverse the oxygen-rich alveoli into the more micro-aerophilic bloodstream suggests the ability of Mn-SodA to neutralize cytosolic oxidative stress is a determinant of pneumococcal pathogenesis and that is dispensable once the bacteria reach the bloodstream. This finding highlights the importance of understanding the niche-specific contribution of virulence genes to pathogenesis.

Mn2<sup>+</sup> accumulation in the cytosol increases *S. pneumoniae* resistance to oxidative stress (Rosch et al., 2009). This finding was expected because Mn2<sup>+</sup> is an antioxidant and is the cofactor for SodA, which eliminates ROS from the cytosol. The high accumulation of Mn2<sup>+</sup> in the *mntE* mutant can, however, be detrimental: increased internal concentrations of Mn2<sup>+</sup> dysregulate the transcriptional profile of the pneumococcus, altering multiple cellular pathways (Rosch et al., 2009). Although the results of *in vitro* studies demonstrated that the *mntE* pneumococcus is viable even under higher levels of oxidative stress, the results of *in vivo* pathogenicity studies indicate a fitness tradeoff for these benefits. The *mntE* mutant has a significantly reduced ability to colonize the nasopharynx and establish systemic infection (Rosch et al., 2009). Therefore, communication at a transcriptional level in response to internal and external Mn2<sup>+</sup> levels is of critical importance during disease progression.

#### **TARGETING MANGANESE HOMEOSTASIS FOR VACCINES**

Because of both its surface location and important roles in virulence, PsaA has attracted considerable interest as a potential vaccine antigen for the pneumococcus. The current polyvalent pneumococcal vaccine is based on the polysaccharide capsule, of which there are more than 90 serotypes (Robbins and Schneerson, 1983; Robbins et al., 1983). Since the vaccine only protects against 23 of the most common serotypes, non-vaccine-type pneumococci have emerged that are capable of causing pneumonia and invasive diseases. Therefore, one major goal in the pneumococcal field is to develop a protein-based vaccine that targets one or more surface-exposed pneumococcal proteins. Candidate proteins must be present in all serotypes and elicit a strong serotypeindependent immune response. Immunization of mice with PsaA conferred protection against invasive pneumococcal disease in a serotype-independent manner (Talkington et al., 1996). Inclusion of PsaA in multicomponent protein-based vaccines has also been efficacious for protection against both colonization and invasive disease (Briles et al., 2000; Ogunniyi et al., 2000). Hence, inclusion of such surface-exposed, metal-acquisition proteins may play an important role in the development of the next generation of pneumococcal vaccines.

## **IRON**

In the 1990s and early 2000s, substantial information was available about how Gram-negative pathogens acquired iron from their hosts (Crosa et al., 2004). Two main systems had been discovered: (1) small molecular siderophores that are synthesized and secreted by the bacteria to sequester free iron atoms or iron from host proteins such as transferrin; and (2) bacteria expressed surface proteins that bound hemin and imported the porphyrin into the cytosol for iron utilization. However, it remained unclear how Gram-positive organisms acquired iron through the thick cell wall and a single membrane.

#### **HOST SOURCES OF IRON USED BY PNEUMOCOCCI**

In 1993, Tai and others began to uncover how the pneumococcus acquires iron from its host during systemic bacteremia (Tai et al., 1993). Free iron levels in the blood are <sup>∼</sup>10−<sup>18</sup> <sup>M</sup>−<sup>1</sup> in the mammalian host, with most iron sequestered within the tetrapyrrole ring of heme, which is bound to hemoglobin (Hb) in erythrocytes (De Domenico et al., 2008; Heinemann et al., 2008). Initial analysis focused on determining whether pneumococcal culture supernatants contain siderophores as do those from multiple Gram-negative and Gram-positive pathogens, including *Escherichia coli*, *Yersinia pestis*, *Haemophilus influenzae*, *Bacillus anthracis*, and *Staphylococcus aureus* (Crosa et al., 2004; Honsa and Maresso, 2011; Haley and Skaar, 2012). However, no siderophores were present in the supernatants of pneumococcal cultures grown in low-iron media, suggesting that the pneumococcus does not produce iron-scavenging siderophores for iron acquisition (Tai et al., 1993).

The possibility existed that the pneumococcus could acquire iron via hemin sources or other iron-atom acquisition systems such as transferrin-iron capture, as seen in *Neisseria meningitidis* and similar pathogens (Gray-Owen and Schryvers, 1996; Pintor et al., 1998). This hypothesis was tested by using pneumococci grown in EDTA-treated media whereby growth was inhibited due to low iron levels. Supplementation with iron-loaded (holo) transferrin or lactoferrin did not restore growth, suggesting that while the pneumococcus is a mucosal pathogen, it does not use these two host iron sources (Tai et al., 1993). However, when cells were grown in hemin or Hb, bacterial growth recovered to WT levels, providing the first evidence that the pneumococcus may exploit host hemin stores for iron during growth in the bloodstream. Furthermore, this was the first evidence that any Gram-positive pathogen could use hemin as an iron source.

Tai et al. continued to study possible hemin-binding and acquisition functions of the pneumococcus (Tai et al., 1997). They used a non-encapsulated strain of pneumococcus to detect heme-binding activity by cell wall proteins. As increasing concentrations of hemin were incubated with live pneumococci, the absorbance at 405 nm, a well-defined method to detect hemin in a sample (Berry and Trumpower, 1987), increased by nearly a 1:1 ratio with ∼70% of hemin bound to the pneumococcal surface. Furthermore, pretreating cells at 75◦C did not inhibit hemin binding, suggesting that hemin binding does not require metabolically active cells. Although this heating may destroy proteins that could be responsible for hemin binding, treating pneumococci with proteinase K also did not inhibit hemin association with the bacterial cell surface. In an attempt to identify the polypeptides responsible for hemin binding, batch affinity chromatography was performed, with hemin-coated agarose beads used to capture potential heminbinding surface proteins (Tai et al., 1997). Several protein bands were seen, with the dominant species being 43 kDa, and adding free hemin to the cell lysate before column filtration eliminated the elution of the 43-kDa band. This 43-kDa protein was found in all pneumococcal serotypes tested and was predicted to be cell surface–exposed via fractionation results; hence, it was the most-promising candidate for a hemin-binding factor at the pneumococcal surface. Recently, researchers preliminarily identified two pneumococcal membrane proteins that bound hemin and Hb and were essential for viability (Romero-Espejel et al., 2013). As such these candidates show promise for the future understanding of how the pneumococcus acquires these complexes.

Elucidation of the details of iron transport can sometimes be confounded by the biochemistry of hemin and hemoglobin. One such difficulty can be non-specific binding of hemin to the bacterial cell surface, which can occur due to the hydrophobic nature of hemin. Another potential issue is that Hb can begin to dissociate during prolonged incubation: that is, heme will be oxidized to produce hemin and will dissociate from Hb subunits (Hargrove et al., 1997). Also, in some reported instances, hemin-binding proteins transiently bind porphyrin, and this event can be followed by a rapid dissociation of apo-hemin from the protein (Tsuruga and Shikama, 1997). As such, care must be taken to address these factors when investigating hemin and Hb utilization.

#### **THE IRON IMPORT MACHINERY OF PNEUMOCOCCUS**

The ability to scavenge free iron atoms and the protein complexes responsible for this activity had not been discovered in the pneumococcus until two possible iron-import machineries were identified (Brown et al., 2001a). In this study, it was hypothesized that possible virulence determinants, which include iron importers, could be found in pathogenicity-associated islands. A previously performed signature-tagged mutagenesis screen used insertion-duplication mutants that were analyzed for their ability to survive and replicate in murine models of pneumonia and bacteremia. One gene, *smtA*, was attenuated for virulence, and was annotated as a Fe3+/-dicitrate ABC transporter (**Figure 1**) (Lau et al., 2001). This gene was predicted to encode a transmembrane permease and was initially termed the *pit1BCDA* operon (pneumococcal iron transporter 1, now referred to as *piuBCDA*) (**Figure 1**) (Brown et al., 2001a). A second locus also discovered in this study was termed *pit2ABCD* (now referred to as *piaABCD*). Recently, PiaA was crystallized, revealing the ability of this membrane protein to bind ferrichrome, a hydroxamate siderophore (Cheng et al., 2013). While previous data determined that the pneumococcus does not produce siderophores, this new PiaA data may suggest that *S. pneumoniae* is capable of stealing holo-siderophores produced by other bacteria in the human host. This could possibly act as an iron source, and has been previously reported to be an iron-acquistion mechanism in other pathogenic bacteria (Hibbing et al., 2010).

For both the *piu* and *pia* loci, a single operon encoded a putative lipoprotein receptor, a putative ATPase, and two transmembrane permease proteins. All mutants had impaired growth abilities in cation-free media, with the double *pia/piu* mutant having the largest defect. Growth defects were reversed by the addition of FeCl3 and FeCl2 but not by that of ferritin or lactoferrin. Also, Hb partially restored the growth of the individual mutants but not that of the double mutant. This partial rescue of growth in the presence of Hb may be due to the ability of Hb or hemin to be imported through the Piu/Pia transporters. Of note is that during the growth experiments, hemin may also be released from Hb via spontaneous dissociation, and the oxidized iron in hemin could be dissociated under these conditions (Hargrove et al., 1997; Tsuruga and Shikama, 1997). Therefore, the Piu/Pia transporters could target this fresh heme-iron pool for import.

To corroborate the finding that one or both iron transporters have a role in iron import, the iron-dependent antibiotic streptonigrin was used to determine bacterial sensitivity (Brown et al., 2001a). This bacteriocidal antibiotic requires free iron in the cytosol of bacteria; therefore, bacteria are resistant to this drug when their iron import systems are disrupted. The individual mutants both showed ∼10-fold less sensitivity to streptonigrin than did the WT strain, suggesting that the mutants had less iron accumulate within the cytosol of the single/double mutants. The double mutant was even more resistant to the iron-dependent killing via streptonigrin. These data indirectly show that the loss of one or both iron import systems reduces iron import. This study also demonstrated direct iron uptake into the cytosol and indicated that significantly lower levels of iron accumulate within the cytosol (Brown et al., 2001a). These data clearly indicate the role of both the Piu and Pia systems in mediating iron uptake by the pneumococcus.

A third operon was discovered to contain genes encoding proteins similar to the already characterized iron import systems (Hoskins et al., 2001; Brown et al., 2002). The new putative iron import system was named *pitADBC* (**Figure 1**). The *pitA* gene encoded a lipoprotein iron receptor; *pitD* encoded an ATPase, and *pitB* and *pitC* encoded transmembrane permease proteins. At an amino acid level, PitA has more homology to *Streptococcus equi* and *Streptococcus pyogenes* PitA-homologs than to the pneumococcal PiuA and PiaA. The single *pitA*, *piuB*, and *piaA* mutants have WT-like growth characteristics in several growth media (Brown et al., 2002). However, growth is delayed in chelated media, and exogenous FeCl3 partially restores this growth defect. In contrast, adding *pitA* to the double *piu/pia* mutant further reduces growth in all media tested, and this growth was partially restored in cation-free media by adding FeCl3 (Brown et al., 2002). These data indicate that some degree of specificity exists among these partially redundant systems.

### **REGULATION OF IRON TRANSPORT**

Although iron import systems are needed for the pneumococcus to acquire iron, this acquisition process must be tightly regulated at a transcriptional level for optimal detection of and response to the environmental stimuli encountered during infection. Because the pneumococcus inhabits multiple body sites, it is extremely important to detect extracellular and intracellular nutrient levels. Two-component systems (TCSs) can regulate such events, and studies to determine the role of TCSs within the pneumococcus revealed the existence of 13 such systems and one orphan response regulator, RitR (Lange et al., 1999; Throup et al., 2000; Tettelin et al., 2001). The results of studies initiated by Throup et al. and expanded upon by Ulijasz et al. show that the RitR (repressor of iron transport) regulator is required during murine lung infection (Throup et al., 2000; Ulijasz et al., 2004). Additional data show that this regulator may repress iron acquisition genes in high-iron environments.

Global transcriptional profiling efforts to determine the genes under control of RitR identified 54 genes, with 17 repressed and 37 activated in the pneumococcal WT R800 strain (Ulijasz et al., 2004). Notable changes in gene expression for this review were the following: (i) *piuB* and *piuA* (but not *piuC* or *piuD*), which showed the highest degree of differential gene expression and were repressed in WT R800; (ii) increased expression of Dpr homologs, which are iron-storage peroxide-resistance proteins, and the iron-binding alcohol dehydrogenase AdhE, both of which are proposed to protect bacterial cells against H2O2 damage (Echave et al., 2003; Pulliainen et al., 2003); (iii) HemH, which is responsible for the final step in heme synthesis (Panek and O'Brian, 2002); and (iv) MutY, an iron-sulfur protein that is required for A/G DNA mismatch repair, which occurs during oxidative stress (Michaels et al., 1992; Grollman and Moriya, 1993; Samrakandi and Pasta, 2000). Therefore, it seems that RitR is important for the controlled expression or repression of genes involved in oxidative stress response and protection.

To further analyze the effect that RitR has on the iron-uptake system Piu, streptonigrin sensitivity was measured to indirectly detect iron uptake (Ulijasz et al., 2004). When grown in irondeplete media with no iron supplementation, the WT R800 and *ritR* strains grew to similar levels and were resistant to streptonigrin. However, when Fe2<sup>+</sup> or hemin was added to the media, only the *ritR* mutant had increased susceptibility to streptonigrin killing (Ulijasz et al., 2004). These findings suggest that when RitR is absent, iron uptake is dysregulated and the metal hyperaccumulates in the cytosol. However, adding Fe3<sup>+</sup> and Hb did not increase the susceptibility of either strain to streptonigrin, suggesting that the pneumococcus cannot use these iron sources under these conditions. These Hb results are in contrast to those previously discussed in this review (Tai et al., 1993). Additionally, the *ritR* strain is hyper-susceptible to H2O2 damage but not to the superoxide anion. The fact that RitR did not alter the expression of SodA is most likely responsible for maintenance of the resistance to oxidative stress caused by the superoxide anion. However, without a catalase, the pneumococcus would become increasingly susceptible to H2O2 damage, which would be further amplified by the higher intracellular Fe2<sup>+</sup> concentrations that are present when RitR is absent.

Because RitR is a regulator of gene transcription that may be affected by differing Mn and Fe levels, Ong *et al*. analyzed gene expression of WT and *ritR* strains grown in media with differing Fe:Mn ratios (Ong et al., 2013). The WT strain expressed two-fold more *ritR* in high-Fe media than in high-Mn media. This is consistent with reports that iron upregulates *ritR* (Ulijasz et al., 2004). The expression of *spxB*, which generates H2O2, was higher in the *ritR* mutant when grown in high-Fe media than when grown in high-Mn media. This finding supports the detection of increased H2O2 and oxidative stress in the mutant grown in high-Fe media (Ong et al., 2013). Also, because high Fe itself can produce oxidative stress, the researchers analyzed whether levels of NADPH, which can detoxify ROS, are changed in high-Fe environments. Additionally, *gnd*, which is involved in pneumococcal NADPH generation and is directly upstream of *ritR*, is expressed at a higher level in WT and *ritR* mutant cells grown in high-Fe media than it is in those grown in high-Mn media (Lanie et al., 2007; Ong et al., 2013). This expression pattern is further enhanced in the *ritR* mutant. Compared to the WT and *ritR-*complemented strains, the *ritR* mutant also expresses elevated levels of *zwf*, another gene essential for NADPH generation, when grown in high-Mn media. Additionally, the WT and complemented strains express 12-fold higher levels of the *psaA* gene in high-Fe media than in high-Mn media. However, the *ritR* mutant did not have increased *psaA* gene expression, suggesting that RitR may have a role in the expression of *psaA*.

Iron may also serve as a signal for a number of pneumococcal processes. Proteomic analysis of pneumococci cultured under iron-limiting conditions revealed altered expression of PsaA and numerous proteins involved in cellular stress responses and biofilm formation (Nanduri et al., 2008). The role of iron in biofilm formation has also been directly demonstrated, as supplementation of Fe3<sup>+</sup> enhanced biofilm formation, and iron chelation severely impaired formation of these structures (Trappetti et al., 2011). The central regulator in the case of iron-dependent biofilm formation appears to be LuxS, which also controls expression of the iron importer PiuA (Trappetti et al., 2011). LuxS has been implicated in numerous cellular processes and is central for coordinating the formation of biofilms both on inert surfaces and on human respiratory cells (Vidal et al., 2011, 2013). These findings underscore the importance of iron in the regulation of diverse cellular processes in the pneumococcus.

#### **ROLE OF IRON TRANSPORTERS IN PNEUMOCOCCAL PATHOGENESIS**

To determine the roles of Piu and Pia iron transporters in virulence, both pneumonia and a systemic infection were studied in murine models (Brown et al., 2001a). No differences were seen in survival between WT and individual-mutant strains. However, all mice infected with the double mutant survived the pneumonia infection. In contrast, the double mutant still caused 90% mortality during systemic infection, although the time-to-death was significantly delayed. Mixed infections were used to determine a competitive index between the WT strain and each mutant strain to better understand the roles of each iron import system in virulence. Here, the *pia* deletion was more attenuated in both the pneumonia and systemic infectious routes, suggesting that this iron importer is more important for pathogenesis. Also, the double mutant was extremely attenuated for virulence, with no bacteria recovered from the spleen 24 h after IP injection and few recovered from lungs after intranasal inoculation (Brown et al., 2001a). These data suggest that Piu and Pia transporters function independently yet synergistically and that, although each can compensate for the loss of the other, Pia may be more important for the viability of the pneumococcus during invasive infection.

These differences in virulence attenuation suggest that (1) the single *piu* or *pia* mutants are still virulent, most likely due to the function of the second import system; and (2) the iron requirements and availability in the lungs differ greatly from those in the bloodstream (**Figure 1**). It may be that the pneumococcus requires both iron transporters during the initial infection in the lungs for invasion and systemic infection to occur. However, the precise iron requirements and iron level availability during nasal colonization remain to be determined. Also, the possibility of a third iron-uptake transporter system, which would explain the low attenuation of virulence of the single mutants in this study, was highlighted in a 2002 paper from the same group (Brown et al., 2002).

In further support of the iron-uptake defect, the single mutations in all three iron importers caused similar sensitivity to streptonigrin, and each was more resistant than was the WT (Brown et al., 2002). Furthermore, the triple mutant was highly resistant to high doses of the drug, up to 20µg/mL. Also, 55FeCl3 uptake was severely impaired in the triple mutant compared to the *piu/pia* mutant strain. The small amount of iron import in the triple mutant may be from another unknown cation importer non-selectively pumping in iron. Since the iron import was additive, it was not surprising that the deletion of *pit* did not further decrease virulence in a pneumonia mouse model of infection but did slightly impair the mutant's ability to cause systemic infection. Also, adding the *pit* mutation to the double *piu/pia* strain did not increase the virulence attenuation in either form of infection. Finally, *piaA* was shown to be transcribed at a higher rate than any *pit/piu* components, consistent with the view that this is the dominant iron import system (Brown et al., 2002). Together, these data show the relative contributions and redundancies of the three iron import systems during pneumococcal infection.

The role of RitR in the pathogenesis of the D39 strain was tested via systemic intraperitoneal infection (Ong et al., 2013). Although the mice infected with *ritR* had slightly increased survival times, all mice eventually succumbed to infection. Furthermore, no differences were detected in the concentration of bacteria in the blood 24 h after infection with either the WT or mutant strain. This outcome was also seen in an intranasal challenge mimicking pneumonia. These data suggest that RitR is not needed for pneumococcal survival in systemic or pneumonia murine models of infection, contrasting those previously published (Ulijasz et al., 2004). The differences in the strains used (i.e., encapsulated D39 vs. non-encapsulated R800) as well as the infection model system may account for these contrasting virulence results.

As discussed, Ong et al. determined that RitR regulated iron import and manganese, which was needed for protection against oxidative stress that can occur when iron levels are high (Ong et al., 2013). To further understand the impact of RitR on virulence, the virulent encapsulated D39 strain and an isogenic *ritR* mutant were analyzed for their growth in media with differing Fe:Mn ratios. The data suggested that RitR expression is essential for the growth of pneumococci in high-Fe:low-Mn media and that Mn2<sup>+</sup> may provide a protective advantage when Fe2<sup>+</sup> levels are in excess (Ong et al., 2013). Therefore, the authors hypothesized that the Mn-dependent SodA may mediate growth restoration when Mn2<sup>+</sup> levels increase, protecting the bacteria against superoxides generated in the cytosol when the concentration of Fe2<sup>+</sup> is high. A *sodA/ritR* double mutant was constructed and analyzed in growth experiments. In high-Fe:low-Mn media, exogenous Mn2<sup>+</sup> still restored growth, suggesting that the Mnmediated protection is not attributed to the oxidative protection of SodA. Regardless, these data suggest that Mn2<sup>+</sup> can rescue the growth of the *ritR* mutant in high-Fe by reducing intracellular iron, and subsequently, oxidative stress.

#### **TARGETING IRON IMPORTERS FOR VACCINES**

The pneumococcal ABC iron transporter systems elicit a strong antibody response during systemic infection, and these responses can protect against a pneumococcal infection (Brown et al., 2001b; Jomaa et al., 2005). This strategy has been used against many pathogens: in fact, iron import systems have previously been the target of a vaccine for *Staphylococcus aureus* (Ebert et al., 2010; Kim et al., 2010; Joshi et al., 2012; Pancari et al., 2012; Zapotoczna et al., 2013). Therefore, studies established by Brown et al. and expanded upon by Jomaa et al. used two of the iron import systems, Piu and Pia, as possible vaccine candidates (Brown et al., 2001b; Jomaa et al., 2005, 2006). Because PiuA and PiaA proteins are found in all pneumococcal serotypes tested, each protein was used (individually and together) to immunize mice for antibody responses and protection from subsequent pneumococcal challenge (Brown et al., 2001b). Specific antibody responses were detected against both proteins, with both PiuA and PiuA antibodies having cross-reactivity for each other. Although all of the mice immunized with adjuvant-only succumbed to the infection, both PiuA- and PiaA- immunized mice displayed improved survival, demonstrating that immunization with the iron import proteins increases survival rates and confers significant protection against systemic infection. As expected, immunization with both antigens further reduced the mortality rate. Passive immunization with antisera against PiaA-PiuA also significantly delayed time-to-death, indicating that immunization is at least partially antibody-mediated (Jomaa et al., 2005, 2006). Together, these data demonstrate that the lipoprotein components of two iron import systems can be used as a protein-based vaccine to protect against systemic pneumococcal infection.

In 2006, Whalan et al. determined that the *piaA* gene was 100% conserved in all typical pneumococci tested, which included 27 different serotypes, but was not found in any oral streptococci, including the closely related *Streptococcus mitis* (Whalan et al., 2006). However, *piuA*, was found in only 20 pneumococci serotypes, with a very low level of nucleotide divergence (0.3%), and was also found in *S. mitis* and *S. oralis*, with a higher level of divergence (10%). Regardless, these data further support the idea that PiuA and PiaA could be viable protein-vaccine candidates.

Evidence emerged in 2005 that PiuA and PiaA elicit a strong immune response in humans: patients with laboratoryconfirmed pneumococcal septicemia had elevated levels of antibodies directed against these two antigens in the convalescent phase of the infection but not in the acute phase (Whalan et al., 2005). This finding suggests that humans are exposed to PiuA and PiaA during systemic infection and mount antibody responses against these antigens, as occurs in infected mice. It also suggests that these two iron import proteins have the potential to be included in a protein-based vaccine. In the patient sera tested, eight different pneumococcal serotypes had specific antibody responses against PiuA and PiaA, demonstrating serotype-independent cross-reactivity. Also, both lipoproteins were immunogenic in healthy 7-month-old infants, suggesting that immunogenicity can be quickly established after birth, most likely through normal pneumococcal colonization of the nasopharynx (Whalan et al., 2005). These experimental and clinical observations indicate that targeting iron import may prove to be an effective vaccine strategy against the pneumococcus.

### **COPPER**

Copper is toxic to a multitude of microorganisms, including bacteria, fungi, and viruses. Accordingly, operating rooms have begun using copper surfaces and surgical tools to reduce the risk of nosocomial infections (Salgado et al., 2013). To resist copper toxicity, bacteria have developed highly efficient copper export systems, many of which have important roles during infection as recently reviewed (Samanovic et al., 2012).

### **THE cupA COPPER EFFLUX SYSTEM**

*S. pneumoniae* has a highly conserved *cop* operon containing three genes: *copY*, the operon regulator; *cupA*, a copper transport protein with homology to a cupredoxin; and *copA*, the P-type ATPase copper exporter (**Figure 1**) (Shafeeq et al., 2011b). Several additional proteins have been identified as putative copper homeostasis proteins in *S. pneumoniae* (CutC, CtpC, and CtpE). Although these putative copper homeostasis proteins are upregulated during infection, they are not upregulated during copper stress, putting their precise function in *S. pneumoniae* into question (Shafeeq et al., 2011b). One possibility is that the relative abundance of other trace metals during infection is distinct to the *in vitro* media conditions used, resulting in increased expression. The precise function of these other proteins in copper homeostasis and pathogenesis remains unknown. It should also be noted that the mechanism of copper import in the pneumococcus has yet to be elucidated.

#### **REGULATORY CONTROL OF COPPER EFFLUX**

Transcriptional control of the *cop* operon in pneumococci is under the tight regulatory control of CopY, which represses the *cop* operon by binding the promoter region of the operon (Shafeeq et al., 2011b). Data obtained from other organisms show that once CopY binds copper, its affinity for DNA is greatly decreased, thus allowing transcription of the operon (Portmann et al., 2006). One atom of a metal inside the bacterial cell corresponds to ∼1.5 nM concentration, assuming a bacterial volume of <sup>∼</sup>10−<sup>15</sup> liters per cell. CopY's affinity for copper has been estimated to be in the zeptomolar range (10−21) in other bacterial species, equating to less than one molecule of copper per bacterium and supporting the case that free intracellular copper is extremely detrimental to bacterial (Changela et al., 2003). Additionally, in other bacterial species, CopY controls expression of lactate oxidase, which can function to scavenge molecular oxygen (Barre et al., 2007). LctO is also involved in hydrogen peroxide production in streptococci (Kietzman and Caparon, 2010). It is unknown whether the pneumococcal CopY controls *lctO* expression or plays a role with SpxB, which is encoded immediately downstream of the *cop* operon.

### **MOLECULAR MECHANISM OF COPPER EFFLUX**

Recent structural data indicate that copper export is mediated by CupA transporting copper from the protein's low-affinity site to its high-affinity site (Shafeeq et al., 2011b; Fu et al., 2013). This system provides an extremely elegant model for the efficient trafficking of intracellular copper for efficient efflux because both CupA and CopA are membrane-anchored proteins (Fu et al., 2013). It remains unclear whether CupA and CopY interact to transfer the copper and precisely how the membrane-bound CupA acquires free copper inside the bacteria. The results of homology studies suggest that CupA has cupredoxin activity, reducing soluble Cu2<sup>+</sup> to insoluble Cu1+, but direct evidence has not been shown (Fu et al., 2013). Pneumococcus can produce millimolar amounts of hydrogen peroxide, which reacts with Cu1<sup>+</sup> to form water and free radicals via Fenton chemistry (Pericone et al., 2003). In theory, free-radical production could be circumvented by CupA sequestering Cu1<sup>+</sup> after reduction and directly transferring it to CopA for export from the bacteria or by active uptake of glutathione, which converts hydrogen peroxide to water, thus relieving the stress of the free-radical formation by removing both precursors (Potter et al., 2012). The observation that glutathione uptake deficient pneumococci are more susceptible to copper stress supports this notion (Potter et al., 2012).

One of the major underlying questions about intracellular copper accumulation is the precise mechanism of toxicity. Recent data suggest that copper alone is not sufficient to cause oxidative damage in bacteria; therefore, it is not unreasonable that copper's toxic effects are mediated through another pathway (Macomber et al., 2007). Copper is very stable as represented in the Irving-Williams series, so it could displace metals in crucial bacterial enzymes, leading to their inactivity (Milicevic et al., 2011). Accordingly, copper negatively affects iron-sulfur clusters of dehydratases, inhibits branched chain amino acid biosynthesis, photosystem oxidase, and displaces manganese in SOD as additional mechanisms of toxicity (Batinic-Haberle et al., 1997; Macomber and Imlay, 2009; Azzouzi et al., 2013). Ribonucleotide reductase protein, NrdF, and coupled factor NrdE are both necessary for the aerobic nucleotide synthesis pathway in pneumococci and may interact with copper, as may other proteins involved in aerobic nucleotide synthesis, such as NrdI (Sun et al., 2011). Thus, it is also possible that copper is causing toxicity by inhibiting nucleotide synthesis or other essential metabolic processes.

### **COPPER AND PATHOGENESIS**

Proteins involved in copper homeostasis have been implicated at multiple stages of pneumococcal pathogenesis. The inability of the pneumococcus to functionally export copper is detrimental to the cell, with *copA* mutants being more susceptible to copper toxicity than are wild-type bacteria (Shafeeq et al., 2011b; Fu et al., 2013). Mice intranasally infected with *copA* mutants have an increased survival rate, likely due to the mutant's inability to grow in the host's nasopharynx (Shafeeq et al., 2011b). The results of signature-tagged mutagenesis screening and transposon sequencing implicate CopA involvement in pneumococcal fitness in both the nasopharynx and the lung (Hava and Camilli, 2002; van Opijnen and Camilli, 2012). Virulence data for the *copY* and *cupA* mutants are unavailable, however, transposon sequencing results indicate that no significant defects in either lung infection or nasopharyngeal colonization would occur when these genes are disrupted (van Opijnen and Camilli, 2012). This finding is in agreement with previous findings that *copY* mutants have wild-type tolerance of copper, *cupA* mutants have a similar tolerance to that of *copA* mutants depending on the media, and *cupA* and *copY* mutations have little or no effect on virulence, respectively (Shafeeq et al., 2011b; Fu et al., 2013).

The attenuation of the *copA* mutant during infection may be partially explained by the activity of the ATP7a copper transporter expressed in macrophages and other cells because this strain is unable to export copper. ATP7a has been implicated in the elimination of pathogens via importing excess copper into the phagolysosome to mediate more-effective bacterial killing (White et al., 2009). Deletion of ATP7a in murine models resulted in severe growth defects and premature death, making investigation into susceptibility to infection difficult (Wang et al., 2012b). Advances into the role of ATP7a will be facilitated by the recent cell line–specific conditional knockouts of this transporter in murine systems (Wang et al., 2012a,b). Such tools will allow for refined investigations into the role of this copper transporter during the progression of infection. Another major question that remains unanswered is why the pneumococcus uptakes copper at all. Copper-dependent enzymes have not been found, yet the amount of intracellular copper and the universal conservation of dedicated export machinery indicate that a copper acquisition system may exist. Future studies of the precise molecular interactions that facilitate efficient copper export and of the precise role of *cop* operons and other predicted copper homeostasis pathways will provide insight into the essential copper homeostasis pathways.

## **ZINC**

In contrast to copper's toxicity in *S. pneumoniae*, zinc has both beneficial and detrimental effects in bacteria, with several classes of bacterial proteins, such as metalloproteases and transcriptional regulators, relying on zinc for function although high concentrations of zinc are toxic (Guerra et al., 2011; McDevitt et al., 2011; Milicevic et al., 2011; Bek-Thomsen et al., 2012; Menon and Govindarajan, 2013). In a recent review, Shafeeq et al. comprehensively outline the literature on zinc's homeostasis, transport, and involvement in the pathogenesis of streptococci (Shafeeq et al., 2013). During infection, the phagolysosomes of macrophages either sequester zinc, reducing the bacteria's ability to use zinc for essential cellular processes, or overwhelm bacteria with zinc, leading to toxicity (Kehl-Fie and Skaar, 2010; Botella et al., 2011). Switching between these innate immune responses may be due to the perceived level of toxicity of the bacteria, however, further investigation would be needed to understand the exact mechanisms (Botella et al., 2011). To account for both situations inside the host, *S. pneumoniae* have zinc influx and efflux pumps.

#### **ZINC ACQUISITION**

*S. pneumoniae* encodes five proteins directly involved in zinc acquisition: zinc importers AdcA and AdcAII, permease AdcB, ATPase AdcC, and transcription regulator AdcR (**Figure 1**) (Dintilhac and Claverys, 1997; Dintilhac et al., 1997; Bayle et al., 2011). The transporters AdcA and AdcAII have overlapping specificity for zinc *in vitro*, and the combined *adcA*/*adcAII* mutant is deficient in zinc uptake. Furthermore, strains having deletions of both genes have deficiencies in growth in low-zinc environments and have severe colonization defects in intranasal and intraperitoneal infection models (Bayle et al., 2011). Deletion of the permease AdcB also confers deficiency in growth in lowzinc environments, with a slight defect in growth under normal conditions as well.

The Pht family of proteins (PhtA, PhtB, PhtD, and PhtE) may also be involved in zinc homeostasis. These proteins are upregulated in the presence of excess zinc but are under the control of AdcR (Ogunniyi et al., 2009; Shafeeq et al., 2011a). The single *phtD* mutation does not affect zinc uptake or growth, but the mutation of all four Pht proteins increases growth in the presence of excess zinc, leading to the suggestion that these proteins may be zinc scavengers or involved in zinc uptake. However, the results of direct binding assays involving these proteins and zinc have not been published (Bayle et al., 2011; Rioux et al., 2011).

### **ZINC EFFLUX**

In levels of zinc that are higher than equilibrium, *S. pneumoniae* mediates resistance to zinc toxicity by upregulating proteins such as AdhC, a glutathione-dependent alcohol dehydrogenase, and CzcD, a zinc export system protein in the cation diffusion facilitator family, both of which defend against the reactive nitrogen byproducts of zinc (Kidd et al., 2007; Kloosterman et al., 2007; Kimaro Mlacha et al., 2013; Shafeeq et al., 2013). Gene transcript levels of *czcD* and *adhC* are elevated in more-invasive strains of pneumococcus, such as TIGR4, and although initial reports showed that AdhC was essential for lung infection, it has since been shown that neither *czcD* nor *adhC* contribute to overall pathogenesis (Hava and Camilli, 2002; van Opijnen and Camilli, 2012; Kimaro Mlacha et al., 2013). However, just as it does with zinc import, *S. pneumoniae* may have overlapping functionality in zinc efflux, in which case single mutations may not yield discernible phenotypes under general conditions.

## **OTHER TRANSITION METALS**

Although little is known about the role of cobalt, nickel, and other transition metals not already mentioned in this review, it is likely that these metals can also enter the bacterium, so *S. pneumoniae* likely possesses the ability to export these metals. Several uncharacterized putative E1-E2 P-type ATPases exist in the *S. pneumoniae* genome that could facilitate import or export these additional transition metals. The *czc* locus has been shown to mediate cobalt and cadmium resistance in other organisms, however, there is no direct evidence of such activity in *S. pneumoniae* (Nies, 1992). Nevertheless, *czcD* transcript levels are upregulated in the presence of both cobalt and nickel, implying that CzcD could indeed be exporting these metals. Additionally, Sun *et al*. have described a group of proteins that likely bind cobalt and nickel, with the cobalt-binding proteins found in ribosomal structures and having RNA binding and structural activity and the nickel-binding proteins having ligase activity (Sun et al., 2013). However, the roles of these transporters in pneumococcal metabolism and virulence have not been determined. Cobalamin (vitamin B12) is a cobalt-binding compound that some intestinal bacteria use, however, no evidence exists that *S. pneumoniae* uses vitamin B12 (Giannella et al., 1971). It should also be noted that the pneumococci encode numerous additional ABC transporters and putative efflux systems, although the substrates of these systems and their roles during infection remain unknown.

### **CONCLUSIONS**

The environments encountered by the pneumococcus in the human body during both colonization and infection vary greatly in terms of the bioavailability of trace metals used as co-factors by both host and pathogen. As such, metal homeostasis is vital not only to fight bacterial infections but also to survival of the pathogen. Building upon our understanding of the metal import and export machinery of *S. pneumoniae* not only improves basic science knowledge of metal trafficking and usage in the bacteria but also offers additional therapeutic targets for inhibiting bacterial infections. Because many of the metal acquisition systems of the pneumococcus are both surface-exposed and important for invasive infection, they remain attractive vaccine candidates for protein-based vaccines. Another potential avenue for future therapeutics would be compounds that specifically target these essential bacterial uptake and efflux systems. The feasibility of this approach was recently demonstrated in *Staphylococcus aureus*, whereby using small molecules to activate the regulatory system controlling heme uptake perturbed the central metabolism and was toxic to the bacterium (Mike et al., 2013). Extension of such screens to other essential metal homeostasis systems may provide a new avenue for novel therapeutics to target bacterial pathogens.

#### **REFERENCES**


Yesilkaya, H., Kadioglu, A., Gingles, N., Alexander, J. E., Mitchell, T. J., and Andrew, P. W. (2000). Role of manganese-containing superoxide dismutase in oxidative stress and virulence of *Streptococcus pneumoniae*. *Infect. Immun.* 68, 2819–2826. doi: 10.1128/IAI.68.5. 2819-2826.2000

Zapotoczna, M., Jevnikar, Z., Miajlovic, H., Kos, J., and Foster, T. J. (2013). Iron-regulated surface determinant B (IsdB) promotes *Staphylococcus aureus* adherence to and internalization by non-phagocytic human cells. *Cell. Microbiol.* 15, 1026–1041. doi: 10.1111/cmi.12097

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 14 August 2013; accepted: 19 November 2013; published online: 04 December 2013.*

*Citation: Honsa ES, Johnson MDL and Rosch JW (2013) The roles of transition metals in the physiology and pathogenesis of Streptococcus pneumoniae. Front. Cell. Infect. Microbiol. 3:92. doi: 10.3389/fcimb.2013.00092*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2013 Honsa, Johnson and Rosch. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Role of transition metal exporters in virulence: the example of *Neisseria meningitidis*

## *Cyril Guilhen , Muhamed-Kheir Taha and Frédéric J. Veyrier\**

*Département Infection et Epidémiologie, Institut Pasteur, Unité des Infections Bactériennes Invasives, Paris, France*

#### *Edited by:*

*Mathieu F. Cellier, Institut National de la Recherche Scientifique, Canada*

#### *Reviewed by:*

*Caroline Genco, Boston University School of Medicine, USA Thomas Kehl-Fie, University of Illinois, USA*

#### *\*Correspondence:*

*Frédéric J. Veyrier, Département Infection et Epidémiologie, Institut Pasteur, Unité des Infections Bactériennes Invasives, 28 Rue du Dr. Roux, 75015 Paris, France e-mail: veyrier@pasteur.fr*

Transition metals such as iron, manganese, and zinc are essential micronutrients for bacteria. However, at high concentration, they can generate non-functional proteins or toxic compounds. Metal metabolism is therefore regulated to prevent shortage or overload, both of which can impair cell survival. In addition, equilibrium among these metals has to be tightly controlled to avoid molecular replacement in the active site of enzymes. Bacteria must actively maintain intracellular metal concentrations to meet physiological needs within the context of the local environment. When intracellular buffering capacity is reached, they rely primarily on membrane-localized exporters to maintain metal homeostasis. Recently, several groups have characterized new export systems and emphasized their importance in the virulence of several pathogens. This article discusses the role of export systems as general virulence determinants. Furthermore, it highlights the contribution of these exporters in pathogens emergence with emphasis on the human nasopharyngeal colonizer *Neisseria meningitidis*.

**Keywords: virulence factors,** *Neisseria meningitidis***, metals, exporter, efflux**

#### **INTRODUCTION**

For decades, it has been known that transition metals played a role in regulating host pathogen relationships (Weinberg, 1971; Finkelstein et al., 1983). Bacterial pathogens must acquire these metals in order to survive in the host during an infection. Metals such as Fe, Mn, Zn, Ni, Cu, Co, and Mo (Schaible and Kaufmann, 2005) have an incomplete "d" orbital which permits different states of oxidation, e.g., Fe2<sup>+</sup> and Fe3+. These metals often serve essential roles in protein structural stabilization or as enzymes cofactors (Barondeau and Getzoff, 2004). However, the unique chemistry of these metals can also provoke inappropriate redox reactions with O− <sup>2</sup> and H2O2, (Fenton's reaction), generating highly damaging hydroxyl radicals (OH and OH−) that can ultimately lead to the cell death (Stadtman, 1990). It is perhaps this duality that has driven the selection of sophisticated bacterial strategies to orchestrate transition metal homeostasis by sensing, acquiring, storing, or when necessary, exporting these essential but potentially lethal metals.

The stringent and complex requirements of bacterial pathogens for metals have been exploited by the immune system to limit bacterial growth. The majority of described examples demonstrate that the immune system uses starvation strategies that consist in decreasing metal availability (mainly Fe2+, Zn2+, and Mn2+) (Canonne-Hergaux et al., 1999; Corbin et al., 2008) to restrict bacterial growth. However, recent reports suggest the existence of an immune strategy whereby the bacteria are poisoned with an overload of metal, principally Zn2<sup>+</sup> and Cu+*/*2+(White et al., 2009). This latter finding also corroborates the fact that deletion of bacterial efflux pumps often impairs the virulence of pathogens (Stahler et al., 2006; Rosch et al., 2009; Botella et al., 2011; Li et al., 2011; Veyrier et al., 2011; Padilla-Benavides et al., 2013). It is therefore important to call attention to these efflux systems as virulence factors, as they have received less attention than metal importer systems. This article offers a brief perspective of the different families of metallo-exporters and a discussion of their general importance in the virulence of bacterial pathogens with emphasis on *Neisseria meningitidis*, an obligate human respiratory symbiont.

#### **METAL EFFLUX SYSTEMS**

The concentrations of metals can vary dramatically in the host organism during the course of a bacterial infection, and pathogens have developed a large panel of exporters to regulate their intracellular metal concentrations. Currently five main classes of bacterial exporters (**Figure 1**) have been described:

1. The Resistance-Nodulation-Cell division (RND) type transporters are integral membrane proteins mediating the efflux of a broad variety of substrates with a subset exporting metals. This subgroup is named heavy-metal efflux RND (HME-RND). This tripartite transporter utilizes the proton motive force to drive the efflux of the substrates (Nies, 1995; Goldberg et al., 1999). The RND pump (annotated A in **Figure 1**) is an integral membrane protein with a hydrophilic periplasmic component, connected to a trimeric outer membrane factor (C in **Figure 1**). This outer membrane channel allows the efflux of metal into the extracellular space (Paulsen et al., 1997). The third part of the RND transporter complex is composed of several units of a periplasmic membrane protein (B in **Figure 1**). It serves as an adaptor that forms a ring around the outer membrane proteins and the pump, thereby stabilizing contact between the two other components (Murakami et al., 2002; Akama et al., 2004a,b).

Several RND exporter systems have been identified to date, including CzcABC from *Cupriavidus metallidurans* which mediates the efflux of Co2+, Zn2+, and Cd2<sup>+</sup> with different affinities. The deletion of *czcC* (the outer membrane component) resulted in a decrease of Cd2<sup>+</sup> and Co2<sup>+</sup> efflux whereas the lack of *czcA* or *czcB*, the pump and fusion protein, respectively, induced a complete loss of efflux activity (Nies and Silver, 1989; Nies et al., 1989; Goldberg et al., 1999). Other characterized RND systems include CnrABC from *Alcaligenes eutrophus* which mediates the efflux of Co2<sup>+</sup> and Ni2<sup>+</sup> (Liesegang et al., 1993), CznABC from *Helicobacter pylori* which mediates resistance against high concentrations of Ni2+, Zn2+, and Cd2<sup>+</sup> (Stahler et al., 2006) and CusABC from *E. coli* which mediates the efflux of Cu+ and Ag+ (Long et al., 2012).


transport various metals in both prokaryotes and eukaryotes (Haney et al., 2005). The first description of a CDF protein was YiiP from *E. coli* and its function, the efflux of Cd2<sup>+</sup> and Zn2+, is coupled to H<sup>+</sup> antiport. In addition, Fe2<sup>+</sup> was also suggested to be exported through YiiP (Grass et al., 2005) but subsequent studies have shown that Fe2<sup>+</sup> transport is not as efficient as for Cd2<sup>+</sup> and Zn2<sup>+</sup> (Wei and Fu, 2005; Hoch et al., 2012). YiiP and other members of the family are usually composed of six transmembrane domains followed by a metallochaperone-like cytoplasmic domain that regulates metal transport activity (Lu et al., 2009). Crystal structures revealed an inward-facing homodimeric structure with four Zn2<sup>+</sup> binding sites per monomer, designated Z1–Z4 (Lu and Fu, 2007; Coudray et al., 2013). Several homologs with specificity for Zn2<sup>+</sup> have been described, including ZitB in *E. coli* (Chao and Fu, 2004), ZitA in *M. tuberculosis* (Nies, 2003), CzcD in *B. subtilis* (Guffanti et al., 2002) or *S. pneumoniae* (Kloosterman et al., 2007).

A subclass of CDF exporters, MntE, with a preference for Mn2<sup>+</sup> has also been described. In general Mn has been viewed as completely beneficial for the bacteria. For instance, it has been established that Mn plays a role in resistance to superoxide and hydrogen peroxide in several bacteria including *S. pneumoniae* (Yesilkaya et al., 2000; McAllister et al., 2004) *Bradyrhizobium japonicum* (Hohle and O'Brian, 2012) and *Neisseria gonorrhoeae* (Seib et al., 2006) among others. Accordingly Mn2<sup>+</sup> importers, e.g., MntH and MntABC, have received the majority of attention until recently, when MntEdependant Mn2<sup>+</sup> export was described in *S. pneumoniae* (Rosch et al., 2009).

4. Several recent studies have also shown that MntE is not the only type of Mn2<sup>+</sup> exporter and the existence of a new and distinct family corresponding to the fourth type of exporters, was revealed: the MntX (Transporter Mediating Manganese Export) family (**Figure 1**) (Li et al., 2011; Veyrier et al., 2011). Little is known about the MntX family transport mechanism but secondary structure and topological predictions suggest an inverted repeat of three transmembrane segments, i.e., DUF204 (Veyrier et al., 2011). Unlike the other families, MntX is found exclusively in the bacterial kingdom, indicating the family may have a relatively recent origin following the genetic fusion of two DUF204 domains (Veyrier et al., 2011). For the moment, homologs of MntX in *N. meningitidis* (Veyrier et al., 2011), *Xanthomonas* sp. (Li et al., 2011; Veyrier et al., 2011), and *E. coli* (Waters et al., 2011) have all been described to export principally Mn2<sup>+</sup> with some secondary affinity for other divalent metals.

5. The fifth exporter family is composed of a subclass of the 2- TM-GxN family (CorA, Cobalt Resistance protein A) that was first identified as Mg2<sup>+</sup> transporters (**Figure 1**) (Smith et al., 1993). However, some members are dedicated to the export of other divalent cations, principally Zn2+. ZntB of *Salmonella enterica* is involved in the transmembrane flux of Zn2<sup>+</sup> and Cd2<sup>+</sup> (Worlock and Smith, 2002). This protein, which may multimerize, harbors two transmembrane domains and a long cytoplasmic region that facilitates acquisition and subsequent delivery of cations to the transport channel.

#### **METAL EFFLUX AND VIRULENCE**

As stated before, metal chelation is used by the immune-system to restrict bacterial growth and, by definition, the use of exporters may not help the bacteria in this situation. From the above description, the majority of exporters from bacterial pathogens are dedicated to export of three transition metals (Zn, Cu, and Mn) that are found in substantial amount in the human body. Interestingly, Zn2<sup>+</sup> levels are increased during inflammation (20) and have long been recognized to regulate the immune system (13). In addition, Zn2<sup>+</sup> is also present in phagosomes containing bacteria (35). Thus, it is postulated that pathogenic bacteria uses Zn2<sup>+</sup> export systems to face the fluctuating levels of Zn2<sup>+</sup> during infection of the human body. The same may apply for copper, as it has been recently demonstrated that animals use copper as an anti-microbial weapon by inducing oxidative stress (26). Similarly, Cu+ is imported into the phagosome via the protein ATP7A (White et al., 2009). It is therefore not surprising that pathogens lacking Zn2<sup>+</sup> or Cu<sup>+</sup> exporters have impaired virulence. As an example, it has been established that a *copA* mutant strain showed decreased virulence in a mouse model of pneumococcal pneumonia and a decreased ability to survive in the mouse nasopharynx (NP), indicating that Cu+ homeostasis plays an important role in *S. pneumoniae* physiology and virulence (26). All together, these studies suggest that both metals are used by the immune system to intoxicate bacteria (Botella et al., 2012).

The role of Mn2<sup>+</sup> during infection is less understood as both beneficial and adverse effects have been reported. Nevertheless, all studies point to a general but important role of Mn2<sup>+</sup> export in bacterial pathogenesis. All the pathogens tested to date have a decrease in their virulence when deprived of their Mn2+ exporters. In *S. pneumonia*e, it was demonstrated that the lack of MntE, belonging to the CDF family, reduced virulence by diminishing both nasal colonization and blood invasion, resulting in decreased mouse mortality (Rosch et al., 2009). The same applies to *Xanthomonas oryzae* pv. *oryzae* in a plant model of infection or *N. meningitidis* in a mouse sepsis model of infection after inactivation of their Mn2<sup>+</sup> exporter from the MntX family (Li et al., 2011; Veyrier et al., 2011). These data could suggest the existence of a host immune strategy based on Mn2<sup>+</sup> poisoning. This hypothesis is somewhat discordant with the recent description of a host Mn2<sup>+</sup> and Zn2<sup>+</sup> chelator, calprotectin (Corbin et al., 2008). This host factor is capable of inhibiting bacterial growth in a Mn2+-dependent manner (Damo et al., 2013), thereby fulfilling an important role in the protection of the host against infection by bacterial pathogens such as *S. aureus* (Corbin et al., 2008; Damo et al., 2013). Furthermore, the poisoning hypothesis is also in disagreement with the fact that deletion of Mn2<sup>+</sup> importers decreases the virulence of several pathogens (Boyer et al., 2002; Anderson et al., 2009; Champion et al., 2011; Perry et al., 2012). For this reason, further study of this phenomenon will be important to understand the exact role that Mn2<sup>+</sup> plays during infection (poison, nutrient or both).

#### **METAL EXPORT: THE EXAMPLE OF** *N. meningitidis*

The NP defines the upper part of the pharynx from the end of nasal cavities (choanoe) to the upper surface of the soft palate. On the lateral parts it communicates with the Eustachian tubes by the pharyngeal ostium whereas the posterior part is composed of the pharyngeal tonsils (adenoids). This compartment is open and serves as a habitat for many microorganisms which are collectively called the NP microbiota (or flora). In this sense, the NP is the ecological niche for many bacterial pathogens such as *N. meningitidis*, *S. pneumoniae*, *Haemophilus influenzae*, and *Moraxella catarrhalis*. While carriage is usually asymptomatic, it can occasionally evolve into local infections of the upper-respiratory tract (pharyngitis, laryngitis, bronchitis, sinusitis, and otitis) or an invasive infection leading to life threatening diseases, such as invasive pneumonia, septicemia, and meningitis. Consequently, this leads to major morbidity and mortality as well as public health and economic burdens.

*N. meningitidis* is exclusively found in humans and frequently isolated from the upper respiratory tract of asymptomatic carriers (overall 10% of the general population). It is also the causative agent of life threatening invasive infections such as septicemia and meningitidis. The carriage of *N. meningitidis* is low in children (around 4–7%) where the principal neisserial colonizer (around 15%, Cartwright et al., 1987) is a closely related, non-pathogenic species, *Neisseria lactamica*. The prevalence of *N. meningitidis* increases after 10 years old with a peak at 19 years old (around 24%) and decreases throughout adulthood (13% in 30-year old to 8% in 50-year old) (Christensen et al., 2011).

The importance of Fe import systems for *N. meningitidis* virulence has been previously demonstrated following deletion of genes coding for several transporters (Genco et al., 1991; Genco and Desai, 1996; Larson et al., 2002; Renauld-Mongenie et al., 2004; Hagen and Cornelissen, 2006). A complementary approach has been used in which its virulence was enhanced by providing a compatible Fe source (Oftung et al., 1999; Zarantonelli et al., 2007). The role of exporters in the virulence of this nasopharyngeal pathogen is less-well established. The emergence of data concerning the role of exporters as virulence determinants, and the new concept of bacterial metallo-intoxication by the immune system, should encourage future research on this topic.

Our group has recently identified a novel Mn2+-exporter, MntX. We showed the *mntX* gene is expressed during sepsis in a mouse model and required for full virulence (Veyrier et al., 2011). We have further shown that MntX is required to maintain the Fe/Mn ratio thus avoiding molecular replacement between these two metals (Veyrier et al., 2011). Meningococci acquire Fe from host sources such as albumin, transferrin, or Fe-citrate which have also been shown to bind Mn. Therefore, a portion of these molecules is complexed with Mn in the host (concentration in the µM range) (Michalke et al., 2007). We speculate, based on the well-described needs of *N. meningitidis* for Fe (Genco et al., 1991; Genco and Desai, 1996; Larson et al., 2002; Renauld-Mongenie et al., 2004; Hagen and Cornelissen, 2006), that the intensive import of Fe could result in non-specific import of other divalent metals, which consequently must be exported. In this sense, we observed a specific Mn2+-export by MntX of *N. meningitidis* whilst the homologous exporter of *X. campestris* was also able to export Fe2+to some extent (Veyrier et al., 2011). In this case, metallo-exporters may be required to maintain the optimal ratio between different metals (e.g., Mn/Fe and Mn/Zn). Non-specific metal uptake should be also considered as a complementary hypothesis to Mn-intoxication by the host immune system. As another alternative, it has been reported that *N. meningitidis* harbors a specific Mn-dependant hemolysin called HrpA (Michalke et al., 2007). It is therefore possible that MntX (and other exporters) delivers Mn2<sup>+</sup> to *N. meningitidis*-specific extracellular virulence factors such as HrpA.

Although *N. gonorrhoeae* is closely related to *N. meningitidis* it generally resides asymptomatically in the female genitourinary tract. This ecosystem is more anaerobic than the NP and is also occupied by H2O2 producing lactobacilli. These features of the genitourinary tract are known to increase bacterial requirement for intracellular Mn2<sup>+</sup> and that may explained the high proportion of strains of *N. gonorrhoeae* harboring a premature stop codon mutation in the *mntX* gene (Veyrier et al., 2011).

### **METAL EFFLUX SYSTEMS AND THE EVOLUTION OF** *N. meningitidis*

The discovery of MntX highlights the importance of metal efflux system in the virulence of *N. meningitidis*. Importantly, the genome of *N. meningitidis* harbors other putative metal exporters. **Figure 2A** presents the genes with homologies to putative exporters detected in the genomes of *N. meningitidis* and two other major Gram-negative pathogens of the NP: *M. catarrhalis* and *H. influenzae*. Although the genomes have similar sizes, *N. meningitidis* seems to harbor more efflux systems. Only one gene was common to all three species, NMB1325, which shares a high similarity with HI0290 (81%) and with MCR\_1049 (68%). As Fe importers are often present in horizontally transferred pathogenicity islands, we wondered if this could be the case for additional metallo-exporters, and if some of them could have been specifically acquired by *N. meningitidis*. As this bacterium is

a human specific nasopharyngeal pathogen, without possibility of survival in the external environment, acquisition of such exporter by horizontal gene transfer (HGT) would support a role of these exporters in the emergence of *N. meningitidis*.

With the exception of NMB1732, all the putative exporters identified have homologs in other *Neisseria* species, and therefore cannot represent examples of recent HGT. NMB1732 is rarely present (if ever) in the genomes of other *Neisseria* species (such as *N. gonorrhoeae*) nor in other closely related non-neisseria strains (e.g., *Kingella*). In addition, all the isolates of *N. meningitidis* sequenced to date (∼200) harbor this gene, coding for a protein from the CDF family. As a consequence, this *N. meningitidis* specific gene is used by the Centre National de Reference des Meningocoques (CNRM) that is located within our laboratory, at the Institut Pasteur in Paris, to definitively distinguish *N. meningitidis* from all the other *Neisseria* species by PCR. Surprisingly, this gene and the surrounding region have an unusually high identity (99%) with a region of the genome of the non-closely related *M. catarrhalis* (**Figure 2B**) which shares the same ecosystem. Moreover, the gene is also present in other *Moraxella* species. Altogether, these findings suggest a possible transfer of the CDF exporter NMB1732 from *M. catarrhalis* to *N. meningitidis*. Our hypothesis is reinforced by the fact that, with the exception of NMB1732 and the adjacent pseudogene, the organization of the locus (including *tonB*) is conserved between *N. meningitidis* and other closely related *Neisseria* species (**Figure 2C**). The importance of MntX for *N. meningitidis* virulence and the acquisition by HGT and the conservation of NMB1732, a putative CDF exporter, highlight the pathogen's needs for metal exporters and the role that these exporters have played in the emergence of pathogens.

#### **CONCLUSION**

The existence of exporters and their transfer between species has been known for a long time in the context of bacterial living in the environment. In the last few years, bacterial metallo-exporters have also been demonstrated to play a role in the context of infection. The immune system uses Cu+*/*2<sup>+</sup> or Zn2<sup>+</sup> to poison bacteria and export systems are used to detoxify this overload. The role of Mn is not yet completely understood, but MntE and MntX are the proof of concept that Mn2<sup>+</sup> exporters are important for pathogenesis. Nevertheless, further research is required to understand the role of exporters in emergence and adaptation of pathogens and how these efflux systems can be used to thwart the host immune system defenses.

#### **REFERENCES**


Weinberg, E. D. (1971). Role of iron in host-parasite interactions. *J. Infect. Dis.* 124, 401–410. doi: 10.1093/infdis/124.4.401


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 16 October 2013; paper pending published: 08 November 2013; accepted: 05 December 2013; published online: 23 December 2013.*

*Citation: Guilhen C, Taha M-K and Veyrier FJ (2013) Role of transition metal exporters in virulence: the example of Neisseria meningitidis. Front. Cell. Infect. Microbiol. 3:102. doi: 10.3389/fcimb.2013.00102*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2013 Guilhen, Taha and Veyrier. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Common themes and unique proteins for the uptake and trafficking of nickel, a metal essential for the virulence of *Helicobacter pylori*

#### *Hilde de Reuse1 \*, Daniel Vinella1 and Christine Cavazza2*

*<sup>1</sup> Unité Pathogenèse de Helicobacter, Département de Microbiologie, Institut Pasteur, ERL CNRS 3526, Paris, France*

*<sup>2</sup> Metalloproteins Group, Institut de Biologie Structurale Jean-Pierre Ebel, UMR 5075, CEA, CNRS, Université Joseph Fourier-Grenoble 1, Grenoble, France*

#### *Edited by:*

*Frédéric J. Veyrier, Institut Pasteur, France*

#### *Reviewed by:*

*David Pignol, Commissariat à l'énergie Atomique, France Alberto Danielli, University of Bologna, Italy Wolfgang Fischer, Max von Pettenkofer-Institut der Ludwig-Maximilians-Universitaet, Germany*

#### *\*Correspondence:*

*Hilde de Reuse, Unité Pathogenèse de Helicobacter, Département de Microbiologie, Institut Pasteur, ERL CNRS 3526, 28 Rue du Docteur Roux, 75724 Paris, France e-mail: hdereuse@pasteur.fr*

Nickel is a virulence determinant for the human gastric pathogen *Helicobacter pylori*. Indeed, *H. pylori* possesses two nickel-enzymes that are essential for *in vivo* colonization, [NiFe] hydrogenase and urease, an abundant virulence factor that contains 24 nickel ions per active complex. Because of these two enzymes, survival of *H. pylori* relies on an important supply of nickel, implying a tight control of its distribution and storage. In this review, we will present the pathways of activation of the nickel enzymes as well as original mechanisms found in *H. pylori* for the uptake, trafficking and distribution of nickel between the two enzymes. These include (i) an outer-membrane nickel uptake system, the FrpB4 TonB-dependent transporter, (ii) overlapping protein complexes and interaction networks involved in nickel trafficking and distribution between urease and hydrogenase and, (iii) *Helicobacter* specific nickel-binding proteins that are involved in nickel storage and can play the role of metallo-chaperones. Finally, we will discuss the implication of the nickel trafficking partners in virulence and propose them as novel therapeutic targets for treatments against *H. pylori* infection.

**Keywords: nickel, urease maturation, hydrogenase,** *Helicobacter pylori***, metal trafficking**

## **INTRODUCTION**

For many organisms, acquisition of essential metal ions that are present at low concentrations in their environment is a critical process. Once acquired, the correct metal has to be delivered and incorporated into specific target enzymes through dedicated protein complexes comprising chaperones and so-called accessory proteins and when in excess the metals have to be stored as their non-physiological high intracellular concentrations are toxic.

The proteins involved in import, cellular storage, distribution, and incorporation of metal ions into enzymes are collectively referred to as "metal trafficking proteins". In bacteria, tremendous efforts have been devoted to decipher the mechanisms of iron uptake, trafficking and intracellular homeostasis control. Much less information is available for the acquisition and homeostasis of nickel, which is nevertheless an essential element for several bacteria. Nickel is the cofactor of at least nine enzymes involved in diverse cellular processes such as energy metabolism or virulence, including [NiFe]-hydrogenase, urease, Ni-SOD, COdehydrogenase (Higgins et al., 2012; Boer et al., 2013). Nickel is ubiquitously found in the environment and present at low concentrations in vertebrates. Although no enzymes or co-factors that include nickel were identified in higher organisms so far, animal experiments with nickel-deficient diets suggest a physiological role for nickel in these organisms (Denkhaus and Salnikow, 2002). It is tempting to speculate that nickel is required in higher organisms to sustain growth of the microbiote. Finally, different mechanisms have been proposed to explain cellular nickel toxicity, among which replacement by nickel of the metal of essential metalloproteins or indirect generation of oxidative stress (Macomber and Hausinger, 2011).

As for iron, specific and controlled nickel transport, efflux and trafficking processes are necessary as well as specific nickel storage proteins and nickel-responsive transcriptional regulators (such as NikR, for a review see Dosanjh et al., 2009). The gastric pathogen *Helicobacter pylori* presents interesting nickel management strategies, some being common to other bacteria and others being unique to this organism (**Figure 1**). *H. pylori* is therefore a fascinating model organism to study nickel uptake and trafficking as well as the link between virulence and these pathways. These aspects will be discussed in this mini-review.

## **NICKEL, A VIRULENCE DETERMINANT FOR** *Helicobacter pylori*

*H. pylori* is a gram-negative bacterial pathogen of major importance that colonizes the stomach of about half of the human population worldwide. Gastric infection by *H. pylori* causes gastritis and peptic ulcer disease. In addition, decades of persistent infection by *H. pylori* favors the development of gastric cancer (Wroblewski et al., 2010). *H. pylori*, is until now the sole bacterium recognized as a class I carcinogen and causes 800,000 deaths worldwide annually. It is the only bacterium that can multiply in the stomach, a hostile acid niche (median pH 2). Nickel can be regarded as a virulence determinant for *H. pylori* since it is the co-factor of urease, an enzyme essential for resistance to acidity. Urease is a major virulence factor essential for colonization of animal models. [NiFe] hydrogenase, the only other nickel-enzyme

found in *H. pylori* is important for murine colonization probably because it enables the use of H2 as an energy source. Urease represents 6–10 % of total *H. pylori* proteins. In addition, we measured a total intracellular nickel concentration of approximately 60 nM in *H. pylori*, corresponding to 50 times that of *Escherichia coli* (Schauer, 2007). Thus, a constant and important supply of nickel is required for the survival of *H. pylori* within the stomach, implying a tight control of its acquisition, distribution and storage. The properties of urease and hydrogenase will be briefly presented followed by the description of the nickel trafficking pathways.

## **UREASE AND HYDROGENASE UREASE**

Urease catalyzes the hydrolysis of urea to produce ammonia and carbamate, the latter being spontaneously degraded into a second molecule of ammonia and bicarbonate (Boer et al., 2013). These compounds act as buffers to maintain a neutral cytosolic pH in *H. pylori* (Stingl and De Reuse, 2005). The first crystal structure of a bacterial urease, from *Klebsiella aerogenes*, revealed a dinuclear nickel active site deeply buried in the alpha subunits (Jabri et al., 1995). The two nickel ions are bound to the protein *via* two histidines (His), an aspartate and a bridging carboxylate group of a carbamylated lysine. The *H. pylori* urease is composed of the UreA and UreB subunits. Its structure has also been solved (Ha et al., 2001) revealing a giant 1.1-MDa dodecameric complex (four trimers of UreA-B heterodimers) containing as much as 24 nickel ions. This enzyme has the highest affinity to its substrate ever described for a urease.

### **HYDROGENASE**

[NiFe]-hydrogenases catalyze the reversible heterolytic cleavage of dihydrogen. The active site, buried in the large subunit of a heterodimeric protein, contains a Fe(CO)(CN)2 unit and a nickel ion (Fontecilla-Camps et al., 2007). Protein coordination is mediated by two Fe-Ni bridging and two nickel-binding terminal Cys thiolates (Volbeda et al., 1995). The catalytic nickel ion has both a terminal coordination site and a bridging coordination site. *H. pylori* possesses a membrane-bound respiratory [NiFe] hydrogenase (Maier et al., 1996) that catalyzes the oxidation of dihydrogen. It is important for colonization of the mouse model possibly because dihydrogen, a fermentation by-product of the gut microorganisms, is used by *H. pylori* as an energy substrate (Olson and Maier, 2002; Maier, 2005).

## *IN VIVO* **UREASE ACTIVATION AND ACCESSORY PROTEIN COMPLEXES**

*In vivo* activation of urease requires dedicated accessory proteins that function through protein complexes to help the specific incorporation of nickel at the right site in a coordinated manner (Carter et al., 2009). In *H. pylori,* the *ureA*-*ureB* structural genes are followed by the accessory gene cluster *ureEFGH*. Using Tandem Affinity Purification, we isolated from *H. pylori* cells a complex comprising UreA-B and the complete activation complex UreH-UreF-UreG-UreE (Stingl et al., 2008). A computational model of this complex was recently proposed (Biagi et al., 2013).

UreE is a nickel metallochaperone that interacts with UreG (Bellucci et al., 2009). UreG is an intrinsically unstructured GTPase that dimerizes upon binding of a metal ion (Zambelli et al., 2005, 2009; Musiani et al., 2013). It has been suggested that binding of the UreF-UreH complex induces conformational changes in urease, allowing nickel ion and carbon dioxide to access the active site. UreF was shown to gate the GTPase activity of UreG to enhance the fidelity of urease metallocenter assembly (Boer and Hausinger, 2012). A recent crystal structure of the *H. pylori* UreG-UreF-UreH complex reveals how UreF and UreH facilitate UreG dimerization and how it assembles its metal binding site (Fong et al., 2013). The addition of nickel and GTP to the UreG-UreF-UreH complex causes release of the UreG dimer that binds nickel at the dimeric interface. *In vitro*, nickel-charged UreG dimer was shown to activate urease in the presence of UreF/UreH. How nickel is transferred from UreE to the binding site of UreG and how the complete activation complex interacts with urease has still to be determined.

#### *IN VIVO* **HYDROGENASE ACTIVATION AND INTERCONNECTIVITY BETWEEN THE UREASE AND HYDROGENASE MATURATION PATHWAYS IN** *H. pylori*

[NiFe] hydrogenase activation by nickel incorporation also requires dedicated accessory proteins that are conserved in *H. pylori* (Leach and Zamble, 2007; Maier et al., 2007). HypCDEF are necessary for the synthesis and correct insertion of the Fe(CO)(CN)2 moiety in apo-hydrogenase. Once the iron center gets assembled and inserted, nickel is delivered to the enzyme through the concerted action of the metallo-chaperone HypA and the GTPase HypB that were characterized in *H. pylori* (Xia et al., 2012). In *E. coli*, the nickel-binding protein SlyD is an essential additional partner of hydrogenase maturation, presumably involved in stimulating the release of nickel from HypB (Leach et al., 2007).

A unique particularity of nickel trafficking in *H. pylori* is the interconnectivity between the urease and hydrogenase maturation pathways. Indeed, *H. pylori* mutant characterization revealed that HypA and HypB are required not only for hydrogenase maturation but also for full urease activation (Olson et al., 2001). In agreement with this, our *H. pylori* interactome analysis evidenced that HypB is physically associated to the urease maturation complex (Stingl et al., 2008). This interactome also contained SlyD (Stingl et al., 2008) shown to interact with HypB *in vitro* and *in vivo* in *E. coli* (Cheng et al., 2013). However, the role of SlyD in *H. pylori* cells and in urease/hydrogenase activities remains unclear. Benanti and Chivers (2009) showed that in *H. pylori* strain 26695, urease activity was strongly diminished in a double *slyD*/*hypA* mutant as compared to a single *hypA* mutant, but this effect was not observed in strain G27. While SlyD may serve as a nickel reservoir to activate urease, the properties of nickel storage proteins might differ depending on the *H. pylori* genetic context. To be mentioned, in the closely related organism *Campylobacter jejuni*, hydrogenase activity is not modified in a *slyD* mutant (Howlett et al., 2012).

Using optical-biosensing methods, it was found that the *H. pylori* HypA and UreG proteins compete with each other for UreE recognition, suggesting that the function of HypA in urease activation relies on nickel delivery or exchange rather than on catalytic activities (Benoit et al., 2012). Indeed, purified recombinant HypA is sufficient for the recovery of urease activity in *H. pylori* cell lysates from a *hypA* deletion mutant (Herbst et al., 2010). This can be related to the observation that unlike many of its orthologs, the *H. pylori* UreE dimer only binds one equivalent of Ni2<sup>+</sup> (Bellucci et al., 2009).

While the accessory proteins for both urease and hydrogenase maturation are conserved in *H. pylori*, several particularities of nickel trafficking in this organism have emerged. At least three nickel-binding proteins have important functions in nickel trafficking in *H. pylori* and a novel nickel uptake system was discovered. We believe that *H. pylori* acquired these additional partners to deal with its exceptionally high intracellular nickel concentration, the fluctuating nickel availability and pH in the stomach and with a critical need to coordinate nickel distribution between two essential enzymes.

#### **NICKEL TRANSPORT AND EFFLUX**

In Gram-negative bacteria, energized transport of metabolites such as iron-siderophore complexes through the outer membrane (OM) relies on the TonB machinery and on TonB-dependenttransporters (TBDTs). The first nickel transport system across a bacterial OM was described in *H. pylori* by our group (Schauer et al., 2007). Its expression is repressed by NikR in the presence of nickel. This transport requires the FrpB4 TBDT and is acidinduced, allowing *H. pylori* to optimize urease activity by nickel incorporation under conditions where urease activity needs to be maximal. Additional mechanisms certainly exist allowing, for example, nickel entry at neutral pH. FecA3, another *H. pylori* TBDT whose synthesis is under nickel control (Romagnoli et al., 2011), is probably an alternative nickel-uptake system (Ernst et al., 2006). It is not clear under which form nickel is recognized by the TBDT. By analogy with siderophores, it is possible that a nickelophore, *i.e*., a small organic chelator of nickel, is required for its transport. A nickelophore was shown to be required for nickel binding into the *E. coli* NikA protein: it can be either a small organic ligand (Cherrier et al., 2008) or a (L-His)2 (Chivers et al., 2012; Lebrette et al., 2013).

In *H. pylori*, nickel transport across the cytoplasmic membrane can be mediated by NixA, a high-affinity and low-capacity nickel transporter (Fulkerson and Mobley, 2000) of the NiCoT family, which expression is repressed by nickel (Wolfram et al., 2006). As a NixA mutant retains half of urease activity and is still able to colonize the mouse model (Nolan et al., 2002), alternative ways of nickel entry must exist. Other *Helicobacter* species present different combinations of similar nickel transport systems. In *Helicobacter mustelae*, nickel uptake and urease activation depend on NikH, a TBDT different from FrpB4 (Stoof et al., 2010b) and on the FecDE/CeuE ABC transport system (Stoof et al., 2010a). *Helicobacter hepaticus* possesses genes regulated by NikR that are homologous to those encoding the *E. coli* nickel ABC transporter and to *nikH* of *H. mustelae* (Benoit et al., 2013b). The *fecDE/ceuE* genes are conserved in *H. pylori* and might be an additional nickel transport system across the inner membrane.

Again, the possible variety in nickel uptake mechanisms underscores the utmost importance of nickel uptake in *H. pylori* and closely related *Helicobacter* species.

Having crossed the inner membrane, nickel has to be directed to its proper targets while avoiding potential damages caused by free metal ions. If nickel is in excess with respect to *H. pylori* cellular needs, it must either be stored or exported from the cell. Only one nickel export system has been described in *H. pylori*, a proton-driven RND-type metal efflux-pump encoded by the *cznABC* genes. Inactivation of this pump increases *H. pylori* sensitivity to nickel, cadmium and zinc and impairs colonization of the gerbil stomach (Stahler et al., 2006), underlining the importance of metal homeostasis for *H. pylori* virulence.

#### **UNUSUAL NICKEL CHAPERONES AND STORAGE PROTEINS IN** *H. pylori*

In *H. pylori*, HspA is the sole member of the highly conserved and essential GroES co-chaperonine family (Suerbaum et al., 1994). The *hspA* gene transcription is activated by NikR with nickel (Muller et al., 2011). HspA protein is particular by the fact that it contains a His- and Cys-rich C-terminal extension that was shown to bind nickel ions *in vitro* (Kansau et al., 1996). Deletion of this extension is viable and impairs the maturation of hydrogenase but not that of urease (Schauer et al., 2010). We concluded that HspA could constitute a nickel storage pool specifically used for hydrogenase maturation. How nickel is mobilized from HspA and whether HspA provides nickel to other proteins remain to be determined. A more general role of HspA in nickel storage/detoxification is suggested by its abundance and by the fact that deletion of its C-terminal extension decreases the intracellular nickel content and increases nickel sensitivity (Schauer et al., 2010).

*H. pylori* also possesses two proteins of remarkable amino-acid composition that are conserved in *H. pylori* and have no orthologs outside the *Helicobacter* species. Hpn and Hpn-2 (or Hpn-like) are two small proteins (7 and 8 kDa) that are extremely rich in His-residues: 47 and 25 % of the total residues, respectively. Hpn-2 contains additional poly-Glutamine stretches representing 40% of the total residues. These two proteins were shown *in vitro* to bind nickel and to form multimers (Gilbert et al., 1995; Ge et al., 2006; Rowinska-Zyrek et al., 2011; Zeng et al., 2008, 2011). Like *hspA*, transcription of these genes is upregulated by NikR with nickel (Muller et al., 2011). Because *hpn* and *hpn-2* mutants were found to be more sensitive to high exogenous nickel concentrations than a wild type strain, these abundant proteins were suggested to be involved in nickel storage and detoxification *via* sequestration of excess nickel (Mobley et al., 1999; Seshadri et al., 2007). Seshadri et al. (2007) reported that both proteins compete with the nickel-dependent urease maturation machinery under low nickel conditions. However, previous data showed wild-type urease activity in a *hpn* deletion mutant (Gilbert et al., 1995). While Hpn and Hpn-2 proteins are certainly central in the nickel trafficking pathways of *H. pylori* and are possibly directly or indirectly involved in urease activation, their respective roles in these processes remain to be established. Recently, purified Hpn was shown to form *in vitro* amyloïd-like fibrils that are toxic when applied to cultured gastric epithelial cells (Ge et al., 2011). The existence and function of these fibers and the effect of nickel on their formation has yet to be demonstrated *in vivo*.

#### **UREASE, NICKEL, AND VIRULENCE**

The two nickel metalloenzymes (urease and [NiFe] hydrogenase) are determinant in *H. pylori* colonization capacity. In addition to its role in acid resistance, urease fuels *H. pylori* with ammonium for nitrogen assimilation (Williams et al., 1996). The produced ammonia is cytotoxic either alone or in conjunction with neutrophil metabolites (Sommi et al., 1996). Urease activity is also important for survival into macrophages, evasion from phagocytosis, and complement-mediated opsonisation. Purified urease protein stimulates activation of macrophages, dysregulates tightjunctions and induces cytokine production from gastric epithelial cells. These effects might be related to the large amounts of urease-bound nickel delivered to the host cells, considering the 24 nickel ions per active urease complex.

*H. pylori* displays a chemotactic repulsive response to nickel that might help its orientation during stomach colonization (Sanders et al., 2013). In addition, using a mouse model fed with Ni-deficient chow, a weak colonization defect was observed with the double *hpn-hpn-2* mutant (Benoit et al., 2013a). This suggests a role of Hpn and/or Hpn-2 in nickel incorporation during host colonization. Incubation of gastric epithelial cells with purified recombinant *H. pylori* SlyD protein disturbs cell proliferation, apoptosis, and enhances cell transformation and invasion (Kang et al., 2013). It was thus proposed that SlyD contributes to the gastric pathogenicity of *H. pylori*. Further studies with *slyD*-deficient strains are needed to establish the role of SlyD *in vivo*. Finally, purified recombinant HspA protein induces the expression of pro-inflammatory cytokines in human cells (Lin et al., 2006). Here again, nickel delivery to hosts cells mediated by nickel-binding proteins might cause these effects.

## **CONCLUSION**

*H. pylori* possesses original properties for nickel uptake and trafficking that are directly related to its virulence and reflect the absolute need for this organisms to manage high nickel concentrations. Some of the factors that were discovered in *H. pylori* seem to be present in other organisms (nickel TBDTs in other *Helicobacter* species) and such transporters are predicted in other species (Schauer et al., 2008).

Several fascinating questions remain to be answered. Are the functions of Hpn and Hpn-2 proteins restricted to nickel storage or do they constitute urease-dedicated *H. pylori* chaperones? How large is the variety of nickel transporters? Are there nickelophores needed for nickel uptake and, if so, is this nickelophore synthesized by *H. pylori* or acquired from the environment? Given the properties of *H. pylori* and the absence of nickel-enzymes in the human body, it is tempting to propose the nickel trafficking pathways of *H. pylori* as targets for the development of alternative antibacterial drugs.

## **ACKNOWLEDGMENTS**

We would like to thank Frédéric Fischer from the Unité de Pathogenèse de *Helicobacter* for his careful and constructive comments and corrections on this mini-review and Julien Gallaud for the figure. We thank Janssen for financial support.

## **REFERENCES**


chemotactic response to zinc and nickel. *Microbiology* 159, 46–57. doi: 10.1099/mic.0.062877-0


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 16 October 2013; paper pending published: 01 November 2013; accepted: 21 November 2013; published online: 09 December 2013.*

*Citation: de Reuse H, Vinella D and Cavazza C (2013) Common themes and unique proteins for the uptake and trafficking of nickel, a metal essential for the virulence of Helicobacter pylori. Front. Cell. Infect. Microbiol. 3:94. doi: 10.3389/fcimb.2013.00094 This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2013 De Reuse, Vinella and Cavazza. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Fungal zinc metabolism and its connections to virulence

## *Charley C. Staats 1,2\*, Lívia Kmetzsch1, Augusto Schrank1,2 and Marilene H. Vainstein1,2*

*<sup>1</sup> Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil*

*<sup>2</sup> Departamento de Biologia Molecular e Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil*

#### *Edited by:*

*Frédéric J. Veyrier, Institut Pasteur, France*

#### *Reviewed by:*

*Dario S. Zamboni, Universidade de São Paulo, Brazil Aaron Mitchell, Carnegie Mellon University, USA*

#### *\*Correspondence:*

*Charley C. Staats, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, 43421, Caixa Postal 15005, Porto Alegre RS 91501-970, Brazil e-mail: staats@cbiot.ufrgs.br*

**INTRODUCTION**

Zinc is fundamental for all domains of life, as it composes the catalytic and structural center of a large array of proteins. Thus, the "zinc quota," defined as the cellular zinc content required for optimal growth (Outten and O'halloran, 2001), must be finely tuned. There is a huge variation in the number of zinc atoms per cell in different organisms (10<sup>5</sup> for *Escherichia coli*, 10<sup>7</sup> for yeast, and 108 for mammalian cells). However, given cell size variation, zinc concentration is kept in close limits (0.1–0.5 mM) (Eide, 2006). The zinc quota is maintained by the activity of specific membrane transporters or by zinc-binding proteins that mediate zinc uptake or storage. Zinc-depleting conditions are known to reduce fungal growth (Lulloff et al., 2004) and evidence suggests that host cells employ sequestration of zinc to hamper fungal development. This is exemplified by the reduced zinc levels in macrophages infected with *Histoplasma capsulatum* (Winters et al., 2010), and neutrophils infected with *Cryptococcus neoformans* enhance the production of calprotectin, a zinc-binding protein (Mambula et al., 2000). However, excess cellular zinc can generate an imbalance in oxidative metabolism (Pagani et al., 2007). Indeed, macrophages have developed a strategy to kill phagocytosed bacterial cells by zinc overload in the phagosomal environment and the consequent generation of high levels of reactive oxygen species (ROS) in the invading microorganisms (Botella et al., 2011). Here, we focused on the proteins involved in zinc metabolism in the fungal pathogens *Aspergillus fumigatus*, *Candida albicans, C. neoformans*, and *C. gattii* as compared to the well-characterized zinc metabolism-associated proteins from *S. cerevisiae*. Moreover, a critical appraisal on the participation of zinc metabolism and associated proteins in the establishment of fungal infections is presented.

#### **ZINC-ASSOCIATED BIOLOGICAL PROCESSES IN FUNGI**

There is little labile zinc inside cells; the estimated concentration of zinc in cells is in the picomolar to the nanomolar range

Zinc is a ubiquitous metal in all life forms, as it is a structural component of the almost 10% of eukaryotic proteins, which are called zinc-binding proteins. In zinc-limiting conditions such as those found during infection, pathogenic fungi activate the expression of several systems to enhance the uptake of zinc. These systems include ZIP transporters (solute carrier 39 family) and secreted zincophores, which are proteins that are able to chelate zinc. The expression of some fungal zinc uptake systems are regulated by a master regulator (Zap1), first characterized in the yeast *Saccharomyces cerevisiae*. In this review, we highlight features of zinc uptake and metabolism in human fungal pathogens and aspects of the relationship between proper zinc metabolism and the expression of virulence factors and adaptation to the host habitat.

**Keywords: zinc ZIP transporters, zinc metabolism, zinc deprivation, ZAP transcription factor, fungal virulence**

(Eide, 2006), and it is assumed that virtually all cellular zinc is associated with zinc-binding proteins. Bioinformatic analyzes to evaluate the presence of canonical sequences related to zinc binding have revealed that 5–6% of the predicted proteomes of prokaryotes consist of zinc-binding proteins, while this proportion reached 9% in eukaryotic proteomes (Andreini et al., 2009). Using the fungal model *S. cerevisiae*, with a manually curated annotation of the genome and considerable biochemical information (Cherry et al., 2012), it is possible to infer the fraction of the proteome that can bind zinc. In fact, analyzes of predicted gene products from different organisms using the term "zinc ion binding" as a query of Gene Ontology databases revealed that some fungal species have a proportion of zinc-binding proteins that corresponds to ∼5% of the proteome (**Figure 1**). Considering only the zinc-binding proteins from *S. cerevisiae*, a large proportion of these proteins (25%) are associated with biological processes related to transcriptional regulation. An even higher proportion of these proteins have the ability to bind DNA as accessed by Gene Ontology analysis (**Figure 1**). These numbers and the accumulation of a significant number of experimental reports point to the central role of zinc in gene expression regulation.

Consistent with the plethora of zinc-binding proteins associated with the regulation of gene expression in *S. cerevisiae,* a large number of these zinc-binding proteins consist of zinc finger transcription factors, the largest family of transcriptional regulators. Analysis of the Fungal Transcription Factor Database (Park et al., 2008) revealed that fungal species are characterized by a high diversity of zinc finger transcription factors, ranging from 116 in *C. albicans* to 311 in *A. fumigatus* (**Figure 1**). The vast majority of these transcription factors belong to the widely phylogenetically distributed Zn2Cys6. In *S. cerevisiae*, members of this family are involved in the regulation of several biological processes, including sugar and amino acid metabolism, nitrogen utilization, mitosis, and meiosis (Macpherson et al., 2006). However,

other biological processes are associated with zinc-binding proteins. This is consistent with the enzymatic activity present in some zinc-binding proteins, including ligase, peptidase, and oxidoreductase activities (**Figure 1**). Yet in *S. cerevisiae*, the Cu/Zn superoxide dismutase and the alcohol dehydrogenase (Adh1) proteins are the most abundant zinc-binding proteins (Eide, 2006).

Some zinc-binding proteins are also involved in fungal virulence. Superoxide dismutases (Sods) are the central enzymes in fungi associated with the detoxification of ROS generated by host cells during host-pathogen interactions (Huang et al., 2009). In this view, specific Sods from pathogenic fungi are assumed to be virulence determinants. *C. albicans* expresses six superoxide dismutase isoforms (Sod1—Sod6), four of which are annotated as copper/zinc-dependent enzymes (Sod1, Sod4, Sod5, and Sod6) (Frohner et al., 2009). Functional analysis has revealed that Sod1, Sod4, and Sod5 are necessary for the proper detoxification of ROS by *C. albicans*, as SOD-null mutants displayed growth defects in the presence of ROS-generating compounds. In addition, these mutants displayed increased susceptibility to macrophage killing and reduced virulence (Hwang et al., 2002; Martchenko et al., 2004; Frohner et al., 2009). The *A. fumigatus* genome contains four genes encoding Sods, two of which are annotated as copper/zinc-dependent (Sod1 and Sod4). *A. fumigatus* cells lacking the *SOD1* gene are hypersensitive to menadione, a ROS generating agent, but the virulence of cells lacking this gene is not affected (Lambou et al., 2010). In *C. neoformans*, two Sodencoding genes have been described. The *SOD1* gene encodes a copper/zinc-dependent Sod required for full virulence in animal models of cryptococcosis and for survival inside macrophages (Cox et al., 2003).

Zinc-binding metalloproteases have also shown to be involved in virulence. Distinct species of pathogenic fungi secrete proteases during the infection. These proteases are classified into aspartic proteases, serine proteases, and metalloproteases (Yike, 2011). The deuterolysin (M35) family of metalloproteases is characterized by the presence of two zinc-binding histidines and a catalytic glutamate in their catalytic centers (Markaryan et al., 1994). The roles of metalloproteases secreted by pathogenic fungi are largely associated with tissue degradation. This is evident for the Mep3 metalloprotease from *Microsporum canis* (Brouta et al., 2001). The ADAM proteases (from A Disintegrin And Metalloproteinase) belongs to the M12 family of metalloproteases according to the MEROPS database (Rawlings et al., 2012). These proteins are produced as pro-enzymes that must be secreted and activated prior to performing their biological functions. ADAM proteases have been implicated in several aspects of cell biology including adhesion, migration, proteolysis, and signaling (Edwards et al., 2008). The presence of two copies of putative ADAM coding sequences in the genome of *A. fumigatus* indicates a possible contribution for this family in virulence in this fungus. However, no functional characterization was performed yet to evaluate whether ADAM proteases can be associated to virulence in *A. fumigatus.*

## **FUNGAL ZINC UPTAKE**

Fungal cells must acquire zinc for proper development during their life cycle, even when they are saprophytes or during the infection process. To hamper pathogen growth, mammalian hosts typically reduce the levels of free zinc and other metals (Kehl-Fie and Skaar, 2010). The concentration of zinc in human tissues varies dramatically, ranging from 10μg/g (lungs) to 83.2μg/g (liver). In body fluids, the zinc concentration ranges from 0.2 to 8.7 μg/mL (Lech and Sadlik, 2011). Thus, pathogenic fungi have developed efficient strategies to uptake zinc to overcome the limits imposed by host.

The initial characterization of zinc transport mechanisms in fungi was done in *S. cerevisiae* and revealed the central role of the ZIP (Zrt-, Irt-like protein) family of zinc transporters (Eide, 2006). The name of this family, also known as SLC39 (solute carrier 39), refers to the first members to be functionally characterized, the *S. cerevisiae* zinc transporters Zrt1 and Zrt2 and the *Arabidopsis thaliana* iron transporter Irt1 (Eide, 2004). ZIP family transporters are associated with zinc transport into the cytoplasm across cellular membranes, either from the extracellular space or from within organelles. ZIP transporters are characterized by eight putative transmembrane regions, and the amino- and carboxyl-termini are often located on the extracellular or luminal side of membranes (Eide, 2004). A histidine-rich region present between transmembrane regions three and four is necessary for zinc selectivity, as demonstrated for the TjZNT1 ZIP transporter from the nickel hyperaccumulator plant *Thlaspi japonicum* (Nishida et al., 2008).

The *S. cerevisiae* Zrt1 is a high-affinity zinc transporter that is expressed when cells are cultivated in low-zinc media (Zhao and Eide, 1996a), while the low-affinity transporter Zrt2 mediates the uptake of zinc, cooper, and iron (Zhao and Eide, 1996b). Additional non-specific zinc transporters are also associated with zinc uptake, as*zrt1/zrt2* double-mutants are capable of growing in low-zinc conditions (Zhao and Eide, 1996b). These transporters include the low-affinity iron transporter Fet4 and the phosphate transporter Pho84, which mediate the uptake of zinc by phosphate chelation of this metal (Waters and Eide, 2002; Jensen et al., 2003). Inside cells, zinc is shuttled to different compartments, including the nucleus, endoplasmic reticulum and vacuole, by the activity of specific transporters not related to the ZIP family (Eide, 2009). Zrt3 is a ZIP transporter found in the membranes of vacuoles that accumulate zinc, the so-called "zincosomes." The function of Zrt3 is associated supplying zinc to the cytoplasm from zincosomes (Simm et al., 2007).

The number of ZIP genes in the genomes of *A. fumigatus*, *C. albicans*, *C. neoformans,* and *C. gattii* ranges from four to nine. Genome annotation based evidences suggest that many of these transporters show high similarity to *S. cerevisiae* Zrt1 or Zrt2 proteins, suggesting that both low- and high-affinity zinc uptake systems exist in these pathogenic fungi. In *A. fumigatus,* three ZIP zinc transporters have been characterized. The expression of *zrfA* and *zrfB* genes is activated by low levels of zinc or iron. In addition, the expression of these transporters in response to zinc deprivation occurs mainly in an acidic environment. Null mutants for these genes, as well as double mutants lacking both genes, showed a reduced ability to grow under zinc deprivation (Vicentefranqueira et al., 2005). Further analysis showed that the expression of these genes is under control of the pH homeostasis regulator PacC (Amich et al., 2009). A third zinc transporter from *A. fumigatus*, which is encoded by the gene *zrfC*, is expressed during zinc-deprivation conditions when the fungus is grown in alkaline pH conditions. Null mutants of this gene are severely reduced in their ability to grow during zinc deprivation (Amich et al., 2010). In *C. gattii*, zinc deprivation by the chelator N,N,N,N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) induced the expression of the putative zinc transporters encoded by genes *ZIP1*, *ZIP2*, and *ZIP3* (Schneider et al., 2012).

Some fungal species possess additional mechanisms to sequester zinc from host cells and tissues in a process analogous to iron chelation by secreted siderophores. *C. albicans* secretes the antigenic protein Pra1, a zinc-binding protein that is able to scavenge zinc from tissues invaded by the fungus. Moreover, molecular docking experiments revealed that Pra1 could interact with the zinc transporter Zrt1. Pra1 participates in proper endothelial colonization by *C. albicans* (Citiulo et al., 2012) and is associated with evasion of immune cells (Luo et al., 2011; Soloviev et al., 2011). In addition, orthologs of the gene encoding Pra1 are found in diverse fungal pathogens (Citiulo et al., 2012) including *A. fumigatus* (the zinc-regulated *aspf2* gene (Amich et al., 2010). However, no orthologs could be found in *C. neoformans* or *C. gattii.* These zinc-sequestering genes are generally clustered with Zrt1 orthologs in these fungi in a highly syntenic fashion (Citiulo et al., 2012), representing a conserved mechanism for zinc acquisition during host-fungal interactions.

#### **EFFECTS OF ZINC DEPRIVATION ON FUNGAL CELLS**

Zinc chelation is able to reduce fungal growth in both rich and defined media (Lulloff et al., 2004). In fact, zinc chelation is assumed to occur during infection and is an important strategy developed by immune cells to hamper pathogen growth (Corbin et al., 2008). Zinc restriction by host cells is achieved by lowering metal availability via the activity of the host zinc transporters or the expression of zinc-binding proteins. An example of a zincbinding protein that is expressed to reduce the bioavailability of zinc is calprotectin, a member of the S100 family of metalbinding proteins (Goyette and Geczy, 2011). Calprotectin was found to reduce the growth of diverse fungal species *in vitro* (Lulloff et al., 2004). Moreover, this protein is produced by neutrophils in order to reduce the development of *A. fumigatus* (McCormick et al., 2010), *C. albicans* (Urban et al., 2009), and *C. neoformans* (Mambula et al., 2000). Neutrophils are able to kill invading pathogens by phagocytosis, secreting anti-microbial molecules and forming neutrophil extracellular traps (NETs). NET formation is derived from a distinct mechanism of cell death that is characterized by the loss of intracellular membranes and further structural derange of the plasma membrane. As a result of this loss of membrane functionality, NETs are composed of nucleosomes and a set of cytoplasmic and granular interacting proteins (Brinkmann and Zychlinsky, 2012). NETs formed in response to *A. fumigatus* and *C. albicans* infection contain calprotectin, and the presence of this protein in such structures is fundamental for the proper antifungal activity of neutrophils (Urban et al., 2009; McCormick et al., 2010).

The direct effects of zinc deprivation on fungal cells are poorly understood. Assays employing *S. cerevisiae* revealed that, by an unknown mechanism, cells that are exposed to zinc deprivation experience increased levels of ROS (Wu et al., 2007). However, yeast cells employ different strategies to cope with the stress caused by zinc deprivation. As revealed by transcriptomic and functional analyzes in *S. cerevisiae,* low zinc conditions lead to alterations in lipid synthesis, methionine, and sulfate metabolism and to oxidative stress tolerance (Iwanyshyn et al., 2004; Wu et al., 2007, 2009). Furthermore, a genome-wide functional analysis employing a *S. cerevisiae* mutant library that encompasses more than 4500 gene knockout mutants revealed that almost 400 different gene products are necessary for proper growth in zinc-limiting conditions. Among these gene products are those associated with the oxidative stress response, endoplasmic reticulum function, peroxisome biogenesis, histone deacetylation, and zinc uptake (North et al., 2012). Zinc deprivation induced by TPEN also induced accumulation of intracellular ROS in *C. gattii* cells (Schneider et al., 2012). Moreover, proteomic profiling of the dimorphic fungus *Paracoccidioides brasiliensis* exposed to TPEN also revealed an increased expression of proteins involved in stress tolerance, suggesting that zinc deprivation induces stress in these cells (Parente et al., 2013). Thus, it is reasonable to suggest that zinc deprivation hampers fungal development by restricting the activity of zinc-binding proteins and by submitting the fungal cells to different kinds of stress.

## **REGULATION OF ZINC HOMEOSTASIS**

The transcriptional responses to zinc deprivation of fungal cells are regulated by the Zap1 transcription factor. The first characterization of the role of Zap1 in regulating zinc homeostasis was performed in *S. cerevisiae* (Zhao and Eide, 1997). This major zinc metabolism regulator contains seven C2H2 zinc finger domains (ZF). While the domains ZF3–ZF7, located to C-terminal region, are directly associated with the recognition and binding of zincresponsive elements (ZRE) in the promoters of Zap1-regulated genes, ZF1 and ZF2 lie in the zinc-responsive element present in the activation domain (AD) of this transcription factor. Zinc binding to ZFs in AD2 of Zap1 represses the activity of this transcription factor and therefore inhibits the expression of Zap1 regulated genes (Bird et al., 2003; Herbig et al., 2005). However, zinc can also influence the binding of Zap1 to its ZREs by modulating of the activity of ZF3-ZF7 (Frey et al., 2011). Zap1 activates the expression of more than 60 genes in response to low-zinc environment, including genes that code for zinc transporters (*ZRT1, ZRT2*, ZRT3, *ZRC1*, and *FET4*), proteins related to stress (*CTT1, TSA1*, and *HSP26*), as well as regulating its own expression (Lyons et al., 2000; Wu et al., 2008).

Orthologs of *S. cerevisiae* Zap1 were functionally characterized in three pathogenic fungal species. The *A. fumigatus zafA* expression is induced in zinc-limiting media and repressed by zinc. In addition, null mutants of *zafA* have a reduced ability to grown in low-zinc media as a direct effect of diminished zinc transport. Consequently, such mutants displayed a complete lack of virulence in murine models of aspergillosis. In addition, no conidial germination could be observed in mice infected with mutant cells lacking *zafA* (Moreno et al., 2007).

The *C. albicans* Csr1/Zap1 transcription factor was also shown to influence growth in zinc-deprivation conditions and affect important virulence traits in this pathogenic yeast. While cells lacking the *CSR1/ZAP1* gene displayed reduced filamentation, the same mutants showed increased β-glucan content in their biofilm matrices. Moreover, the biofilms produced by such cells had a predominance cells in the yeast form when compared to wild-type biofilms (Kim et al., 2008; Nobile et al., 2009). The characterization of Csr1/Zap1 targets in biofilm-inducing conditions by chromatin immunoprecipitation analysis showed that this transcription factor recognizes the promoters of approximately 60 genes including the *ZRT1, ZRT2*, and *ZRT3* genes (zinc transporters) and the *CSR1*/*ZAP1* gene itself (Nobile et al., 2009). Further characterization of Csr1/Zap1 revealed that this transcription factor also regulates cell-cell signaling during biofilm development by regulating the expression of Dpp1, a farnesol synthesis protein. Farnesol is an important mediator of biofilm formation as it acts as a quorum-sensing molecule that functions as an inhibitor of the yeast-to-hyphae transition in biofilms (Ganguly et al., 2011).

The Zap1 ortholog from *C. gattii* also regulates the expression of zinc uptake systems. Null mutants of this gene were defective in growth in zinc-deprivation conditions, accumulated intracellular ROS, were hypersensitive to reactive nitrogen species, and displayed attenuated virulence in murine inhalation models of cryptococcosis. Transcriptomic profiling of wild type and *zap1-*null mutants exposed to TPEN revealed that more than 500 genes were differentially expressed between these cell types. The large majority of these differentially expressed gene products were found to be related to adaptive responses to zinc deprivation, but inferred true Zap1 targets were also found including the zinc transporters encoded by *ZIP1* and *ZIP2* (Schneider et al., 2012).

A direct comparison between of *C. albicans, C. gattii,* and *S. cerevisiae* Zap1-dependent transcription profiles revealed a common Zap1 regulon (**Table 1**). Among positively regulated genes, figure out the genes for the zinc acquisition systems, while a large proportion of negatively regulated genes consist of zinc-binding proteins (Wu et al., 2008; Nobile et al., 2009; Schneider et al., 2012). These proteins include a large family of alcohol dehydrogenases, which are most likely the most



*aLog2 Fold Change values retrieved from microarray expression data of WT and zap1- S. cerevisiae cells exposed to low zinc concentrations (61 nM of ZnCl2).(Lyons et al., 2000).*

*bLog2 Fold Change values retrieved from microarray expression data of complemented mutant and zap1-/ zap1-C. albicans biofilms (Nobile et al., 2009).*

*cLog2 Fold Change values retrieved from RNA-Seq expression data of WT and zap1- C. gattii cells exposed to a zinc chelator (10 μM of TPEN).(Schneider et al., 2012).*

abundant zinc-binding proteins in the cell. This trend represents a conserved fungal strategy to shuttle zinc into essential proteins specialized for zinc conservation. This mechanism is characterized by the down-regulation of the expression of abundant zinc storage proteins, such as zinc-dependent alcohol dehydrogenases, in zinc-deprivation conditions. In this way, zinc can easily be mobilized to other zinc-dependent proteins necessary for proper development under these harsh conditions (Eide, 2009).

#### **CONCLUSIONS AND FUTURE STUDIES**

Zinc influences diverse mechanisms of fungal pathogenesis by directly associating with virulence determinants (i.e., metalloproteases or Sods) or by regulating the expression of many

### **REFERENCES**


I., et al. (2011). Mycobacterial p(1)-type ATPases mediate resistance to zinc poisoning in human macrophages. *Cell Host Microbe* 10, 248–259. doi: 10.1016/j.chom.2011.08.006


during endothelial invasion. *PLoS Pathog.* 8:e1002777. doi:

**ACKNOWLEDGMENTS**

CNPq, FINEP, and CAPES.

proteins required for infection. The regulation of zinc acquisition by the Zap1 transcription factors is fundamental for fungal pathogenesis in mammalian hosts. A broader functional characterization of Zip transporters in fungi, including plant and insect fungal pathogens, will elucidate the pivotal role of pathogen zincbinding proteins during the infectious process. As active zinc deprivation by hosts represents an important antifungal mechanism, development of chelating strategies to control *in vivo* fungal development may be a plausible chemotherapeutic alternative.

This work was supported by grants from the Brazilian agencies


*Acta* 1763, 711–722. doi: 10.1016/j.bbamcr.2006.03.005


Vainstein, M. H., and Staats, C. C. (2012). Zap1 regulates zinc homeostasis and modulates virulence in *Cryptococcus gattii*. *PLoS ONE* 7:e43773. doi: 10.1371/journal.pone.0043773


induced by zinc limitation. *Proc. Natl. Acad. Sci. U.S.A.* 93, 2454–2458. doi: 10.1073/pnas.93. 6.2454


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 26 July 2013; accepted: 26 September 2013; published online: 14 October 2013.*

*Citation: Staats CC, Kmetzsch L, Schrank A and Vainstein MH (2013) Fungal zinc metabolism and its connections to virulence. Front. Cell. Infect. Microbiol. 3:65. doi: 10.3389/fcimb. 2013.00065*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2013 Staats, Kmetzsch, Schrank and Vainstein. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Competition for zinc binding in the host-pathogen interaction

## *Mauro Cerasi 1, Serena Ammendola1 and Andrea Battistoni 1,2\**

*<sup>1</sup> Dipartimento di Biologia, Università di Roma Tor Vergata, Rome, Italy*

*<sup>2</sup> Istituto Nazionale Biostrutture e Biosistemi, Consorzio Interuniversitario, Rome, Italy*

#### *Edited by:*

*Frédéric J. Veyrier, Institut Pasteur, France*

#### *Reviewed by:*

*Klaus Hantke, Universität Tübingen, Germany Robert D. Perry, University of Kentucky, USA*

#### *\*Correspondence:*

*Andrea Battistoni, Via della Ricerca Scientifica, Dipartimento di Biologia, Room 372, Università di Roma Tor Vergata, Rome 00133, Italy e-mail: andrea.battistoni@ uniroma2.it*

Due to its favorable chemical properties, zinc is used as a structural or catalytic cofactor in a very large number of proteins. Despite the apparent abundance of this metal in all cell types, the intracellular pool of loosely bound zinc ions available for biological exchanges is in the picomolar range and nearly all zinc is tightly bound to proteins. In addition, to limit bacterial growth, some zinc-sequestering proteins are produced by eukaryotic hosts in response to infections. Therefore, to grow and multiply in the infected host, bacterial pathogens must produce high affinity zinc importers, such as the ZnuABC transporter which is present in most Gram-negative bacteria. Studies carried in different bacterial species have established that disruption of ZnuABC is usually associated with a remarkable loss of pathogenicity. The critical involvement of zinc in a plethora of metabolic and virulence pathways and the presence of very low number of zinc importers in most bacterial species mark zinc homeostasis as a very promising target for the development of novel antimicrobial strategies.

**Keywords: zinc uptake, ZnuABC, antibacterial therapies, metal cofactor, host-pathogen interaction,** *Salmonella enterica***, zinc transporter, nutritional immunity**

### **ZINC: CHEMICAL PROPERTIES AND ROLE IN BACTERIAL PROTEINS**

Among transition metals, zinc is likely the one which is used as a structural or catalytic cofactor in the wider number of proteins. The widespread use of zinc in proteins can be related to its peculiar chemical properties (Andreini et al., 2008). Unlike the other biological relevant transition metals (Fe2+, Mn2+, Cu2+, Ni2+) the zinc ion (Zn2+) has a filled *d* orbital and, therefore, it is redox stable. Zinc mainly participates to catalytic reactions by acting as a Lewis acid able to accept electron pairs or, as an alternative, by attracting or stabilizing negative charges of the substrates. Moreover, zinc binding to proteins is facilitated by its capability to form stable chemical bonds with nitrogen, oxygen and sulfur atoms and assume different coordination numbers. As a consequence zinc can be found in a large variety of distinct chemical environments, which may significantly modulate its reactivity. However, a potential problem of zinc is that it binds to proteins stronger than the other divalent metals (Irving and Williams, 1948) and, therefore, cells maintain the intracellular pool of "free" metal at very low levels to prevent its unspecific binding to proteins (Colvin et al., 2010).

Different studies have attempted to measure the amount of zinc in bacteria. It has been shown that microorganisms have a remarkable capability to modify their intracellular zinc content in response to variations in the environmental availability of the metal (Outten and O'Halloran, 2001; Garmory and Titball, 2004) and that the total cellular zinc in bacteria growing in rich media is in the submillimolar range (10−<sup>4</sup> M), i.e., a concentration comparable to that usually observed in most eukaryotic cells (Eide, 2006). More complex is to obtain a careful evaluation of the intracellular pool of metal ions not tightly bound to proteins. *In vitro* studies carried out with purified zinc-responding transcriptional regulators have initially suggested that cellular "free" zinc levels are in the femtomolar range, i.e., around 10−<sup>15</sup> M (Outten and O'Halloran, 2001). However, recent studies involving proteinbased ratiometric biosensors have established that *in vivo* the concentration of intracellular exchangeable zinc is around 20 pM, i.e., 2 <sup>×</sup> <sup>10</sup>−11M (Wang et al., 2011). Picomolar values of "free" zinc have been reported also in several eukaryotic systems (Colvin et al., 2010).

Interestingly, although the zinc concentration in bacterial cells is close to that of iron, a significant fraction of iron may be found in association to proteins such as ferritins, bacterioferritins or DPS (Andrews et al., 2003), whereas *bona fide* zinc-storage proteins are present only in a few bacteria (Blindauer et al., 2002). An experimental attempt to explore the complexity of the bacterial zinc proteome has shown that more than 3% of the proteins expressed in *Escherichia coli* contain zinc (Katayama et al., 2002), whereas bioinformatics investigations have revealed that about 5% of all bacterial proteins contain recognizable zinc-binding sites (Andreini et al., 2006). This means that an *E. coli* cell with about 4300 protein-encoding genes contains more than 200 zincbinding proteins. These figures, however, are not sufficient to have an accurate idea of the actual importance of this metal in the physiology of a bacterial cell. In fact, in addition to being an essential cofactor in a large number of enzymes involved in central metabolic pathways, zinc is bound to several proteins involved in the management of gene expression, including some ribosomal proteins (Hensley et al., 2011), RNA polymerases (Scrutton et al., 1971), tRNA synthetases (Miller et al., 1991), sigma factor interacting proteins (Campbell et al., 2007) and zinc responding transcriptional factors (Chivers, 2007). Moreover, zinc is involved in other crucial processes, including DNA repair (Kropachev et al., 2006), response to oxidative stress (Ortiz De Orue Lucana et al., 2012), antibiotic resistance (Meini et al., 2013) and production of virulence-related proteins (Ammendola et al., 2008). It follows that changes in the intracellular concentrations of zinc can have pleiotropic effects on the composition of the bacterial proteome, involving changes in the expression and activity of zinc-containing proteins as well as of proteins which do not employ this cofactor.

#### **BACTERIAL ZINC UPTAKE SYSTEMS AND RESPONSE TO ZINC SHORTAGE**

Although in bacteria exposed to high levels of zinc the metal may enter through a large number of unspecific channels, only a few metal transporters are known to mediate the specific uptake of zinc (Hantke, 2005) (**Figure 1**). Some recent studies on the pneumococcal PsaA protein involved in manganese uptake have provided interesting hints to understand the mechanisms ensuring specificity in transition metal import (McDevitt et al., 2011; Counago et al., 2013). PsaA may bind either manganese or other first-row transition metals, but, due to the propensity of zinc to form stable complexes with proteins (Irving and Williams, 1948) it competitively affects Mn2<sup>+</sup> binding and locks the protein in a conformation which prevents the entry either of zinc or of manganese. This observation provides an explanation for the ability of zinc to inhibit pneumococcal growth (McDevitt et al., 2011) and an elegant example of the strategies used by living cells to guarantee the correct uptake of specific metal ions (Waldron and Robinson, 2009).

In several Gram-negative bacteria growing in metal replete conditions, zinc uptake is thought to be primarily mediated by ZupT, a constitutively expressed low affinity transporter belonging to the ZIP (ZRT-, IRT-like Protein) protein family (Grass et al., 2002). This metal permease has a broad metal specificity, but it displays a clear preference for zinc over other divalent metals (Grass et al., 2005). ZupT depends on the proton motive force to energize zinc import (Karlinsey et al., 2010; Taudte and Grass, 2010).

The response to zinc paucity is controlled through the coordinated expression of a set of genes regulated by the transcriptional factor Zur, which may bind two or more zinc ions, depending on the species (Outten et al., 2001; Lucarelli et al., 2007; Shin et al., 2011). One atom of zinc serves a structural role, whereas the other atom(s) favors the folding of a DNA-binding domain enabling the protein to tightly bind to a consensus sequence located in the promoter of said genes. In contrast, when the intracellular zinc content decreases, zinc-devoid Zur is no longer able to stably interact with DNA and to repress transcription. The number of known Zur–regulated genes changes in different bacteria, but in all species they include a small operon encoding for the components of ZnuABC, a high affinity zinc importer of the ABC family, and one or more genes encoding for paralogs of zinccontaining ribosomal proteins (Panina, 2003; Graham et al., 2009; Li et al., 2009; Lim et al., 2013). The ZnuABC uptake system is composed of three proteins: the ZnuB channel, the ZnuC ATPase component which provides the energy necessary for ion transport through the inner membrane, and ZnuA, a soluble protein which captures Zn(II) in the periplasm with high efficiency and delivers it to ZnuB (Patzer and Hantke, 1998). In some bacteria there is also an accessory component of the ZnuABC transporter, ZinT, which is known to form a complex with ZnuA in presence of zinc and is thought to enhance ZnuA ability to recruit zinc (Petrarca et al., 2010; Ilari et al., 2013). A similar zinc uptake system (AdcABC) can be found in pneumococci and some other Gram-positive bacteria, where the lipoprotein AdcA is characterized by two structural domains that show clear sequence and structural homology with ZnuA and ZinT, respectively (Dintilhac et al., 1997; Panina et al., 2003).

An interesting facet of the Zur-mediated response to zinc shortage is the substitution of ribosomal proteins containing zinc with homologous proteins lacking the zinc-binding motif. This change in ribosomal structure reduces the metal requirements of bacterial cells, as the majority of intracellular zinc is thought to be associated to ribosomes (Hensley et al., 2011). Moreover, the production of zinc-independent ribosomal proteins may be useful to mobilize a relevant amount of metal from pre-existing ribosomes and facilitates the adaptation to zinc-limiting conditions (Gabriel and Helmann, 2009). From this point of view, the ribosome may be described as a zinc storage protein complex. Additional paralogs of zinc-containing proteins have been identified in several bacteria (Haas et al., 2009) and include a homolog of the transcriptional factor DksA which is involved in the control of the bacterial response to stress and starvation (Blaby-Haas et al., 2011).

The outer membrane of Gram-negative bacteria allows the passive diffusion of low molecular weight molecules. However, a mechanism of nutrient uptake solely based on diffusion may be hardly able to ensure the adequate absorption of elements which are poorly available in the environment. In recent years, an outer membrane TonB-dependent receptor involved in zinc uptake has been identified in *Neisseria meningitidis* and some other Gramnegative bacteria (Stork et al., 2010). This protein, denominated ZnuD, mediates either zinc or heme uptake and is regulated either by Zur or by Fur (Kumar et al., 2012; Pawlik et al., 2012). The pneumococcal surface protein PhtD has been proposed to play a functionally similar role in favoring zinc uptake through AdcAII (Loisel et al., 2011). No outer membrane zinc receptors have been so far identified in Enterobacteria or in other Gram-negative bacteria. However, it has been observed that apo-ZinT can be extruded outside the cell, suggesting that it could have some role in the acquisition of zinc from the environment (Ho et al., 2008; Gabbianelli et al., 2011).

It should also be noted that a few bacterial species have been shown to express more than one high affinity zinc uptake systems. This is the case of *Listeria monocytogenes* which expresses two ABC-type zinc importers (ZnuABC and ZurAM), both contributing to full virulence (Corbett et al., 2012) and of nontypeable *Haemophilus influenzae,* where the zinc binding system ZevAB facilitates growth in zinc-limiting conditions and lung colonization in infected mice (Rosadini et al., 2011). Similarly, disruption of *znuA* in *Pseudomonas aeruginosa* results in a very limited growth defect under zinc-limiting conditions (Ellison et al., 2013), possibly due to the expression of a zinc-importing P-type ATPase (Lewinson et al., 2009).

The bacterial outer membrane is thought to be permeable to hydrophilic solutes of *<*600 dalton (Nikaido and Vaara, 1985) and, therefore, zinc concentration in the periplasmic space is largely dependent on zinc availability in the environment. Under zinc replete conditions (left), the metal is imported

expression of the importer ZnuABC. Under conditions of zinc shortage (right), apoZur is unable to bind DNA and the high affinity zinc importer ZnuABC is expressed. *Neisseria meningitidis* expresses a Zur-regulated TonB-dependent outer membrane protein, ZnuD, involved in zinc uptake.

### **ZINC IN THE HOST-PATHOGEN INTERACTION**

Whereas the competition for iron acquisition has been recognized as a key element of the host pathogen interaction for a long time, only in recent years the efficient uptake of other divalent metals has emerged to play a comparable role (Kehl-Fie and Skaar, 2010). In particular the importance of zinc has become clear through a series of investigations which have established that deletion of the *znuABC* genes not only decrease bacterial ability to growth in *in vitro* environments poor of this metal, but also dramatically affects their pathogenicity. Bacterial pathogens which have been shown to critically depend on ZnuABC to infect their hosts include *Acinetobacter baumannii* (Hood et al., 2012), *Brucella abortus* (Kim et al., 2004; Yang et al., 2006), *Campylobacter jejuni* (Davis et al., 2009), pathogenic *E. coli* strains (Sabri et al., 2009; Gabbianelli et al., 2011), *H. ducreyi* (Lewis et al., 1999), *Moraxella catarrhalis* (Murphy et al., 2013), *Pasteurella multocida* (Garrido et al., 2003), *Salmonella enterica* (Campoy et al., 2002; Ammendola et al., 2007) and *Yersinia ruckeri* (Dahiya and Stevenson, 2010). In contrast, while being required for zinc uptake *in vitro*, ZnuABC does not contribute to *Y. pestis* virulence (Desrosiers et al., 2010) and provides only a limited advantage to *Proteus mirabilis* during urinary tract infections (Nielubowicz et al., 2010). It is not yet clear whether these bacteria possess additional zinc importers or if they show limited zinc requirements during infections.

Probably, the previous underestimation of the importance of zinc in the interaction between bacteria and their hosts can be largely attributed to the apparent abundance of this element in all tissues. In fact, high levels of zinc are present either within cells or in the plasma, where most of the metal is loosely associated to proteins (Zalewski et al., 2006) and, therefore, potentially available for invading microorganisms. However, it should be noted that a typical feature of the early response to the infection is the rapid fall of plasma zinc concentration, accompanied by zinc accumulation in the liver. Redistribution of zinc among the various tissues is regulated by a lipopolysaccharide-induced cytokines cascade (with IL-6 playing a central role) which stimulates increased synthesis of acute phase proteins, such as metallothionein, and the hepatic uptake of the metal through the induction of the solute carrier 39 (SLC39) protein ZIP14 (Liuzzi, 2005). In view of the importance of the ZnuABC transporter in bacterial zinc uptake during infections, this feature of the acute phase response appears as an adaptive mechanism intended to deprive pathogens of zinc.

The role of the ZnuABC transporter has been investigated in details in *S. enterica* serovar Typhimurium (*S.* Typhimurium). Expression of *znuABC* is repressed in *S.* Typhimurium cultivated in synthetic media containing zinc concentrations as low as 1µM. In contrast, the *znuABC* operon is strongly induced in bacteria recovered from the spleens of infected mice or from cultured epithelial or macrophagic cells (Ammendola et al., 2007). These observations suggest that zinc bound to proteins is not easily available for invading bacteria and that ZnuABC is required to have rapid access to the pool of "free" zinc. More recently, it has been shown that during gut infections ZnuABC confers resistance to the antimicrobial protein calprotectin (Liu et al., 2012). Calprotectin is a neutrophilic protein of the S100 family of calcium binding proteins, that is abundantly released at sites of infection to control the multiplication of pathogens by the sequestration of zinc and manganese (Kehl-Fie and Skaar, 2010). In support to the experimental evidence that calprotectin starves bacteria for metal ions, structural studies have confirmed that calprotectin possesses two distinct binding sites for transition metals, one of which is specific for zinc and the other one may accommodate either zinc or manganese (Brunjes Brophy et al., 2013; Damo et al., 2013). Bacteria expressing ZnuABC are able to resist to such antimicrobial strategy and this favor their growth over competing microbes in the inflamed gut (Gielda and Dirita, 2012; Liu et al., 2012). Taken together, these studies suggest that zinc acquisition through the ZnuABC transporter is essential for the colonization of *Salmonella* in mice, and provide a parallelism between the mechanisms of iron and zinc sequestration in the host-pathogen relationships. It is worth noting that calprotectin is not the unique S100 protein involved in zinc sequestration. In fact, a comparable function has been proposed for the antibacterial protein psoriasin (S100A7), which protects human skin from *E. coli* infections (Glaser et al., 2005).

Although, the above mentioned studies have suggested that zinc sequestration is a strategy widely used by vertebrates to control microbial infections, a few recent observations have revealed an alternative way to use zinc in host defense. In fact, it has been shown that human macrophages control mycobacteria by elevating zinc levels in the bacteria-containing phagosomes (Botella et al., 2011). This process is dependent on reactive oxygen species generated by the phagocytic NADPH oxidase and involves the mobilization of zinc from intracellular stores. Mycobacterial resistance to zinc intoxication in macrophages relies on their ability to induce the expression of heavy metal efflux P-type ATPases, which prevent the intracellular accumulation of zinc at toxic levels. Structurally homologous zinc efflux pumps have been identified in a large number of bacteria, including *E. coli*, where the P-type zinc exporter ZntA has been proved to be critical for zinc tolerance (Beard et al., 1997; Rensing et al., 1997) and for the maintaining of appropriate levels of intracellular zinc (Wang et al., 2012). Whereas mycobacteria lacking the efflux pump CtpC or *E. coli* cells devoid of ZntA display a reduced ability to survive in human macrophages, disruption of CtpC does not affect the ability of *M. tuberculosis* to infect mice (Botella et al., 2011).

To add to the complexity, a mobilization of zinc in the opposite direction to that found in response to mycobacteria has been observed in murine macrophages infected with the fungus *Histoplasma capsulatum* (Subramanian Vignesh et al., 2013). In this case, phagosomes are deprived of zinc and the metal accumulates in the Golgi or in the cytoplasm, in association to metallothioneins. Further studies are needed to understand whether these different responses depend on the cross-talk between each specific microorganism and phagocytic cells, or whether the ability to poison bacteria through an excess of zinc is a prerogative of human macrophages.

#### **ZINC HOMEOSTASIS AS A TARGET FOR ANTIBACTERIAL THERAPIES**

Different studies have proposed that ABC transporters could be effective targets for the development of novel antibacterial drugs or vaccines (Garmory and Titball, 2004; Counago et al., 2012). In this view, ZnuABC appears as a particularly promising candidate.

Whereas a large number of pathogens produce multiple metal import systems that enable the uptake of different iron forms (reduced or oxidized, "free" or bound to proteins or to heme), in most bacteria there is only one high affinity zinc importer. Moreover, it has been shown that *Salmonella* strains lacking the whole *znuABC* operon display the same dramatic loss of virulence of strains producing ZnuB and ZnuC, but lacking ZnuA (Petrarca et al., 2010). This finding indicates that the ability of ZnuA to effectively compete for zinc binding with other periplasmic proteins is critical to ensure zinc import in the cytoplasm (Berducci et al., 2004) and suggests that drugs targeting the soluble component of the transporter could block zinc import. This is a very interesting possibility because ZnuA is a suitable substrate for biochemical and structural studies (Banerjee et al., 2003; Chandra et al., 2007; Wei et al., 2007; Yatsunyk et al., 2008; Ilari et al., 2011; Castelli et al., 2013). It is worth noting that several molecules able to interfere with zinc uptake in *Candida albicans* have been recently identified through the screening of a small-molecule library of 2000 compounds (Simm et al., 2011), thus providing a proof of concept that it is possible to pharmacologically target zinc homeostasis in pathogenic microorganisms.

Bacterial mutant strains unable to produce the ZnuABC transporter are also putative candidate for the development of live– attenuated vaccines. It has been shown that a *S*. Typhimurium *znuABC* strain is able to induce short lasting infections in mice which induce a solid and durable immune-based protection against virulent strains of *S.* Typhimurium (Pasquali et al., 2008; Pesciaroli et al., 2011). The same strain proved to be attenuated, immunogenic and protective also in pigs (Gradassi et al., 2013; Pesciaroli et al., 2013). Similarly, it has been shown that vaccination with *Brucella znuA* mutants protects mice from *Brucella* infections (Yang et al., 2006; Clapp et al., 2011). In addition, it has been shown that ZnuD is able to elicit the production of antibodies triggering complement-mediated killing of several *Neisseria meningitidis* serogroup B strains, suggesting that it is a promising candidate for the generation of an effective vaccine (Hubert et al., 2013). Taken together, these studies suggest that zinc homeostasis offers interesting options to generate vaccines against different pathogenic bacteria.

#### **REFERENCES**


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Zalewski, P., Truong-Tran, A., Lincoln, S., Ward, D., Shankar, A., Coyle, P., et al. (2006). Use of a zinc fluorophore to measure labile pools of zinc in body fluids and cell-conditioned media. *BioTechniques* 40, 509–520. doi: 10.2144/06404RR02

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 01 October 2013; accepted: 11 December 2013; published online: 24 December 2013.*

*Citation: Cerasi M, Ammendola S and Battistoni A (2013) Competition for zinc binding in the host-pathogen interaction. Front. Cell. Infect. Microbiol. 3:108. doi: 10.3389/ fcimb.2013.00108*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2013 Cerasi, Ammendola and Battistoni. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## *Gaëlle Porcheron1,2‡, Amélie Garénaux1,2‡, Julie Proulx1,2, Mourad Sabri 1,2† and Charles M. Dozois 1,2,3\**

*<sup>1</sup> INRS-Institut Armand Frappier, Laval, QC, Canada*

*<sup>2</sup> Centre de Recherche en Infectiologie Porcine et Aviaire, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada*

*<sup>3</sup> Groupe de Recherche sur les Maladies Infectieuses du Porc, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada*

#### *Edited by:*

*Frédéric J. Veyrier, Institut Pasteur, France*

#### *Reviewed by:*

*Michael L. Vasil, University of Colorado School of Medicine, USA Laura Runyen-Janecky, University of Richmond, USA*

#### *\*Correspondence:*

*Charles M. Dozois, INRS-Institut Armand Frappier, 531, boul. des Prairies, Laval, QC, H7V 1B7, Canada e-mail: charles.dozois@iaf.inrs.ca*

#### *†Present address:*

*Mourad Sabri, Département de Biochimie de Microbiologie et Bio-Informatique, Faculté des Sciences et de Génie, Université Laval, Quebec, QC, Canada ‡These authors have contributed equally to this work.*

For all microorganisms, acquisition of metal ions is essential for survival in the environment or in their infected host. Metal ions are required in many biological processes as components of metalloproteins and serve as cofactors or structural elements for enzymes. However, it is critical for bacteria to ensure that metal uptake and availability is in accordance with physiological needs, as an imbalance in bacterial metal homeostasis is deleterious. Indeed, host defense strategies against infection either consist of metal starvation by sequestration or toxicity by the highly concentrated release of metals. To overcome these host strategies, bacteria employ a variety of metal uptake and export systems and finely regulate metal homeostasis by numerous transcriptional regulators, allowing them to adapt to changing environmental conditions. As a consequence, iron, zinc, manganese, and copper uptake systems significantly contribute to the virulence of many pathogenic bacteria. However, during the course of our experiments on the role of iron and manganese transporters in extraintestinal *Escherichia coli* (ExPEC) virulence, we observed that depending on the strain tested, the importance of tested systems in virulence may be different. This could be due to the different set of systems present in these strains, but literature also suggests that as each pathogen must adapt to the particular microenvironment of its site of infection, the role of each acquisition system in virulence can differ from a particular strain to another. In this review, we present the systems involved in metal transport by Enterobacteria and the main regulators responsible for their controlled expression. We also discuss the relative role of these systems depending on the pathogen and the tissues they infect.

**Keywords: metal transporters, iron, copper, zinc, manganese, Enterobacteria, regulation, virulence**

## **INTRODUCTION**

Metal ions such as iron, copper, zinc, and manganese, are involved in many crucial biological processes and are necessary for the survival of all living organisms. They are ubiquitously found in all organisms, nearly exclusively as constituents of proteins, including enzymes, storage proteins and transcription factors (Hood and Skaar, 2012). Due to the unique redox potential of some of these transition metals, many serve important roles as cofactors in enzymes and it is estimated that 30–45% of known enzymes are metalloproteins whose functions require a metal co-factor (Klein and Lewinson, 2011). However, transition metals are toxic at high intracellular concentrations, as they perturb the cellular redox potential and produce highly reactive hydroxyl radicals. Therefore, all organisms require mechanisms for sensing small fluctuations in metal levels to maintain a controlled balance of uptake, efflux, and sequestration and to ensure that metal availability is in accordance with physiological needs. This ability to sense metal ions is particularly important for bacterial pathogens to invade their hosts and cause disease. The ability of bacteria to colonize specific environments depends on their ability to obtain required nutrients. The strict requirement for these elements during pathogenesis is due to their involvement in processes ranging from bacterial metabolism to virulence factor expression (Waldron and Robinson, 2009; Kehl-Fie and Skaar, 2010). However, during infection, the host also produces proteins that are able to chelate metal ions and thus, can restrict the availability of essential metals from invading pathogens. Moreover, the toxicity of metals such as copper can be used as a host defense mechanism to promote bacterial killing. Nutrient limitation by the host and nutrient acquisition by pathogenic bacteria are therefore, crucial processes in the pathogenesis of bacterial infectious diseases. The host and the bacterial pathogen might thus, be envisioned as living through a constant competition for the essential metal nutrients. As a result of this competition, bacteria have developed sophisticated acquisition systems to scavenge essential metals from the environment. These include constitutively expressed or inducible low- and high-affinity transport systems for chelated or free metals. Moreover, efflux systems are used to eliminate the excess metal ions which might become toxic for the bacterial cell. Acquisition systems are up-regulated during metal starvation, and efflux pumps are activated when metals are in excess (Wakeman and Skaar, 2012).

The *Enterobacteriaceae* comprise a large family of Gramnegative bacteria that include pathogenic species such as pathogenic *Escherichia coli* and *Shigella* spp., *Salmonella enterica*, *Klebsiella pneumoniae,* and others. While *E. coli* is a member of the commensal intestinal flora, some *E. coli* strains have evolved pathogenic mechanisms to colonize humans and animals. *E. coli* strains can cause either intestinal infections (caused collectively by different types of Intestinal Pathogenic *E. coli* [IPEC]) or extraintestinal infections (caused by Extraintestinal Pathogenic *E. coli* [ExPEC]). Eight pathotypes of IPEC are currently described [see Clements et al. (2012) for review]. ExPEC strains contain 3 major pathotypes: UroPathogenic *E. coli* (UPEC), Neonatal Meningitis *E. coli* (NMEC), and Avian Pathogenic *E. coli* (APEC). These strains are responsible for urinary tract infections, meningitis in neonates and avian respiratory tract infections, respectively. ExPEC infections can also lead to septicaemia. ExPEC have an enhanced ability to cause infection outside of the intestinal tract and can infect the urinary tract, the bloodstream, and the cerebrospinal fluid of human and other animal hosts (Dho-Moulin and Fairbrother, 1999; Russo and Johnson, 2000). *Salmonella* is a major pathogen of both animals and humans, and is the cause of typhoid fever, paratyphoid fever, and the foodborne illness salmonellosis. *Salmonella* strains reach the gastrointestinal epithelium and trigger gastrointestinal diseases. They are able to invade the intestinal epithelium and to survive within phagocytes (Liu et al., 2013). *Shigella* species are responsible for bacillary dysentery. To infect their host, they have to be able to survive in the environment (such as contaminated water) as well as inside host epithelial cells (Payne et al., 2006). Seventeen different species of *Yersinia* have been reported, of which three have been shown to be pathogenic to humans and animals. These are *Y. enterocolitica* and *Y. pseudotuberculosis*, and the most virulent and invasive, *Y*. *pestis*. The latter causes highly fatal pneumonic, bubonic and septicemic plague, while the first two are responsible for a wide range of diseases ranging from mild diarrhea to enterocolitis (Mikula et al., 2012). *Klebsiella*, particularly *K. pneumoniae*, frequently cause human nosocomial infections. Nosocomial *Klebsiella* infections most commonly involve the urinary and respiratory tracts (Podschun and Ullmann, 1998). Members of the genus *Serratia*, particularly *Serratia marcesens*, cause important infections in humans, animals, and insects. *S. marcescens* is an opportunistic pathogen causing clinical diseases such as urinary tract infections and pneumonia (Mahlen, 2011). Several species of *Proteus* bacteria infect humans. The most frequently linked with human disease, *Proteus mirabilis*, is the causative agent of nosocomial and urinary tract infections (Jacobsen and Shirtliff, 2011). The genus *Cronobacter* is very diverse and comprises pathogens causing severe meningitis, septicemia, or necrotizing enterocolitis in neonates and infants (Grim et al., 2012). The *Enterobacteriaceae* family contains several other genera that are pathogenic, but they will not be described herein, as studies on metal transporters in these other genera are very limited.

This review will focus on the transport systems of four essential metals, i.e., iron, manganese, zinc, and copper, identified in pathogenic Enterobacteria, and the control between uptake and export of these metals which is needed to ensure physiological needs while countering metal toxicity. Moreover, as metal uptake and efflux are necessary steps for pathogens to invade their hosts, the role of these transporters in virulence of Enterobacteria is described.

#### **METAL TRANSPORT SYSTEMS IRON TRANSPORT SYSTEMS**

Iron is the most abundant transition metal in the host, but free ferrous iron (Fe2+) is extremely poorly available. Risk of infection is reduced by a strategy called "nutritional immunity," consisting in preventing pathogens from acquiring iron. Indeed, extracellular iron, mostly present in its ferric (Fe3+) form, is bound to circulating transferrin. In milk, tears, saliva or in the granules of polymorphonuclear leukocytes involved in mucosal innate immune response, ferric iron is also bound to lactoferrin. These proteins display high affinity for ferric iron. Haem also constitutes an important source of iron. It contains a single Fe2<sup>+</sup> atom encircled by a tetrapyrrole ring. It represents an important cofactor for haemoproteins such as haemoglobin, which is itself contained in circulating erythrocytes, rendering ferrous iron unavailable. If haemolysis occurs, free haemoglobin is bound by haptoglobin and free haem is bound to haemopexin (Johnson and Wessling-Resnick, 2012). In response to infection, a cascade of host signals leads to increased sequestration of iron. Production of Interleukin-6 by immune effector cells is triggered, leading to binding of proinflammatory cytokines to hepatocyte receptors and to increased expression of Acute Phase Proteins (APP) involved in nutritional immunity. Among these, hepcidin reduces release of iron into the circulation, ferritin promotes intracellular iron storage, and haptoglobin binds free haemoglobin (Parrow et al., 2013). Moreover, ferrous iron present in phagosomes is pumped out by Nramp1 (Hood and Skaar, 2012). Pathogenic bacteria use several strategies to acquire iron. These include import of ferrous iron by ATP- or GTP-dependent inner membrane transporters, and TonB-ExbB-ExbD dependent transport of ferric-siderophores, transferrins, haem or haem-bound proteins through specific outer membrane receptors (see **Figure 1**; Braun, 2001; Hood and Skaar, 2012).

### *Fe* **<sup>2</sup><sup>+</sup>** *transporters*

Free Fe2<sup>+</sup> is rarely present in the host, except under conditions where the redox potential or the pH are disturbed, such as ischemia caused by a trauma or following reduction of the environment by proliferating bacteria (Bullen et al., 2005). In bacteria, Fe2<sup>+</sup> enters the periplasm through non-specific porins and is delivered to the cytoplasm through different transporters.

Under anaerobic-microaerophilic conditions, bacteria use the FeoB pathway (Cao et al., 2007). Members of the FeoB-family mediate transport of free Fe2<sup>+</sup> across the inner membrane to the cytoplasm in a GTP-dependent manner. FeoB is located in the periplasmic membrane. It is expressed with FeoA and FeoC

**FIGURE 1 | Iron transporters in Enterobacteria and metal availability in the host during infection.** In a healthy individual, Fe3<sup>+</sup> is stored intracellularly in complex with ferritin (Fn), bound by serum transferrin (TF) or bound by lactoferrin (LTF) at mucosal surfaces. In the blood, Fe2<sup>+</sup> is complexed with haem, which is bound by haemoglobin (Hb) within red blood cells. During infection, haemolytic bacterial cytotoxins damage host cells, leading to the release of ferritin, while hemolytic toxins lyse erythrocytes, liberating Hb, thus, bound by haptoglobin (HP). Free haem is scavenged by haemopexin (HPX). Secreted bacterial siderophores can remove iron from transferrin, lactoferrin and ferritin, and siderophore-iron complexes are then recognized by cognate receptors at the bacterial surface. Similarly, secreted haemophores can remove haem from haemoglobin or haemopexin. Enterobacteria also possess receptors for free haem. Outer membrane receptors for haem

can also transport haem from haemoglobin, and HemR/HmuR can transport haem from haemopexin and haptoglobin. Enterobactin-mediated iron acquisition can be inhibited by the innate immune protein lipocalin-2 (NGAL, Neutrophil Gelatinase-Associated Lipocalin), which binds and sequesters siderophores (Skaar, 2010; Hood and Skaar, 2012). Transporter families are indicated below transporters. One representative outer membrane receptor that transports the ligand to the periplasm represents different siderophore transporters, the identity of the outer membrane receptor being shown at the bottom under the inner membrane transporter in brackets. ABC, ATP-binding cassette; ZIP, Zrt/Irt-like protein; NRAMP, natural resistance associated with macrophage protein; OFeT, oxidase-dependent iron transporter; ILT, iron/lead transporter superfamily; FeoB, ferrous iron uptake family; OM, outer membrane; PP, periplasm; IM, inner membrane; CP, cytoplasm.

of recently characterized functions (Braun, 2001; Kim et al., 2013; Lau et al., 2013). FeoB is produced by the majority of *Enterobacteriaceae* (Table S1; Fetherston et al., 2012; Grim et al., 2012; Hood and Skaar, 2012). In some *E. coli* strains, EfeUOB (YcdNOB) is also involved in Fe2<sup>+</sup> uptake under aerobic, lowpH, low-iron conditions (Cao et al., 2007). As EfeO is able to oxidize ferrous iron, it has also been proposed that EfeU could transport Fe3<sup>+</sup> (Rajasekaran et al., 2010). In *E. coli* K-12, EfeB and its paralog YfeX, widespread in Enterobacteria, may also promote iron extraction from periplasmic haem (Letoffe et al., 2009; Dailey et al., 2011). EfeUOB is also present in *Y. pestis* (Fetherston et al., 2012). An uncharacterized system called FetMP has also been identified in *E. coli* and *Y. pestis* as involved in ferrous iron uptake (Forman et al., 2010).

Inner membrane ATP-Binding Cassette (ABC) transporters can also import Fe2+. The ABC superfamily of transporters consists in several protein complexes which together are capable of transporting various solutes across membranes. They are composed of three or four different subunits, usually encoded by different genes grouped in an operon (Ma et al., 2009; Klein and Lewinson, 2011). The Mn2+/Fe2<sup>+</sup> ABC-transporters of Enterobacteria include the *Yersinia* YfeABCD system and its Sit homolog identified in *S.* Typhimurium, ExPEC, *K. pneumoniae*, *P. mirabilis* and *Shigella* spp. (Table S1; Angerer et al., 1992; Bearden and Perry, 1999; Kehres et al., 2002b; Sabri et al., 2008; Fisher et al., 2009; Himpsl et al., 2010).

#### *Siderophores and low molecular weight iron chelators*

Siderophores are small secreted molecules that display higher affinity for iron than the host iron-binding proteins such as transferrin and lactoferrin. They are synthesized in the cytoplasm and require specific export systems to reach the extracellular space. The export systems usually involve an inner membrane protein from the major facilitator family and an outer membrane channel protein such as TolC (Garénaux et al., 2011). In the extracellular space, iron-bound siderophores are recognized by specific receptors (Braun, 2001; Chakraborty et al., 2007). The TonB-ExbB-ExbD energy transduction system is required for transport of the ligand into the periplasmic space. While the inner-membraneembedded ExbD-ExbB complex tranduces energy from the proton motive force of the cytoplasmic membrane, the TonB protein spans the periplasm to transfer this energy to the outer membrane receptor (Higgs et al., 2002). Then, a specific ABC-transporter mediates entry of the iron-bound siderophores through the inner membrane. A chaperone may be involved in the transfer of the iron-bound siderophore from the outer membrane receptor to an inner membrane ABC-transporter. It is the case for ferric enterobactin and aerobactin siderophores, requiring the FepB and FhuD chaperones, respectively (Koster and Braun, 1990; Chenault and Earhart, 1991). Once in the periplasm, ferric iron can be directly reduced to its ferrous state by reductases, so as to be released from the siderophores and transferred to iron-dependent proteins. As such, in some cases, apo-siderophores can be recycled to the extracellular space without the need for new siderophore biosynthesis. In other cases where the redox potential of the ferri-siderophore is too high, degradation by specific esterases is required before the reductases can release iron (Miethke et al., 2011).

Nearly all *E. coli* and *K. pneumoniae* strains produce enterobactin (Bachman et al., 2011; Garénaux et al., 2011). Enterobactin (or enterochelin), is internalized through the FepA receptor and FepCDG ABC-transporter thanks to the FepB chaperone (Chenault and Earhart, 1991). Some strains also produce a set of pathogen-specific siderophores potentially comprising salmochelins, aerobactin or yersiniabactin (Bachman et al., 2011; Garénaux et al., 2011). Salmochelins, glycosylated forms of enterobactin, are internalized through the IroN receptor and the same ABC-transporter as enterobactin, FepCDG. Aerobactin uptake involves the IutA receptor as well as FhuBC ABCtransporter, while yersiniabactin uptake occurs via the Psn/FyuA receptor and the YbtPQ ABC-transporter (Koster and Braun, 1990; Perry and Fetherston, 2011). The types of siderophores produced are dependent on the pathotypes of the strains. While ExPEC strains are able to synthesize up to four different siderophores, IPEC such as O157:H7 strains only produce enterobactin. Some non-O157 IPEC strains and commensals may also produce aerobactin (Kresse et al., 2007), and IPEC enteroaggregative strains also contain yersiniabactin- and aerobactin-encoding genes (Okeke et al., 2004). Likewise, all *Shigella* isolates produce siderophores, namely enterobactin, salmochelins or aerobactin, but the types of siderophores produced vary from one species to another (Payne et al., 2006). Salmochelins were first identified in *Salmonella*, which also produces enterobactin. Some *Salmonella* strains also produce yersiniabactin or aerobactin (Carniel, 2001; Muller et al., 2009; Izumiya et al., 2011). Siderophores produced by the different pathogenic Enterobacteria are summarized in Table S1.

Other siderophores have been identified in Enterobacteria that do not produce enterobactin. The genome of *Cronobacter* species contains a non-functional enterobactin gene cluster and a plasmid-encoded aerobactin cluster renamed cronobactin (Grim et al., 2012). Virulent *Yersinia* species are known to produce yersiniabactin as well as the alternative yersiniachelin and pseudochelin siderophores (Rakin et al., 2012). *P. mirabilis* produces proteobactin and a yersiniabactin-like siderophore (Himpsl et al., 2010).

In addition to the different siderophores produced by *E. coli* strains, it is not unusual to find more than 10 genes encoding distinct siderophore receptors in their genomes. In non-pathogenic *E. coli*, enterobactin can be internalized through 3 different TonBdependent receptors: FepA, Cir, and Fiu (Andrews et al., 2003). The Iha TonB-dependent receptor mediates enterobactin uptake in UPEC (Leveille et al., 2006). Another TonB-dependent receptor identified in UPEC, IreA, is an additional potential siderophore transporter (Russo et al., 2001, 2002). Many Enterobacteria are able to internalize exogenous siderophores, such as the fungal siderophore ferrichrome, internalized through FhuABCD (Andrews et al., 2003). Low molecular weight iron chelators such as citrate can also be used as a source of iron. The typical ferric citrate transport system involves the FecA TonB-dependent receptor, the FecB chaperone and the FecCDE ABC-transporter at the inner membrane (Braun et al., 2003). These transport genes form the *fecABCDE* operon (Mahren et al., 2005). The *S. marcescens* SfuABC, the *K. pneumoniae* KfuABC, the *E. coli* and *Cronobacter sp* EitABC and the *Y. pestis* YfuABC and YiuABC systems transport ferric iron bound to small chelators across the cytoplasmic membrane (Angerer et al., 1990; Gong et al., 2001; Ma et al., 2005; Johnson et al., 2006; Kirillina et al., 2006; Grim et al., 2012).

This redundancy in siderophore-mediated iron acquisition systems suggests that their fundamental role lies in adaptation to different iron-limited niches in which bacteria are competing with other microorganisms in the environment or with host proteins for iron acquisition (Valdebenito et al., 2006).

#### *Haem uptake*

Haem represents one of the most abundant iron sources inside the host. Organization of iron in haem allows iron solubilization, but also enhances its catalytic activity by 5 to 10 times, making it an even more efficient cofactor but increasing its toxicity (Anzaldi and Skaar, 2010). Pathogens have evolved direct haem uptake systems, as well as haemophore systems.

#### *Direct uptake of haem or haemoproteins*

As haem is not freely available, bacteria secrete exotoxins such as haemolysins, proteases or cytolysins to release haem for direct uptake. Certain proteins of SPATE family (serine proteinase autotransporters of *Enterobacteriacae*), degrade haemoglobin to allow pathogenic bacteria to gain access to haem (Krewulak and Vogel, 2008). Haem and haemoproteins bind to specific TonB-dependent cell surface receptors, where haem contained in haemoproteins is extracted and transported to the periplasm. It is further imported by ABC transporters from the periplasm to the cytoplasm. In the cytoplasm, iron is released from haem through degradation by haem oxygenases (Hood and Skaar, 2012). *S. dysenteriae* encodes a haem transport system composed of *shuA* (coding for the haem receptor), *shuT*, *U* and *V* (coding for a periplasmic chaperone and an ABC-transporter) as well as *shuS* (which protects against haem toxicity by allowing its intracellular processing) and *shuWXY* coding for proteins of unknown functions (Wyckoff et al., 2005). Although the *shu* locus is only found in *S. dysenteriae,* other *Shigella* use haem as the sole iron source, suggesting another haem transport system (Payne et al., 2006). The Shu system is found in some *E. coli* strains including IPEC O157:H7 or UPEC CFT073 (Chu), *Y. pestis* (Hmu), *P. mirabilis* (Hmu) and *S. marcescens* (Hem) (Table S1), suggesting that horizontal transfer might have occurred (Thompson et al., 1999; Payne et al., 2006; Hagan and Mobley, 2009; Benevides-Matos and Biville, 2010; Himpsl et al., 2010). In UPEC, an additional haem receptor, Hma, has also been identified (Hagan and Mobley, 2009).

### *Haemophores*

HasA-type haemophores were first identified in *Serratia marcescens* but are also conserved in other Gram-negative bacteria such as *Yersinia pestis* and *Y. pseudotuberculosis* (Table S1; Ghigo et al., 1997; Rossi et al., 2001). Haemophores are proteins with higher affinity for haem than haem-containing proteins such as haemopexin or myoglobin. The *S. marcescens* Haem Acquisition System (=Has) obtains haem through different steps. First, HasA haemophores are secreted through a Type I Secretion System (TISS). The SecB general chaperone is required to maintain HasA in a secretion-competent state and to facilitate its secretion. Once in the extracellular space, HasA acquires haem from the host haem carrier proteins due to its higher affinity, regardless of its redox state (Fe2<sup>+</sup> or Fe3+). Then, haem-containing HasA is recognized by the HasR specific TonBdependent receptor. In the presence of the HasB-ExbB-ExbD energy transduction system, interaction of haem-bound HasA with HasR leads to a conformational change allowing haem transfer to HasR and apo-HasA release in the extracellular space for recycling (Cescau et al., 2007). HasB is a TonB homolog dedicated to HasR (De Amorim et al., 2013). In *S. marcescens*, the Hem haem uptake system is active at low haem concentrations. Even lower iron concentrations are required for activation of the Has haemophore system, suggesting that these two systems might be involved in haem acquisition under different conditions (Benevides-Matos and Biville, 2010).

## *Iron export*

If studies on iron homeostasis in enterobacteria mainly focus on acquisition systems, the potential role of iron efflux in virulence should also be considered. Indeed, in *S.* Typhimurium, oxidative stress caused by an iron overload is encountered after macrophage invasion. Enterobactin production and iron-citrate efflux have been shown to confer oxidative stress resistance in those conditions (Frawley et al., 2013).

#### **COPPER TRANSPORT SYSTEMS**

Cupric copper (Cu2+) is one of the most stable divalent transition metals and displays high affinity for metalloproteins. If equivalent quantities of all divalent metals were present, proteins would probably all bind copper (Waldron and Robinson, 2009). However, bacteria emerged without atmospheric O2. Under these conditions, copper was not soluble. As a consequence, Cubinding proteins represent less than 0.3% of their annotated proteome. As opposed to bacteria, eukaryotic genomes evolved in the presence of copper and present a higher percentage of genes coding for Cu-binding proteins (Dupont et al., 2011). In bacteria, copper is used as a catalyzer for electron transfer reactions in some metalloenzymes involved in electron transfer reactions, such as cytochrome oxidase. It is also used as a cofactor by copperdetoxifying enzymes (Dupont et al., 2011; Hodgkinson and Petris, 2012). However, intracellular copper levels must be finely controlled, due to its toxicity. Under anaerobic conditions, copper is mainly present in the highly reactive cuprous form (Cu1+). Copper directly disrupts protein structures by forming thiolate bonds with iron-sulfur clusters. Degradation of iron-sulfur clusters results in an increase in free iron, which indirectly increases oxidative stress. In addition, copper interacts with polypeptide backbones and interferes with binding of some cofactors to specific amino acids (Dupont et al., 2011; Hodgkinson and Petris, 2012; Park et al., 2012). In minimal culture medium, intracellular copper in *E. coli* is low (10−<sup>6</sup> M), but higher than extracellular concentration (10−<sup>8</sup> M) (Outten et al., 2001).

### *Copper uptake*

It is still unclear how copper enters the bacterial cytoplasm. Uncharacterized energy-independent channels such as OmpC porins probably allow passage of Cu2<sup>+</sup> and Cu1<sup>+</sup> through the outer membrane (Rensing and Grass, 2003). In *E. coli*, permeability to copper is reduced by the ComC outer membrane protein through an unknown mechanism. ComC expression decreases under low copper conditions to allow copper uptake. Homologs of ComC are present in many Gram-negative bacteria, suggesting that it might play an important role in copper homeostasis (Rademacher and Masepohl, 2012). Only Cu1<sup>+</sup> ions enter the cytoplasm by an unknown mechanism, potentially involving Zn2<sup>+</sup> uptake systems or some ATPases (Ma et al., 2009; Hood and Skaar, 2012; Nies and Herzberg, 2013). However, in the case of pathogenic Enterobacteria, copper efflux is more crucial than copper uptake. To prevent copper from competing with natural ligands of metalloproteins, unbound copper is excluded from the cytoplasm. In bacteria, all copper-dependent proteins are located in the periplasm or within the cytoplasmic membrane with binding sites in the periplasm (Ma et al., 2009; Dupont et al., 2011; Hodgkinson and Petris, 2012; Nies and Herzberg, 2013). The copper tolerance response in Gram-negative bacteria involves three different mechanisms: (1) exporting cytoplasmic copper to the periplasm using P1B-type ATPases, (2) detoxifying copper using the CueO multicopper oxidase, or (3) eliminating unbound periplasmic copper by exporting it or sequestering it (Dupont et al., 2011; Hodgkinson and Petris, 2012).

#### *Copper export*

In Gram-negative bacteria, inner membrane heavy metal pumps (P1B-type ATPases) export cytoplasmic copper to the periplasm. These pumps are monomers and their structure includes a group of three cytoplasmic domains responsible for the ATPase activity. The phosphorylation cycle responsible for ATPase activity induces conformational changes resulting in substrate translocation through the inner membrane (Gourdon et al., 2011; Klein and Lewinson, 2011).

In *E. coli*, the CopA ATPase is involved in Cu1<sup>+</sup> export from the cytoplasm to the periplasm (**Figure 2**; Arguello et al., 2011). In *Salmonella*, copper ATPases are not only required for copper resistance, but also for efficient copper availability to cupro-enzymes such as Cu/Zn SodCII under copper-limited conditions (Osman et al., 2013). They might play a role in delivering cytoplasmic copper contained in unidentified storage compounds (Nies and Herzberg, 2013). Once in the periplasm, highly reactive Cu1<sup>+</sup> is detoxified through re-oxidation by the CueO multicopper oxidase to form less harmful Cu2<sup>+</sup> (Dupont et al., 2011; Nies and Herzberg, 2013).

In some enterobacteria such as *E. coli*, the CusCBA heteromultimeric transport complex can transfer periplasmic copper to the extracellular milieu (Outten et al., 2001; Gudipaty et al., 2012). Using the proton-motive force, the CusCBA complex shuttles between three conformational states to act as a peristaltic pump and excrete copper (Dupont et al., 2011). The periplasmic transfer of Cu1<sup>+</sup> from the P1B-ATPases to the CusB adaptor protein is achieved by the CusF chaperone to limit its potential toxic effects (Hodgkinson and Petris, 2012; Nies and Herzberg, 2013). This Cus system is also present in *K. pneumoniae* (Zulfiqar and Shakoori, 2012). Other chaperones have been described in some *E. coli* strains, such as plasmidic PcoC or PcoE (Espirito Santo et al., 2008).

*Salmonella* and *Yersinia* lack the CusCBA system to export Cu1<sup>+</sup> from the periplasm to the extracellular milieu. Alternatively, another periplasmic protein, CueP, sequesters copper to neutralize toxicity (Pontel and Soncini, 2009; Osman et al., 2010; Dupont et al., 2011). This chaperone also helps supply copper to SodCII, (Nies and Herzberg, 2013; Osman et al., 2013). *S.* Typhimurium uses two different P1B-type ATPases, GolT, and CopA (Dupont et al., 2011; Nies and Herzberg, 2013). Initially described as a gold efflux system, GolT is predominantly involved in copper efflux (Osman et al., 2013). *S.* Typhimurium expresses a CueO multicopper oxidase, also called CuiD, which buffers copper toxicity in the periplasm (Arguello et al., 2011; Hodgkinson and Petris, 2012). Periplasmic Cu2<sup>+</sup> is efficiently reduced to Cu1+by NADH dehydrogenase 2 and components of the respiratory chain. Reoxidation by multicopper oxidases ensures Cu2<sup>+</sup> availability for copper-dependent enzymes (Nies and Herzberg, 2013).

Iron and copper homeostasis are linked, as some siderophores interact with copper. *In vitro*, catecholate siderophores increase copper sensitivity in *E.coli*, reducing Cu2<sup>+</sup> to generate toxic Cu1+. The CueO multicopper oxidase is essential, as it allows oxidation of catecholates to create molecules able to sequester Cu2<sup>+</sup> instead of reacting with it (Grass et al., 2004). However, in *S.* Typhimurium, a *cueO* mutant does not display higher sensitivity to siderophores (Achard et al., 2010). Conversely, yersiniabactin production increases copper resistance, as it sequesters Cu2<sup>+</sup> before it interacts with catecholate siderophores to produce highly toxic Cu1<sup>+</sup> (Chaturvedi et al., 2012).

#### **ZINC TRANSPORT SYSTEMS**

Zinc is an essential transition metal in all organisms, playing an important catalytic and structural role in a number of proteins. When relative abundance is considered, zinc represents the second most important transition metal ion in living organisms after iron. In contrast to other transition metal ions, zinc does not undergo redox reactions. Bacteria are predicted to incorporate zinc into 5–6% of all proteins (Andreini et al., 2006). Zinc plays a role in bacterial gene expression, general cellular metabolism and acts as a cofactor of virulence factors. Zinc proteins are involved in DNA replication, glycolysis, pH regulation and the biosynthesis of amino acids, extracellular peptidoglycan and low molecular weight thiols, and as a result, zinc status is linked to maintenance of the intracellular redox buffering of the cell. The apparent concentration of zinc in *E. coli* is 10−<sup>4</sup> M (Outten and O'halloran, 2001). Procuring sufficient zinc to sustain growth during infection is a considerable challenge for bacterial pathogens. Serum levels of zinc are in the micromolar range, and the metal's bioavailability is further restricted because it is tightly bound to proteins. As with iron, mammals sequester zinc systematically and locally in an attempt to deprive invading pathogens of this critical micronutrient (Desrosiers et al., 2010). While zinc is an essential nutrient, excess zinc is toxic to the cell, possibly through inhibition of key enzymes and competition with other relevant metal ions (Wang and Fierke, 2013). Bacterial cells thus need to achieve a delicate balance between ensuring sufficient concentrations of zinc to fulfill essential functions while limiting concentration to prevent toxic effects. In Enterobacteria, zinc homeostasis is mediated primarily by a network of zinc influx and efflux pumps (**Figure 2**; Wang et al., 2012).

### *Zinc uptake*

The transport of Zn2<sup>+</sup> across the outer membrane is not defined in Enterobacteria. Zinc uptake across the cytoplasmic membrane is mediated by two major types of transporters: ZnuACB, which belongs to the cluster C9 family of (TroA-like) ABC transporters, and ZupT, which is a member of the ZIP (ZRT/IRT-like protein) family of transporters that are also present in eukaryotes

where it binds Cu2+. The complex is then released in blood to bring Cu2<sup>+</sup> to tissues. Following phagocytosis of bacteria in macrophages, interferon-γ induces the import of Cu+ inside the phagolysosome to induce bacterial killing. However, pathogenic Enterobacteria have several systems to detoxify their periplasm or cytoplasm (Hood and Skaar, 2012). Transporter families are indicated in brackets. NRAMP, natural resistance associated with macrophage protein; ABC, ATP-binding cassette; MntP, manganese transporter efflux pump family; ZIP, Zrt/Irt-like protein; MFS, Major Facilitator Superfamily; RND, resistance and nodulation; CDF, cation diffusion facilitator; OM, outer membrane; PP, periplasm; IM, inner membrane; CP, cytoplasm.

(Hantke, 2005). ZnuACB is a high-affinity transporter whereas ZupT is a low-affinity uptake system (Hantke, 2005). Under conditions of moderate zinc availability, zinc uptake is carried out by ZupT, whereas it is carried out by ZnuACB in environments characterized by very low zinc availability.

The gene *znuA* encodes for the periplasmic, zinc-binding component of the transporter, *znuB* encodes for the transmembrane component and *znuC* encodes for the ATPase subunit. Zinc uptake mediated by the Znu system requires ATP hydrolysis by dimeric ZnuC to transport Zn2<sup>+</sup> captured by ZnuA through the pore formed by a ZnuB dimer in the cytoplasmic membrane (Patzer and Hantke, 1998, 2000). In Enterobacteria, numerous studies in different strains of *E. coli*, in *S.* Typhimurium, *S.* Enteritidis, *Proteus mirabilis* or in different species of *Yersinia* have demonstrated that *znuA*, *znuC*, *znuB* or *znuACB* mutants had decreased Zn2<sup>+</sup> uptake compared to the wild-type strains (Table S1; Patzer and Hantke, 1998; Campoy et al., 2002; Ammendola et al., 2007; Gunasekera et al., 2009; Sabri et al., 2009; Desrosiers et al., 2010; Nielubowicz et al., 2010; Gabbianelli et al., 2011).

In some bacterial species such as IPEC strain O157:H7 and *S.* Typhimurium, zinc uptake involves another protein, ZinT (formerly known as YodA). This protein is involved in periplasmic zinc binding under zinc-limiting conditions, and studies carried out in *S.* Typhimurium have suggested that ZinT participates in zinc uptake through ZnuACB, by a mechanism involving direct interaction with ZnuA (Petrarca et al., 2010; Gabbianelli et al., 2011).

Proteins of the ZIP family were initially identified as iron or zinc transporters in eukaryotes, but some members were subsequently shown to also transport other metals, such as manganese or cadmium. ZupT is the first characterized bacterial member of this family and was shown to be responsible for zinc uptake in *E. coli* (Grass et al., 2002). Studies on the role of ZupT on Zn2<sup>+</sup> uptake have been performed in non-pathogenic *E. coli* and in the UPEC strain CFT073 (Table S1; Grass et al., 2002; Sabri et al., 2009). The *zupT* gene is constitutively expressed and ZupT can transport iron and cobalt in addition to zinc and possibly manganese. When overexpressed, ZupT can also transport copper (Grass et al., 2002, 2005).

#### *Zinc export*

Zinc detoxification is primarily achieved by the P1B-type ATPase ZntA and the cation diffusion facilitators (CDF) ZitB and YiiP (Table S1; Rensing et al., 1997; Grass et al., 2001; Wei and Fu, 2006). P1B-type ATPases and CDF transporters catalyze metal translocation across the inner membrane, and the substrate is transported from the cytoplasm to the periplasm (Klein and Lewinson, 2011). CDF is a ubiquitous family of metal transporters found in prokaryotes and eukaryotes. Functional analysis of YiiP and ZipT indicated that these two proteins are protonlinked antiporters that utilize the free energy derived from H+ influx to pump cytosolic Zn2<sup>+</sup> out of the cells (Chao and Fu, 2004; Wei and Fu, 2006).

In addition to Zn2+, ZntA is able to transport Cd2<sup>+</sup> and Pb2<sup>+</sup> (Binet and Poole, 2000). It has been proposed in *S.* Typhimurium that the periplasmic C-terminal domain of ZraP, a periplasmic protein with two zinc-binding domains, facilitates modulation of transporters such as ZntA (Appia-Ayme et al., 2012). It has been suggested that ZntA is critical for the survival of *E. coli* in the presence of high zinc concentrations, while ZitB maintains zinc homeostasis under normal growth conditions, i.e., low environmental zinc stress (Rensing et al., 1997; Wang et al., 2012). Overexpression of *zitB* in *E. coli* resulted in a significant increase in zinc tolerance and reduced uptake of zinc, while overexpression of *yiiP* did not confer additional zinc resistance, and deletion of *yiiP* did not alter zinc resistance (Grass et al., 2001).

A recent study identified new zinc exporters in *E. coli*, MdtABC, a RND-type efflux pump, and MdtD, a MFS (Major Facilitator Superfamilly) transporter, as well as a periplasmic protein, Spy, involved in zinc detoxification (Table S1). It has been proposed that the role of Spy in relieving zinc stress may be to facilitate the folding and to protect the integrity of transmembrane and periplasmic transporters that function in zinc export (Wang and Fierke, 2013).

#### **MANGANESE TRANSPORT SYSTEMS**

Manganese plays an essential role in many cellular processes including lipid, protein, and carbohydrate metabolism. It also contributes to protection against oxidative stress, is a cofactor for a number of enzymes in bacteria and other organisms, and can also contribute directly to the catalytic detoxification of reactive oxygen species (ROS) (Horsburgh et al., 2002; Kehres and Maguire, 2003). The total Mn2<sup>+</sup> concentration in *E. coli* is comparable to that of Cu+ and is about 10-fold lower than that of Zn2<sup>+</sup> (Ma et al., 2009). Like iron, manganese is found in two states, Mn2<sup>+</sup> and Mn3+, Mn2<sup>+</sup> being used by biological systems. Contrary to Fe2+, free Mn2<sup>+</sup> is not toxic in a biological environment (Kehres and Maguire, 2003). In some bacteria, Mn2<sup>+</sup> can replace the more reactive Fe2<sup>+</sup> in Fe2+-containing proteins, reducing oxidative damage to these proteins (Hood and Skaar, 2012). The regulation of manganese homeostasis is complex and appears to overlap with peroxide defenses and iron homeostasis in bacteria (Horsburgh et al., 2002).

#### *Manganese uptake*

As for Zn2+, the mechanisms of Mn2<sup>+</sup> transport across the outer membrane are not yet defined in Enterobacteria. For import across the cytoplasmic membrane, two major manganese transporters have been identified: a proton-dependent Nramp-related transport system typified by MntH and an ABC transporter typified by SitABCD and YfeABCD (**Figure 2**; Goswami et al., 2001; Forbes and Gros, 2003). The Nramp (Natural resistanceassociated macrophage protein) transporter family was first described in plants, animals, and yeasts (Cellier et al., 1996). Mammalian Nramp1 and Nramp2 are H+-dependent transition metal divalent cation transporters with physiologically relevant affinities for at least Mn2+, Fe2+, and Zn2<sup>+</sup> (Papp-Wallace and Maguire, 2006).

MntH (for Proton-dependent manganese transporter) has been characterized in many enterobacterial species (Table S1; Makui et al., 2000; Boyer et al., 2002; Zaharik et al., 2004; Runyen-Janecky et al., 2006; Champion et al., 2011; Perry et al., 2012). Affinity studies for Mn2<sup>+</sup> have shown that the apparent K0*.*<sup>5</sup> for Mn2<sup>+</sup> uptake was 0.1μM in *S.* Typhimurium and 0.5 to 1μM in *E. coli*. The K0*.*<sup>5</sup> for Fe2<sup>+</sup> was about 100μM, far higher than physiological concentrations of Fe2<sup>+</sup> (Kehres et al., 2000).

High-affinity Mn2<sup>+</sup> acquisition can also be mediated by ABC transporters. The SitABCD transporter, present in some pathogenic *E. coli* strains, was first described in *S.* Typhimurium (Table S1; Zhou et al., 1999). In *S.* Typhimurium, SitABCD is primarily a Mn2<sup>+</sup> transporter rather than a Fe2<sup>+</sup> transporter. It mediates influx of Mn2<sup>+</sup> with a K0*.*<sup>5</sup> of 0.1μM, while it mediates influx of Zn2+, Cd2+, and Fe2<sup>+</sup> with affinities of 3, 3 and 30μM, respectively. Moreover, it operates optimally in slightly alkaline medium, whereas MntH seems to be more effective in acid medium, which suggests distinct physiological roles (Kehres et al., 2002b). In the APEC strain χ7122, the SitABCD transporter has affinities for Mn2<sup>+</sup> and Fe2<sup>+</sup> of about 4 and 1μM, respectively. However, the affinities changed in accordance with the genetic background of the strain. In a *mntH* mutant, manganese was better transported than iron; in an *aroB feoB* mutant iron was better transported than manganese. SitABCD is thus a highly versatile and adaptable transporter (Sabri et al., 2006). The SitABCD transporter in *S. flexneri* or YfeABCD system in *Y. pestis* is also able to transport Mn2<sup>+</sup> and Fe2<sup>+</sup> (Bearden and Perry, 1999; Runyen-Janecky et al., 2006).

#### *Manganese export*

As manganese is essential for enzymatic catalysis and protection against oxidative stress, molecular mechanisms of manganese toxicity are not yet clear. A manganese efflux pump, MntP, has been recently described in *E. coli* K-12 (Waters et al., 2011), and this transporter seems to be present in other pathogenic Enterobacteria (Veyrier et al., 2011).

### **REGULATION OF GENES ENCODING METAL TRANSPORT SYSTEMS**

Genes encoding metal transporters must be tightly regulated in bacteria. Indeed, metals are toxic when present at high concentrations. Moreover, as uptake systems are not needed in all niches, it would be energetically costly to produce them anytime. They have thus to be tightly regulated to respond to the appropriate nutrient present. Regulation of metal transport systems thus, occurs primarily by metal-responsive transcriptional regulators that repress metal uptake and activate metal efflux when metal is abundant, and activate acquisition when metal is scarce. Repression of metal transport systems is thus a good way to limit metal toxicity in the presence of excess metal and to limit the energy expenses. As a pathogen encounters various environments with various metal availabilities during the course of infection, it should be able to respond precisely to generate the appropriate physiological response. Moreover, intracellular concentrations of iron, copper, zinc, and manganese are higher than extracellular concentrations of these metals. For instance, intracellular concentrations of iron, copper, and zinc in *E. coli* are 100-fold, 1000-fold and 10,000-fold higher than a chemically defined culture medium, respectively (Ma et al., 2009). Within the host, the free serum iron concentration is about 10−<sup>24</sup> M, while bacteria need to maintain an intracellular iron concentration between 10−<sup>5</sup> and 10−<sup>7</sup> M (Garénaux et al., 2011). Bacteria thus, need highly sensitive regulatory factors responding to metal concentration to allow sufficient expression of metal uptake systems, and to ensure their physiological needs in metal nutrients.

#### **REGULATION OF IRON UPATKE SYSTEMS**

The principal regulator of iron transport sytems in Enterobacteria is Fur. The Fur family of regulatory proteins is named for the *E. coli* Fe-regulated uptake repressor Fur which regulates transcription of about 90 coding and non-coding RNAs mainly related to iron homeostasis (Ma et al., 2009). As Fur also represses oxidative stress, acid resistance and virulence genes that are required for survival under infection conditions, it has been suggested that iron deprivation inside the host might constitute a signal triggering virulence factors in pathogens (Payne et al., 2006). Fur-like repressors form homodimers and display negligible affinity for the DNA operator in the apo-form, but act as transcriptional repressors by tightly binding to the DNA operator in the presence of their cognate metal ion effectors (Pennella and Giedroc, 2005; Ma et al., 2009). Fur is activated *in vitro* when iron exceeds 10−<sup>6</sup> M (Waldron and Robinson, 2009). All of the above-mentioned iron acquisition systems are repressed by Fur (**Table 1**).

Some Fur-regulated genes are overexpressed under iron-rich conditions (Braun, 2001). This might occur indirectly through the action of the Fur-regulated small RNA (sRNA) RyhB. sRNAs regulate target mRNAs by direct base pairing to positively or negatively affect their translation and stability (Storz et al., 2011). RyhB, an iron-regulated sRNA, regulates genes involved in iron metabolism. RyhB is repressed by Fur under iron-rich conditions and induced by iron starvation when Fur becomes inactive. RyhB promotes siderophore production and represses ironusing proteins (Masse and Gottesman, 2002; Masse et al., 2005). Excellent reviews focusing on the role of *ryhB* have been published recently (Salvail and Masse, 2012; Oglesby-Sherrouse and Murphy, 2013).

Regulation of synthesis and transport of some siderophores is also under the control of pathway-specific regulators. The yersiniabactin cluster is regulated by YbtA, an AraC-type transcriptional regulator. In the presence of ferri-yersiniabactin, YbtA represses its own transcription but activates transcription of yersiniabactin genes (Perry and Fetherston, 2011). Genes involved in ferric citrate and haemophore uptake are activated by a signaling cascade involving the membrane receptor as well as sigma-antisigma interactions. This signal transduction cascade is dependent on TonB (Braun et al., 2003; Biville et al., 2004; Mahren et al., 2005). In the case of the *fecABCDE* transport genes, the binding of ferric citrate to the FecA receptor creates a signal transmitted to the cytoplasmic membrane regulator FecR, which in turn activates the sigma factor FecI, directing the RNA polymerase to the promoter of the *fecABCDE* operon (Braun et al., 2003; Mahren et al., 2005). Similarly, transcription of the haemophore *has* cluster is directed by HasI sigma factor, itself inactivated by HasS antisigma factor and activated by the HasR receptor in the presence of haem-loaded haemophores (Biville et al., 2004).

Iron homeostasis does not only respond to strict iron concentrations, but also integrates a variety of signals to protect the cells against metal toxicity. Two-component regulatory systems (TCRS) regulate gene expression or protein function by responding to various environmental signals. In *Salmonella*, PhoPQ promotes Fe2<sup>+</sup> uptake via the response regulator RstA that activates transcription of *feoB* in acidic pH (Choi et al., 2009). Some other TCRS do not directly regulate genes involved in metal transport, but their responses to metals can change metal availability to the cell, thus, interfering with metal transporter activation or repression. For instance, the BasS-BasR system, sensing both iron and zinc, modifies the membrane structure by inducing lipopolysaccharide remodeling, activates genes involved in membrane-associated functions (such as porins) and acts on stress-responsive regulatory proteins, such as the CsgD biofilm formation regulator, indirectly conferring resistance to metal ions (Ogasawara et al., 2012). The iron uptake transporter *efeUOB* is repressed at high pH by dephosphorylated CpxR in response


#### **Table 1 | Characterized iron transport systems involved in virulence of Enterobacteria.**


*ND, Not Determined.*

*involved in virulence, involved with another transport system (indicated in the box), not involved.*

to both copper and acidity (Cao et al., 2007). Interestingly, the PmrAB TCRS is the first example of a signal transduction cascade responding to extracytoplasmic Fe3+, and Zn2<sup>+</sup> activates this system in *E. coli* but not in *S. enterica* (Chen and Groisman, 2013). Finally, the presence of oxygen also regulates iron uptake. In *S. flexneri*, anaerobic conditions activated *feoABC* while repressing genes encoding for SitABCD and the aerobactin synthesis and transport system. FeoABC was activated by the anaerobic regulators FnR and ArcA, whereas aerobactin genes were repressed by ArcA. Transcription of *fur* itself was repressed by ArcA under anaerobic conditions (Boulette and Payne, 2007).

#### **REGULATION OF COPPER TRANSPORT SYSTEMS** *Regulation of copper uptake systems*

The ComC outer membrane protein is under the control of the TetR-like ComR repressor. In the presence of high copper concentrations, Cu2<sup>+</sup> binds to ComR and releases it from the *comC* promoter region, leading to its activation (Mermod et al., 2012).

#### *Regulation of copper export systems*

The copper ATPase CopA, the multicopper oxidase CueO and the periplasmic copper-binding protein CueP are regulated by CueR, belonging to the MerR family of regulators (Outten et al., 2000; Stoyanov et al., 2001; Osman et al., 2013). The MerR family contains nearly exclusively transcriptional activators of the expression of genes required for metal efflux or detoxification, or defense against oxidative stress and drug resistance. Both the apo- and effector-bound forms are capable of binding to their operator DNA sequences with similar affinities. However, binding of the metal ion provokes an allosteric change at the DNA-binding domain of the protein, so that only the effector-bound form can significantly optimized RNA polymerase binding and transcription initiation (O'halloran et al., 1989; Brown et al., 2003). CueR has a copper affinity of 10−<sup>21</sup> M (Changela et al., 2003), and is able to induce the expression of its target genes in response to Cu+, Ag+, and Au+ (Ma et al., 2009; Perez Audero et al., 2010). GolS is a CueR-like sensor and the regulator of the P-type ATPase GolT (Osman et al., 2013). GolS and CueR have similar affinities for Cu+, but *in vivo* GolS distinguishes Au+ from Cu+ or Ag+ to activate its target genes (Ibanez et al., 2013; Osman et al., 2013).

The copper efflux genes *cusCFBA* are induced by the CusSR TCRS which responds to extracellular copper concentrations (Gudipaty et al., 2012). The importance of the Cus system is also dependent on the presence of oxygen. In *E. coli*, the Cus system is not required for copper resistance under aerobic conditions, in which the primary line of defense, the CueR regulon, is sufficient (Outten et al., 2001). However, in *K. pneumonia*, the Cus system is activated under both aerobic and anaerobic conditions and the activation level is higher under aerobic conditions (Zulfiqar and Shakoori, 2012). These differences might be explained by the lower capacity of *E. coli* to store copper under anaerobic conditions (Zulfiqar and Shakoori, 2012).

#### **REGULATION OF ZINC TRANSPORT SYSTEMS** *Regulation of zinc uptake systems*

The zinc uptake system ZnuACB is regulated by Zur, belonging to the Fur family, and by SoxR, belonging to the MerR family of regulators. This system is repressed by Zn-Zur in zinc-rich environment and, under zinc depletion conditions, Zur becomes inactive, leading to the activation of *znuACB* (Li et al., 2009). Zur is able to sense zinc at concentrations as low as 10−<sup>15</sup> M *in vitro* (Waldron and Robinson, 2009). SoxR responds to oxidative stress and is part of the SoxRS regulon. SoxS, activated by SoxR, activates the expression of this regulon containing numerous genes such as *znuACB* (Brown et al., 2003; Warner and Levy, 2012). A network biology approach has predicted that RybA sRNA might regulate ZupT uptake system in *E. coli* (Modi et al., 2011).

### *Regulation of zinc export systems*

The ZntA export system is regulated by the MerR-like ZntR regulator. The apo-ZntR dimer binds to the promoter of *zntA* and weakly represses transcription; Zn-ZntR is a transcriptional activator (Wang et al., 2012). ZntR can also bind Pb2<sup>+</sup> and Cd2<sup>+</sup> to activate *zntA* expression (Binet and Poole, 2000). As Zur, ZntR has zinc affinity of 10−<sup>15</sup> M *in vitro* but, *in vivo*, ZntR up-regulates *zntA* transcription in response to nanomolar intracellular concentrations of free zinc (Outten and O'halloran, 2001; Wang et al., 2012). Finally, ZntA is up-regulated under high zinc stress by ZntR to effectively lower the intracellular zinc concentration, while the other export system, ZitB is constitutively expressed to function as a first-line defense against zinc influx (Wang et al., 2012).

In Enterobacteria, responses to zinc are mediated by BasSR, BaeSR, PmrAB, and ZraSR TCRS (Yamamoto and Ishihama, 2005; Appia-Ayme et al., 2012; Chen and Groisman, 2013; Wang and Fierke, 2013). MdtABC and MdtD zinc exporters are upregulated by BaeSR upon exposure to high concentrations of zinc (Wang and Fierke, 2013). Copper and zinc exposure lead to overexpression of Spy through regulation by CpxAR and BaeSR, respectively (Wang and Fierke, 2013), and ZraP is upregulated by ZraSR (Appia-Ayme et al., 2012).

## **REGULATION OF MANGANESE TRANSPORT SYSTEMS** *Regulation of manganese uptake systems*

In Enterobacteria, except for *Yersinia*, the DtxR-like MntR regulator is the primary sensor of manganese abundance. When bound to manganese, it represses the transcription of the uptake systems MntH and SitABCD (Patzer and Hantke, 2001; Ikeda et al., 2005; Papp-Wallace and Maguire, 2006; Runyen-Janecky et al., 2006). Of the other metal cations, only Cd2<sup>+</sup> can compete efficiently for binding to MntR (Lieser et al., 2003). MntR has a 10−<sup>5</sup> M affinity for manganese, value that matches the estimated 10−<sup>5</sup> M intracellular concentration of this metal (Waldron and Robinson, 2009). Mn2<sup>+</sup> is the most potent and most effective cation in *E. coli* and *S.* Typhimurium. In *S.* Typhimurium, Fe2<sup>+</sup> repressed transcription through interaction with MntR in the absence of Fur (but this requires high extracellular concentrations of Fe2+) (Patzer and Hantke, 2001; Kehres et al., 2002a; Ikeda et al., 2005). In *Y. pestis*, in which MntR is absent, Fur represses YfeABCD and MntH in response to iron and manganese (Perry et al., 2012). Control of Fur repression by manganese is not specific to *Y. pestis*, as *E. coli* aerobactin and *fhuF* genes also respond to manganese in a Fur-dependent manner. However, in both cases, Fur repression through manganese binding is just specific to some promoters (Privalle and Fridovich, 1993).

MntH is activated by the LysR-type transcriptional regulator OxyR in the presence of H2O2in *S.* Typhimurium and *Shigella* (Kehres et al., 2002a; Runyen-Janecky et al., 2006). In the presence of oxidative stressors, OxyR activates genes by directly interacting with RNA polymerase to enhance initiation of transcription (Ma et al., 2009). MntH is also part of the peroxide stress response in *E. coli* (Anjem et al., 2009). It has been predicted that RybA and RyhB sRNAs might coordinately regulate *mntH* (Modi et al., 2011).

### *Regulation of manganese export systems*

In addition to repressing gene expression, Mn-MntR activates the manganese efflux pump MntP (Waters et al., 2011).

### **CONCLUSION**

During metal-depletion conditions, some regulators such as Fur, Zur, or MntR (each one responding to its cognate metal) become inactive, leading to the activation of cognate metal uptake systems and entry of metal into the cell. When intracellular metal concentration increases, these regulators are activated and repress metal uptake, and other metal-bound regulators, such as ZntR, and TCRS activate efflux systems expression. This regulation thus allows to obtain sufficient nutrient for biological functions when metal is scarce, and to limit toxicity in the cell in the presence of high concentrations of metal. However, this system is organized in a highly sophisticated network, as one transporter can be regulated by its own regulator and other sensors responding to metal availability and environmental signals such as oxidative stress, pH or oxygen. This diversity of regulation for one transport system allows bacteria to sense, adapt and respond specifically and rapidly to a specific microenvironment. Moreover, one regulator can be implicated in the regulation network of several metals. For instance, Fur is able to regulate gene expression in response to iron or manganese, but is also activated by zinc (Mills and Marletta, 2005). Fur can thus be used to sense more than one metal in bacteria and the different metallo-forms of Fur control different genes by binding preferentially to different DNA sequences. Moreover, elevated levels of other metals could interfere with normal iron regulation by activating Fur inappropriately, thus, shutting down iron import and leading to iron starvation. Interestingly, the zinc and copper-dependent regulators Zur, ZntR, and CueR have very high affinities for their cognate metals, i.e., 10−<sup>15</sup> and 10−<sup>21</sup> M. As zinc and copper intracellular concentrations are higher from these values (10−<sup>4</sup> and 10−<sup>6</sup> M, respectively), this suggests that all cytoplasmic zinc and copper are bound and buffered at these low concentrations. Contrary to these regulators, Fur and MntR have higher affinities for their cognate metals (10−<sup>6</sup> M for iron and 10−<sup>5</sup> M for manganese, respectively), corresponding to the intracellular concentrations of these metals. This suggests that more free iron and manganese are available in the cell for other weaker metal-binding proteins, as free zinc and copper concentrations are restricting by this regulation mechanism.

#### **ROLE OF METAL TRANSPORTERS IN ENTEROBACTERIAL VIRULENCE**

Metal homeostasis plays a key role in host-pathogen interactions, as individuals suffering from iron or copper homeostasis anomalies such as thalassaemia or Menkes disease are more susceptible to infections (Vento et al., 2006; Samanovic et al., 2012). To fight infection, a first line of defense of the innate immune system is the sequestering of transition metals to specialized transfer or storage proteins, such as circulating transferrin or intracellular ferritin, which can sequester approximately 4500 Fe3<sup>+</sup> ions per protein (Klein and Lewinson, 2011; Hood and Skaar, 2012). The major sources of iron available for intracellular pathogens such as *Shigella* are haem proteins and ferritin (Reeves et al., 2000). Elaborate mechanisms of transition metal limitation occur within macrophages. Following phagocytosis, bacteria are confined in the phagosome, where acidic pH, ROS and iron/manganese/zinc depletion combine to create bacteriostatic/bacteriolytic conditions. To deplete iron and manganese, the mammalian transporter Nramp1 pumps these metals out of the phagosome, while zinc is exported out of the macrophage using ZIP8 and ZnTs transporters. To acquire phagosomal metals, bacteria therefore employ high affinity transporters. Moreover, following phagocytosis of bacteria by macrophages, interferonγ induces the import of toxic Cu+ inside the phagolysosome to promote bacterial killing (Kehl-Fie and Skaar, 2010; Klein and Lewinson, 2011; Hood and Skaar, 2012). Finally, S100A7, secreted by keratinocytes, inhibits microbial growth through the chelation of Zn2+. S100A12, expressed by neutrophils, binds both Zn2<sup>+</sup> and Cu2<sup>+</sup> *in vitro*, and Cu2+-S100A12 is involved in the generation of superoxide species. Calprotectin, also expressed by neutrophils, is able to chelate Zn2<sup>+</sup> and Mn2+. Excellent reviews already detail the mechanisms of transition metal chelation at the hostpathogen interface (Kehl-Fie and Skaar, 2010; Hood and Skaar, 2012). The diversity of the above-mentioned metal transporters might be explained by their relative specialization to particular infection sites, i.e., intracellular niches, systemic transition or local sites of infection such as intestinal, urinary or pulmonary tracts. As such, depending on the pathogen, their importance in host defense might vary.

#### **ADHESION TO EUKARYOTIC CELLS AND INTRACELLULAR REPLICATION** *Role of iron transporters*

In *Shigella*, single inactivation of either the Shu haem transport system, the Sit and Feo ferrous iron transporters or enterobactin biosynthesis does not affect invasion or intracellular growth (Reeves et al., 2000; Runyen-Janecky et al., 2003). The IutA aerobactin receptor is not expressed in *Shigella* grown in HeLa cells. However, combined deletion of siderophore (IucD) and ferrous iron acquisition systems (Feo and Sit) leads to decreased growth and spreading in epithelial cells, suggesting a synergistic role of these different systems (Runyen-Janecky et al., 2003). Similarly, in *S.* Typhimurium, a single FeoB mutation does not affect intramacrophage replication, which is impaired in simultaneous absence of SitABCD, MntH, and FeoB, suggesting that ferrous iron and manganese acquisition might play a critical role in virulence (Boyer et al., 2002).

#### *Role of copper transporters*

The CopA ATPase, involved in export of cytoplasmic Cu1<sup>+</sup> to the cytoplasm, is necessary for intramacrophage survival of *E. coli* (White et al., 2009). In *S.* Typhimurium, it is overexpressed upon phagocytosis by macrophages cells, which supports the theory that copper is accumulated in phagosomes. Deletion of *copA* or *golT* in *S.* Typhimurium has no effect on survival in cultured macrophages, whereas deletion of both results in significantly reduced survival (Achard et al., 2012; Hodgkinson and Petris, 2012). On the contrary, a *cueO* mutant shows no defect in survival in macrophages (Achard et al., 2010).

#### *Role of zinc transporters*

Concerning zinc,*znuA* was strongly induced in an *E. coli* O157:H7 strain adhering to Caco-2 cultured epithelial cells and a *znuA* mutant was significantly less able to adhere to Caco-2 cells in competition with the wild-type strain (Gabbianelli et al., 2011). In *S.* Typhimurium and *S.* Enteritidis, *znuA* mutants were impaired for growth in Caco-2 epithelial cells and bacteria starved for zinc displayed reduced multiplication in phagocytes (Ammendola et al., 2007).

#### *Role of manganese transporters*

In *Y. pseudotuberculosis*, a *mntH* mutant was defective in survival and growth in macrophages expressing functional Nramp1, but survived and replicated in macrophages deficient in Nramp. This mutant was also susceptible to killing by H2O2 when grown under manganese-limited conditions (Champion et al., 2011). In *S. flexneri*, a *mntH sitA* mutant was more sensitive to hydrogen peroxide, but not to superoxide generators. Moreover, the mutant had impaired survival in activated macrophage lines, but was able to form the same number and size plaques on Henle cell monolayers, suggesting Sit and MntH are not required for survival in this epithelial cell line. Expression of *sitA* and *mntH* was higher when *Shigella* was in Henle cells compared to LB medium (Runyen-Janecky et al., 2006). Upregulation of *sitABCD* in *Shigella* was also observed by microarray analysis in HeLa cells and human macrophage-like U937 cells (Lucchini et al., 2005). A most recent study in *S. flexneri* demonstrated that when cultured Henle cells were infected with a mixture of wild-type and *sitA* mutant strains, the *sitA* mutant was recovered in lower numbers than the wild-type strain, indicating that Sit provides an intracellular growth advantage (Fisher et al., 2009). In *S.* Typhimurium, *sitABCD* was induced *in vivo* after invasion of the intestinal mucosa (Janakiraman and Slauch, 2000). The *mntH sit* mutant of *S.* Typhimurium Keller strain was defective for replication in Nramp1−*/*<sup>−</sup> RAW 264.7 macrophages. Overexpression of *mntH* in a *mntH sit* mutant improved the intracellular survival of the strain in macrophages (Boyer et al., 2002). In *S.* Typhimurium strain SL1344, a *mntH* mutant showed no defect in invasion of or survival in cultured HeLa or RAW 264.7 macrophages but was more susceptible to killing by H2O2. However, expression of *mntH* was induced several fold after 3h within macrophages (Kehres et al., 2000). Using the same strain, Zaharik et al. demonstrated that *sitA* and *mntH* were upregulated when strains were internalized by Nramp1-expressing macrophages (Zaharik et al., 2004). As a rule, mutants for manganese transporters impaired in their ability to replicate in macrophages were also impaired in their ability to resist oxidative stress.

### **SYSTEMIC INFECTIONS**

#### *Role of iron transporters*

The FeoB ferrous iron transporter is not required for systemic infections caused by APEC or *S. enterica* infections (Tsolis et al., 1996; Sabri et al., 2008). A decrease in infection of intravenously inoculated mice was observed for *S.* Typhimurium, but the mice used in this study were Nramp−*/*−, thus, more susceptible to iron-induced oxidative stress (Boyer et al., 2002). *Y. pestis* is significantly less virulent after deletion of the *yfe*, *feo*, and *mntH* genes in a bubonic plague model, suggesting that iron and manganese transport are important in a subcutaneous model of infection (Fetherston et al., 2012). Pathogen-specific siderophore production is crucial for the establishment of systemic infections. Enterobactin mutants of *S.* Typhimurium are not attenuated in a systemic model of infection (Benjamin et al., 1985). Enterobactin can efficiently sequester iron from transferrin due to its particularly high affinity for ferric iron, as demonstrated in *K. pneumoniae*, in which enterobactin promotes survival in the perivascular space. However, lipocalin-2, also called NGAL or siderocalin, is an innate immune defense protein that can sequester enterobactin (Bachman et al., 2012). Chicken and quail also produce avian homologs of lipocalin-2 (Garenaux et al., 2013). Thus, production of additional siderophores that are not captured by siderocalin, are required for systemic infection by Enterobacterial pathogens (Fischbach et al., 2006). Salmochelins are involved in systemic infection caused by *S.* Typhimurium (Crouch et al., 2008). Salmochelins and aerobactin are both important for chicken systemic infection caused by APEC (Dozois et al., 2003). If yersiniabactin plays a role in bubonic plague caused by *Y. pestis*, it is not required for septicemic plague following intraveinous injection (Fetherston et al., 2010; Sebbane et al., 2010). In addition, neither the Has haemophore nor Hmu were involved in virulence in both bubonic plague or systemic infection models (Thompson et al., 1999; Rossi et al., 2001).

### *Role of copper transporters*

Inside the host, infection leads to increased copper levels in the serum. This could be due to ceruloplasmin secretion by the liver during the acute-phase response. Ceruloplasmin is a serum copper-containing protein that is associated with 85% of the copper circulating inside the host (Hodgkinson and Petris, 2012). In *S.* Typhimurium, CopA and GolT ATPases are not involved in systemic infection (Achard et al., 2012; Hodgkinson and Petris, 2012). By contrast, deletion of *cueO*, resulted in decreased virulence during systemic infection (Achard et al., 2010). This is consistent with the observation that host copper deficiency increases susceptibility to infection by *S.* Typhimurium (Hodgkinson and Petris, 2012). Surprisingly, a UPEC *cueO* mutant displays a hypervirulent phenotype. Indeed, in the absence of CueO, copperstressed cells displayed a mucoid phenotype and an aggregative behavior. This could lead to an increase in capsule production and virulence by evading the host immune response (Tree et al., 2007).

### *Role of zinc transporters*

In *Y. ruckeri*, a *znuACB* mutant was unable to compete with the wild-type strain and survived poorly in rainbow trout kidney (Dahiya and Stevenson, 2010). By contrast, ZnuACB was not important for high-level infectivity and virulence of *Y. pestis* in either subcutaneous or intranasal infection models (Desrosiers et al., 2010). In *S.* Typhimurium, virulence of a *znuC* mutant was attenuated compared to the wild type strain (Campoy et al., 2002). Studies conducted with *S.* Typhimurium and *S.* Enteritidis showed similar results with *znuA* mutants (Ammendola et al., 2007).

#### *Role of manganese transporters*

An *yfeABCD* mutant strain of *Y. pestis* was attenuated in Nramp1+*/*<sup>+</sup> mice following intravenous infection (Bearden and Perry, 1999). Similarly, *yfeAB*, *feoB yfeAB*, *yfe mntH* mutant strains were attenuated in a bubonic plague model (Fetherston et al., 2012; Perry et al., 2012). *Galleria mellonella* larval survival following inoculation with a *Y. pseudotuberculosis mntH* strain was significantly greater than survival following challenge with the wild-type strain (Champion et al., 2011). During intraperitoneal competition experiments with *S.* Typhimurium, the *sitA* mutant was consistently out-competed by the wild-type strain in the spleens and livers of mice (Janakiraman and Slauch, 2000). Moreover, intravenous inoculation of *sit*, *mntH* and *feoB* mutant strains in Nramp1−*/*<sup>−</sup> mice showed that the *mntH* mutant was fully virulent and the *sitABCD* mutant was markedly attenuated. The *mntH feoB*, *sit feoB* and *mntH sit feoB* mutants were completely avirulent (Boyer et al., 2002). Another study showed in Nramp1+*/*<sup>+</sup> mice that the *mntH* and *sitABCD* mutants were significantly attenuated and the *mntH sit* mutant was completely avirulent (Zaharik et al., 2004). Depending on the mouse model used, MntH and SitABCD systems in *S.* Typhimurium seem to be more or less important alone but play an important combined role during infection. In *S. flexneri*, a *sitA* mutant was attenuated in a mouse lung model of virulence (Fisher et al., 2009). An APEC *sitA* mutant demonstrated reduced colonization of the lungs, liver and spleen compared to the wild-type strain. The *mntH sit* mutant demonstrated reduced persistence in blood and reduced colonization in the lungs, liver, and spleen. The *mntH* mutant was as virulent as the wild-type strain (Sabri et al., 2008). The *mntH sit* mutant strain was more sensitive to H2O2 compared to the wild-type strain (Sabri et al., 2006).

#### **LOCAL INFECTIONS**

#### *Gut colonization*

*Role of iron transporters.* The FeoB ferrous iron transporter promoted colonization of the intestine by *S. enterica* in mice (Tsolis et al., 1996). Iron acquisition by siderophores also plays a particularly important role in gut colonization. Enterobactin (and other catecholate siderophores) is involved in gut colonization of the mouse by Gram-negative bacteria (Pi et al., 2012). Enterobactin is produced in large quantities by commensals or pathogens producing no other pathogen-specific siderophores, such as *E. coli* O157:H7. The Iha siderophore receptor is involved in colonization of the intestine by this enterohaemorragic *E. coli*. However, in this case, its virulence potential is related to its adhesin properties (Yin et al., 2009). Other pathogenic strains preferentially produce pathogen-specific siderophores rather than enterobactin to promote colonization (Henderson et al., 2009). As such, aerobactin is involved in mouse intestinal colonization by *E. coli* O104:H4 (Torres et al., 2012). SitA also contributes to *S*. Typhimurium colonization of the small intestine (Janakiraman and Slauch, 2000).

*Role of zinc transporters.* In *S.* Typhimurium, ZnuACB contributes to resistance against host calprotectin-mediated Zn2<sup>+</sup> chelation. This transporter promotes resistance of extracellular *S.* Typhimurium to calprotectin accumulated in the host intestine following infection. Moreover, *S.* Typhimurium exploits calprotectin-mediated Zn2<sup>+</sup> chelation in order to out-compete host microbiota, which is less well adapted to the zinc-limited environment in the infected intestine (Liu et al., 2012).

#### *Urinary tract infections*

*Role of iron transporters.* The Iha siderophore receptor is involved in bladder and kidney colonization by UPEC (Leveille et al., 2006). FyuA, the yersiniabactin receptor, promotes biofilm formation in the bladder (Hancock et al., 2008; Brumbaugh et al., 2013). In *P. mirabilis*, yersiniabactin, unlike proteobactin, also allows better fitness in the bladder and the kidneys in a coinfection model (Himpsl et al., 2010). However, as demonstrated in UPEC, yersiniabactin might be involved in copper sequestration rather than iron acquisition (Chaturvedi et al., 2012).The IutA and IroN receptors also promote bladder colonization (Garcia et al., 2011; Watts et al., 2012). On the contrary, enterobactin production is not involved in kidney colonization (Torres et al., 2001).

Haem is an important iron source in tissues. Competition assays showed that the ChuA haem receptor contributes to iron acquisition in kidneys following urinary tract infection by *E. coli* (Hagan and Mobley, 2009; Garcia et al., 2011). The Hma haem receptor is also involved in kidney colonization by UPEC (Hagan and Mobley, 2009; Garcia et al., 2011). By contrast, a *chuT* deletion mutant in mono-infections in both APEC and UPEC models had no change in virulence (Gao et al., 2012).

*Role of zinc transporters.* In competitive infections, a UPEC *zupT* mutant was not outcompeted by the wild-type strain. In contrast, the *znuA* and *znuA zupT* mutants demonstrated significantly reduced numbers in the bladders and kidneys. In single-strain infections, *znuA* and *znuA zupT* mutants were reduced in the kidneys. Moreover, the double mutant demonstrated decreased motility and less resistance to hydrogen peroxyde (Sabri et al., 2009). A UPEC *znuB* mutant exhibited a defect in biofilm formation under static conditions and in motility (Gunasekera et al., 2009). In *Proteus mirabilis*, a *znuC* mutant displayed reduced swimming and swarming motility. This mutant was outcompeted by the wild-type strain during competitive infections in urine, bladder and kidneys of mice but colonized mice as well as the wild-type during independent infections (Nielubowicz et al., 2010).

*Role of manganese transporters.* In UPEC, *mntH* and *sitABCD* mutants colonize bladder and kidneys as well as the wild type strain. However, the *mntH sit* double mutant displayed lower colonization rates in kidneys. Moreover, the *mntH* and *mntH sit* mutants were more sensitive to H2O2 and plumbagin compared to the wild-type strain (unpublished data from our laboratory).

#### *Pulmonary infections*

*Role of iron transporters.* Salmochelins and aerobactin play important roles in colonization of the lungs during APEC infections (Dozois et al., 2003), whereas haem uptake through the Chu system does not play a significant role (Gao et al., 2012). Salmochelins and yersiniabactin are both required for *K. pneumoniae* infections (Bachman et al., 2011, 2012). In *Y. pestis*, yersiniabactin is important for pneumonic plague but may not be critical for iron acquisition. Indeed, receptor mutants are less attenuated than synthesis mutants and it has been suggested that yersiniabactin may damage pulmonary epithelial cells or affect immune cells (Fetherston et al., 2010; Perry and Fetherston, 2011).

*Role of copper transporters.* Copper accumulation at the sites of infection, such as infected lungs by *M. tuberculosis* has been reported (Hodgkinson and Petris, 2012). As a consequence, copper tolerance might play an important role in the establishment of some pulmonary infections. However for Enterobacteria, thus far no data to support this have been reported.

*Role of manganese transporters.* Yfe and Feo systems are not essential for pneumonic plague, even though enhanced transcription of *yfe* genes was measured *in vivo* in a pneumonic plague model. However, the environment encountered by bacteria in the lungs is limited in manganese, and manganese transporters have been shown to play significant roles in other types of lung infections. Manganese requirements of *Y. pestis* during lung infection might be lower than that of other pathogens (Fetherston et al., 2012).

#### *Bubonic plague*

The Yfu and Yiu ABC transporters are not involved in virulence in a bubonic plague model (Gong et al., 2001; Kirillina et al., 2006). Deletion of the yersiniabactin siderophore-mediated iron acquisition system resulted in complete loss of *Y. pestis* virulence in a bubonic plague model following subcutaneous inoculation (Fetherston et al., 2010). Using a flea-to mouse infection model, it has also been established that yersiniabactin played a critical role during the early stages of the infection (Sebbane et al., 2010).

#### **CONCLUSION**

Information described above has all been summarized in **Tables 1**, **2**. All the metals described in this review are used by the host to develop defense strategies, either by starving the pathogens, or by overloading them. As a consequence, numerous sophisticated acquisition and detoxification systems have evolved in bacteria to cope with metal depletion or overload. Pathogen-specific siderophores are important for local and systemic infections. This is consistent with the fact that they can acquire iron from transferrin present in the blood and the perivascular space, and the fact that they are involved in evasion of lipocalin-2, which contributes to both systemic and mucosal innate immune defense (Chan et al., 2009). While haemophores do not contribute to virulence, other haem uptake systems seem to play a significant role. Conversely, the importance of CopA and GolT ATPases for intramacrophage survival is consistent with a localized cellular copper-mediated toxicity induced by the host under infection conditions. Recent studies have shown that iron overload is also encountered in macrophages at early stages of infection, and that catecholate (enterobactin and salmochelin) siderophores as well as iron export systems help pathogens survive in these conditions (Achard et al., 2013; Frawley et al., 2013). By contrast, uptake systems described for zinc and manganese are important for pathogens at all infection sites. Yet unidentified systems involved in metal acquisition from host proteins such as calprotectin might be involved, and the requirement or stringency for zinc and manganese acquisition may vary for different sites of infection.

#### **METAL ACQUISITION WITHIN THE HOST: A BATTLE IN WHICH PATHOGENIC BACTERIA HAVE THE UPPER HAND?**

To fight bacterial infections, the first line of defense of the host is to restrict access to essential metals, a process termed nutritional immunity. This process allows sequestration of essential metals by host proteins such as ferritin, haemoglobin or transferrin for iron, calprotectin for zinc and manganese, and ceruloplasmin for copper. In addition to extracellular metal restriction mechanisms, host cells can also deplete metals from inside phagosomes. All together, host metal sequestration should limit metal availability to invading pathogens, which should reduce their capacity to replicate and cause infections. However, pathogenic bacteria have acquired multiple mechanisms to counteract this line of defense. First of all, enterobacterial pathogens possess highly specialized and diversified regulation systems that sense intracellular metal concentrations. When a nutrient metal is lacking, these regulators (Fur, MntR, CueR, Zur...) control expression of uptake systems and repress such systems when these metals are replete. Moreover, the regulation of bacterial export systems also allows bacteria to expel metals that are in excess from the cell and hence protect the bacterial cell from metal toxicity. These regulation systems, which respond to specific concentrations of metals, allow the good pattern of expression of metal transport systems to respond to a specific microenvironment. This ensures sufficient metal uptake to allow replication while limiting metal availability in the bacterial cytoplasm to prevent toxicity. Due to the complexity of this metal regulation network, the distinctive functions of each component in the different niches encountered by pathogens during their infection cycle is still unclear. Behind this sophisticated metal regulatory network, pathogenic Enterobacteria also possess diversified metal transport systems to evade nutritional immunity. To obtain metals sequestered by host proteins, pathogenic bacteria are able firstly to produce several uptake systems for a same cognate metal. Secondly, most of these metal uptake systems have high affinity for their cognate metal, which thus, allows for example siderophores to compete with iron sequestration by host proteins such as transferrin. The diversity of bacterial metal transport systems is an important mechanism to ensure sufficient metal uptake and to adapt to different niches inside the host.

A second line of defense developed by the host against infection is to produce proteins that can specifically sequester bacterial iron-uptake systems. Lipocalin-2 is able to bind enterobactin, a siderophore secreted by many Enterobacteria. This second mechanism should represent an effective way to defend against enteric infections. However, enterobacterial pathogens have also evolved to trump lipocalin-2, by producing other types of siderophores that escape lipocalin-2 sequestration.

Finally, the multiplicity of systems produced by pathogenic Enterobacteria to regulate and transport metal nutrients illustrates why these systems are so important for their pathogenesis. Thanks to specific regulation mechanisms, they are able to sense precisely which concentrations and which types of metal are available in the specific niche they infect. They can thus adapt and respond efficiently by activating or repressing uptake or export systems corresponding to metal availability. As the type of metal and its bioavailability differs depending on the host tissue or cellular environment, this may explain why a transport system may be important for virulence of some strains while the same system may not be required for virulence in another strain associated with infection at a different tissue site. For instance, while haem uptake is important in the establishment of UTIs by UPEC (Hagan and Mobley, 2009; Garcia et al., 2011), such systems are of limited importance in bubonic plague caused by *Y. pestis* (Rossi et al., 2001). Overall, pathogenic Enterobacteria seem to


#### **Table 2 | Characterized copper, zinc and manganese transport systems involved in virulence of Enterobacteria.**

*(Continued)*

#### **Table 2 | Continued**


*ND, Not Determined.*

*involved in virulence, involved with another transport system (indicated in the box), not involved.*

have an edge over host defenses and can cause important infections due to metal acquisition. As pathogenic bacteria use tightly controlled regulation systems to respond and adapt to metal nutrient availability, it would be of interest to further elucidate how metal homeostasis is temporally regulated in bacterial cells within the host. This could allow us to specifically target and limit metal sensing by bacterial pathogens. Reducing the bacterial response to nutritional immunity, could lead to dysfunctional metal homeostasis and novel approaches to prevent and treat infections.

#### **CONCLUSION**

Pathogenic bacteria encounter various metal-related stresses during infection or colonization of hosts, whether by metal starvation through metal chelation by host proteins, or by exposure to metal toxicity, for example with Cu+. Moreover, they have to sense metal levels to prevent metal toxicity at high concentrations. Bacteria have therefore developed very sophisticated transport systems for each metal to ensure sufficient uptake while activating efficient export of such metals if they are in excess. As these metals are essential co-factors for bacterial physiology and growth, it is not surprising that metal transporters are implicated in the virulence of pathogenic Enterobacteria. In addition, as each pathogen encounters various host environments and differences in metal availability, a specific transporter may be important for virulence of one bacterial strain, but may not be required for another strain in a distinct host species or site of infection. Historically, empirical disruption of copper homeostasis has constituted a basic hygiene measure for thousands of years (Samanovic et al., 2012). Copper surfaces, copper nanoparticles, use of copper in food supplementation or copper sprays are still used in construction and agriculture. However, this has already resulted in selection for copper-resistant strains (Dupont et al., 2011). In the last decades, a better understanding of the pathogenspecific systems involved in metal homeostasis has allowed the development of vaccination strategies or therapies. Indeed, since metal uptake systems require specific surface receptors that are exposed on the outer membrane, such receptors if immunogenic could provide targets for protective vaccines or serve as a port of entry for therapeutic molecules. Recent studies have shown that mono or multivalent receptor vaccines induce good immune responses and protect against diseases (Alteri et al., 2009; Wieser et al., 2010; Brumbaugh et al., 2013). Moreover, immunization using attenuated bacterial strains lacking metal transport systems may efficiently protect against infection. For example a *S.* Typhimurium *znuACB* mutant strain has been shown to confer good mucosal protection against salmonellosis in mice and pigs (Pasquali et al., 2008; Pesciaroli et al., 2011, 2013; Gradassi et al., 2013). In addition, some bacterial strains produce antibacterial molecules that are recognized by specific siderophore receptors present on competing bacteria. For instance, microcin E492 produced by *K. pneumoniae* enters *E. coli* through its enterobactin receptors (Destoumieux-Garzon et al., 2006). Creating siderophore analogs as "Trojan horses" can be used for the development of novel antimicrobials (Miller et al., 2009). Pesticin, produced by *Y. pestis*, is known to enter through the yersiniabactin FyuA receptor. Based on this observation, a phage lysin active against Gram-negative pathogens has been engineered (Lukacik et al., 2012). A recent study also demonstrated that the probiotic *E. coli* Nissle strain reduces *S*. Typhimurium intestinal colonization by competing for iron, as this strain possesses more siderophores than *Salmonella* (Deriu et al., 2013). Future characterization of transition metal transport processes and their regulation, and determination of how cellular metal content varies and is controlled in pathogenic bacteria will further elucidate new prospects on vaccination or therapeutic development against Enterobacteria and other important bacterial pathogens.

### **ACKNOWLEDGMENTS**

This work was supported by the Fondation Armand-Frappier (Gaëlle Porcheron), the Natural Sciences and Engineering Research Council Canada (NSERC) Discovery Grant (RGPIN 250129-07) (Charles M. Dozois), the Centre de Recherche en Infectiologie Porcine et Aviaire (Charles M. Dozois), the Canada Research Chairs program (Charles M. Dozois), the Natural Sciences and Engineering Research Council Canada (NSERC) Discovery Grant (RGPIN 250129-07) (Charles M. Dozois).

#### **SUPPLEMENTARY MATERIAL**

The Supplementary Material for this article can be found online at: http://www.frontiersin.org/journal/10.3389/fcimb. 2013.00090/abstract

#### **REFERENCES**


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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 23 August 2013; accepted: 18 November 2013; published online: 05 December 2013.*

*Citation: Porcheron G, Garenaux A, Proulx J, Sabri M and Dozois CM (2013) Iron, copper, zinc, and manganese transport and regulation in pathogenic Enterobacteria: correlations between strains, site of infection and the relative importance of the different metal transport systems for virulence. Front. Cell. Infect. Microbiol. 3:90. doi: 10.3389/fcimb.2013.00090*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2013 Porcheron, Garenaux, Proulx, Sabri and Dozois. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Transcriptional regulation by Ferric Uptake Regulator (Fur) in pathogenic bacteria

#### *Bryan Troxell 1† and Hosni M. Hassan2 \**

*<sup>1</sup> Department of Immunology and Microbiology, Indiana University School of Medicine, Indianapolis, IN, USA*

*<sup>2</sup> Prestage Department of Poultry Science, North Carolina State University, Raleigh, NC, USA*

#### *Edited by:*

*Mathieu F. Cellier, Institut National de la Recherche Scientifique, Canada*

#### *Reviewed by:*

*John Helmann, Cornell University, USA Klaus Hantke, Universität Tübingen, Germany Caroline Genco, Boston University School of Medicine, USA*

#### *\*Correspondence:*

*Hosni M. Hassan, Prestage Department of Poultry Science, North Carolina State University, 334C Scott Hall, Campus Box 7608, Raleigh, NC 27695-7608, USA e-mail: hmhassan@ncsu.edu*

#### *†Present address:*

*Bryan Troxell, Prestage Department of Poultry Science, North Carolina State University, Raleigh, USA*

In the ancient anaerobic environment, ferrous iron (Fe2+) was one of the first metal cofactors. Oxygenation of the ancient world challenged bacteria to acquire the insoluble ferric iron (Fe3+) and later to defend against reactive oxygen species (ROS) generated by the Fenton chemistry. To acquire Fe3+, bacteria produce low-molecular weight compounds, known as siderophores, which have extremely high affinity for Fe3+. However, during infection the host restricts iron from pathogens by producing iron- and siderophore-chelating proteins, by exporting iron from intracellular pathogen-containing compartments, and by limiting absorption of dietary iron. Ferric Uptake Regulator (Fur) is a transcription factor which utilizes Fe2<sup>+</sup> as a corepressor and represses siderophore synthesis in pathogens. Fur, directly or indirectly, controls expression of enzymes that protect against ROS damage. Thus, the challenges of iron homeostasis and defense against ROS are addressed via Fur. Although the role of Fur as a repressor is well-documented, emerging evidence demonstrates that Fur can function as an activator. Fur activation can occur through three distinct mechanisms (1) indirectly via small RNAs, (2) binding at *cis* regulatory elements that enhance recruitment of the RNA polymerase holoenzyme (RNAP), and (3) functioning as an antirepressor by removing or blocking DNA binding of a repressor of transcription. In addition, Fur homologs control defense against peroxide stress (PerR) and control uptake of other metals such as zinc (Zur) and manganese (Mur) in pathogenic bacteria. Fur family members are important for virulence within bacterial pathogens since mutants of *fur*, *perR*, or *zur* exhibit reduced virulence within numerous animal and plant models of infection. This review focuses on the breadth of Fur regulation in pathogenic bacteria.

**Keywords: Ferric Uptake Regulator, iron, oxidative stress, gene regulation, pathogenic bacteria**

## **INTRODUCTION**

Transition metals are essential elements in biological systems. Metabolic pathways, DNA synthesis, RNA synthesis, and protein synthesis are dependent on the availability of the appropriate metal cofactor. In support of this, all cells have designated gene products that transport metals to maintain cellular function; however, certain essential metals cause the formation of toxic reactive oxygen species (ROS). In the earliest description of what is now known as the Fenton reaction, iron (Fe) was shown to act catalytically in the oxidation of tartaric acid (Fenton, 1894). The Fenton reaction produces the hydroxyl radical (HO.), a ROS capable of oxidizing macromolecules and lipids (Imlay et al., 1988; Lloyd et al., 1997). Therefore, cells must tightly regulate the concentration of Fe to avoid ROS-mediated cell damage.

Bacteria sense their environment and alter expression of genes that promote survival. This is accomplished by transcription factors that regulate expression of beneficial or detrimental genes. In order to acquire Fe in Fe-limiting environments, bacteria and fungi synthesize and secrete low molecular weight compounds, called siderophores, which have high affinity for binding Fe3+. Most siderophores are produced by the non-ribosomal peptide synthesis (NRPS) pathway and an example is the siderophore enterochelin. The final steps of the pathway are executed by the action of the Ent proteins (encoded by the *entD*, *entF*, and *entCEBA* genes) (Gehring et al., 1998; Salvail et al., 2010). Aerobactin, another siderophore, is sequentially produced by the proteins IucD, IucB, IucA, and IucC (**Figure 1A**) that are expressed in an operon (*iucABCD*). Aerobactin is an example of a siderophore not produced by the NRPS pathway. Transcriptional control of both siderophores is regulated by the concentration of intracellular Fe2<sup>+</sup> (Bagg and Neilands, 1987b); when intracellular Fe2<sup>+</sup> is low, the model bacterial organism, *Escherichia coli* induces siderophore production (Brot and Goodwin, 1968; Bryce and Brot, 1971). The Fe-bound siderophores are subsequently transported into the cell to satisfy an Fe2<sup>+</sup> requirement. Because Fe2<sup>+</sup> transcriptionally controls expression of gene products that promote iron acquisition, Fe2<sup>+</sup> was predicted to be a corepressor for a DNA-binding protein. Isolation of a mutant of *Salmonella enterica* subsp. *enterica* serovar Typhimurium (*S*. Typhimurium) that constitutively expresses iron uptake proteins supported this hypothesis (Ernst et al., 1978). A mutation in Ferric Uptake Regulator (Fur) encoded by the *fur* gene was identified in *E. coli*

**FIGURE 1 | The classic model of Fur repression of iron acquisition (***iucA* **as an example). (A)** Biosynthesis of the siderophore aerobactin requires several genes located in an operon (*iucABCD, iutA*). Expression of the initial gene, *iucA*, is Fur-repressed (De Lorenzo et al., 1987) and production of aerobactin is known to be produced by virulent strains of bacteria, especially strains causing disease in avian hosts (i.e., Avian pathogenic *E. coli* or APEC) (Lafont et al., 1987; Xiong et al., 2012; Ling et al., 2013). The sequential enzymatic activity of IucD, IucB, IucC, and IucA convert L-lysine into aerobactin, a potent Fe-scavenging siderophore. **(B)** There are two Fur-binding sites (FBS) for Fe-dependent regulation of *iucA*. Both FBS are located within the P1 promoter (overlapping the −35 and also the −10 sites). Under conditions of Fe-deprivation (left panel), there is increased transcription (signified by a +1) of the *iucABCD* genes whose protein products form a biosynthetic pathway that produces aerobactin. Under Fe-replete conditions (right panel), Fur binds to DNA at the FBS (green box) and blocks access of the −35 and −10 sites by RNA polymerase (RNAP, blue shape).

mutants that exhibited constitutive expression of iron uptake genes (Hantke, 1981, 1984; Bagg and Neilands, 1985). Fur is a DNA-binding protein that recognizes specific DNA sequences, utilizes Fe2<sup>+</sup> or Mn2<sup>+</sup> as a corepressor, and blocks transcription of target genes (Bagg and Neilands, 1987a; De Lorenzo et al., 1987). Not surprisingly, the transcriptional control of *entD*, *entF*, *entCEBA,* and *iucABCD* is negatively regulated by Fur (De Lorenzo et al., 1987; Brickman et al., 1990; Stojiljkovic et al., 1994; Tsolis et al., 1995; Bjarnason et al., 2003; McHugh et al., 2003; Troxell et al., 2011a).

The collective work supports a simple model for the molecular mechanism of Fur repression that consists of Fur binding to *cis* regulatory elements of a gene and preventing the binding of the RNA polymerase holoenzyme (RNAP) (**Figure 1B**) (De Lorenzo et al., 1987; Escolar et al., 1999, 2000; Hantke, 2001; Lee and Helmann, 2007; Carpenter et al., 2009). As a transcriptional repressor, Fur-Fe2<sup>+</sup> homodimer binds to the operator site of a target promoter (Ernst et al., 1978; Bagg and Neilands, 1985, 1987a; Neilands, 1993; Escolar et al., 1997, 1998). However, Fur can form a multimeric complex with DNA sequences extending beyond the operator site (Escolar et al., 2000; Baichoo and Helmann, 2002; Lavrrar et al., 2002). Initial studies defined the Fur-binding site (the Fur box) as an ≈19 bp DNA sequence with dyad symmetry, GATAATGATAATCATTATC (Calderwood and Mekalanos, 1987, 1988; De Lorenzo et al., 1987; Stojiljkovic et al., 1994). Insertion of this sequence into an operator site in the promoter of a non-Fe2<sup>+</sup> regulated gene results in derepression under Fe2+-limiting conditions (Calderwood and Mekalanos, 1988). In an elegant approach to define Fur regulated genes within bacteria, a high copy number plasmid containing randomly cloned DNA sequences from Gram positive and negative bacteria were transformed into an *E. coli* strain that harbored a single copy of a *fhuF::lacZ* reporter fusion (Hantke, 1987). Fur represses transcription of the *fhuF* gene, which encodes a protein involved in the acquisition of Fe3<sup>+</sup> (Hantke, 1983, 1987). If the cloned DNA fragment on the high copy number plasmid contains a Fur-binding site, then Fur proteins will be titrated away from the promoter of *fhuF* resulting in derepression of the *fhuF::lacZ* fusion, which can be qualitatively detected during growth on MacConkey agar plates or quantified by a β-galactosidase assay. This assay is called the Fur titration assay (FURTA) and has been used to study Fur regulation for nearly 20 years (Stojiljkovic et al., 1994; Tsolis et al., 1995; Baumler et al., 1996; Fassbinder et al., 2000; Osorio et al., 2004; Haraszthy et al., 2006; Jackson et al., 2010; Tanabe et al., 2010). *In toto*, these works solidified the role of Fur as a Fe2+-dependent transcriptional repressor. However, global gene expression studies have identified numerous genes that require Fur for expression (Foster and Hall, 1992; D'Autreaux et al., 2002; Bjarnason et al., 2003; McHugh et al., 2003; Troxell et al., 2011a).

#### **MULTIFACTORIAL ROLES OF Fe2+-Fur REGULATION IN BACTERIA**

Fur is required for the expression of several proteins within the tricarboxylic acid cycle (TCA) and the Fe2+-dependent superoxide dismutase (SodB) (Hantke, 1987; Gruer and Guest, 1994; Dubrac and Touati, 2000, 2002). The disruption of the TCA cycle within *fur* mutants may have a relevant role for the regulation of virulence since mutations within the TCA cycle alter virulence expression in *Staphylococcus epidermidis* and *Vibrio cholera* (Sadykov et al., 2008; Minato et al., 2013). In addition, disruption of the TCA cycle reduces *S*. Typhimurium virulence in mice (Tchawa Yimga et al., 2006; Bowden et al., 2010). The role of Fur in TCA cycle regulation is an example of how Fur regulation is multifactorial; *fur* mutants exhibit many phenotypes not just enhanced expression of siderophores. The molecular mechanism for the Fur's positive activation in the TCA cycle and SodB went unexplained until a landmark publication determined the importance of a highly conserved small untranslated RNA (sRNA) named *ryhB* in activation by Fur (Masse and Gottesman, 2002). *ryhB* is directly repressed by Fur (Vassinova and Kozyrev, 2000; Masse and Gottesman, 2002) and base pairs with target mRNAs, such as *sodB* and the succinate dehydrogenase operon *sdhCDAB*, which results in degradation of the mRNAs thereby reducing expression of the gene products (**Figure 2A**). Deletion of *ryhB* in a *fur* results in restoration of expression of TCA proteins, SodB, and growth on succinate or fumarate minimal medium (Masse and Gottesman, 2002). Because regulation by *ryhB* requires the RNA chaperone protein, Hfq, deletion of *hfq* in *fur* also restores expression of many Fur activated genes (Masse and Gottesman, 2002; Ellermeier and Slauch, 2008; Troxell et al., 2011a). *ryhB* homologs have a role in virulence, are Fur-repressed, and are encoded in the genomes of several Gram negative pathogens

**FIGURE 2 | Models of the Fur-dependent activation of gene expression in bacteria. (A)** Fur activation through "*ryhB*-dependent" mechanism (SodB as an example). Fur is indirectly required for the expression of the FeSOD (SodB) in bacteria through the sRNA *ryhB* (Masse and Gottesman, 2002; Ellermeier and Slauch, 2008). Under conditions of Fe2<sup>+</sup> depletion (top panel), Fur is unable to directly repress transcription of the sRNA *ryhB* (or its paralog). This results in an increase in the level of *ryhB* within the cell. The RNA chaperone Hfq binds to *ryhB* and to the target mRNA of *sodB* (Afonyushkin et al., 2005; Urban and Vogel, 2007), which through the RNase-dependent cleavage (cleavage sites are signified by filled triangles) reduces the half-life of *sodB* mRNA and reduces SodB protein within the cell. The Fur activation of *sodB* is diminished in the absence of Hfq or *ryhB* (Masse and Gottesman, 2002; Ellermeier and Slauch, 2008; Troxell et al., 2011a). When Fur is activated during Fe2<sup>+</sup> replete conditions (bottom panel), transcription of *ryhB* is blocked, which increases the half-life of *sodB* mRNA allowing for enhanced production of SodB protein and FeSOD activity. **(B)** Fur activation through "RNAP recruitment" mechanism (Examples from *S*. Typhimurium and *H. Pylori*). *In vitro* transcription assays

(i.e., *Klebsiella pneumoniae*, *Shigella*, *Vibrio cholera*, *Yersinia pestis, Salmonella*, *Pseudomonas aeruginosa*, *Neisseria meningitidis*, and *Neisseria gonorrhoeae*) (Wilderman et al., 2004; Davis et al., 2005; Mey et al., 2005a; Oglesby et al., 2005; Mellin et al., 2007; Murphy and Payne, 2007; Ellermeier and Slauch, 2008; Ducey et al., 2009; Metruccio et al., 2009; Troxell et al., 2011a; Deng et al., 2012; Huang et al., 2012; Kim and Kwon, 2013; Leclerc et al., 2013). Indirect positive regulation by Fur through negative regulation of the negative regulator, *ryhB*, is the most studied molecular mechanism for Fe2+-dependent activation of gene expression; however, additional evidence demonstrates that Fur may regulate virulence through more complicated mechanisms.

with *H. pylori norB* regulatory sequences (Delany et al., 2004) and *S*. Typhimurium *hilD* regulatory sequences (Teixido et al., 2011) demonstrate an active Fur-Fe2<sup>+</sup> binding to a FBS (signified with a green box) that promotes increased binding of the RNAP (signified with a blue shape) to the promoter and transcription of the target gene (signified with a +1). In both examples, the regulatory sequences of *norB* and *hilD* contain a repression site (signified with a red box) that may overlap the FBS (an ArsR-binding site with *norB*) or be located immediately downstream of the FBS (an H-NS binding site with *hilD*). If Fur-Fe2<sup>+</sup> physically contacts the RNAP is unknown. **(C)** Fur activation through "antirepressor" mechanism (FtnA as an example). In *E. coli*, expression of the *ftnA* gene is Fur activated, but independent of the "*ryhB*-dependent" activation. Under Fe2<sup>+</sup> poor conditions, H-NS binds upstream of the *ftnA* gene and represses transcription (top panel). When Fur is activated, Fur-Fe2<sup>+</sup> binds to several FBS located upstream of *ftnA*, which prevents H-NS nucleation at the *ftnA* promoter and repressing transcription (bottom panel). In this example, Fur is required to block H-NS binding and can physically remove H-NS from the upstream regulatory site, which allows for *ftnA* expression.

For example, in *S.* Typhimurium, transcription of the virulence factor *hilA* requires Fe2<sup>+</sup> through Fur-dependent regulation (Thompson et al., 2006; Ellermeier and Slauch, 2008; Troxell et al., 2011b). Recently, we demonstrated enhanced transcription of *hns* in *fur* and in a modified chromatin immunoprecipitation (ChIP) assay we determined that Fur bound the upstream regulatory region of *hns* in a metal-dependent manner (Troxell et al., 2011b). H-NS is known to repress transcription of *hilA* (Olekhnovich and Kadner, 2006). H-NS is a protein associated with the bacterial nucleoid and is also known as OsmZ, BglY, and PilG (Defez and De Felice, 1981; Spears et al., 1986; May et al., 1990). Deletion of *fur* and *hns* resulted in Fur-independent activation of *hilA*, which supports the indication that Fur regulation of *hilA* was indirect through H-NS (Troxell et al., 2011b). Furthermore, Fur is not required for expression of Fur-activated genes when the repressor H-NS is absent (Nandal et al., 2010; Troxell et al., 2011b) and Fur and H-NS appeared to recognize similar DNA sequences throughout the bacterial chromosome (Prajapat and Saini, 2012). In another example of the multifactorial role of Fur in bacteria, a recent study shows that Fur represses transcription of the *vvhA* gene, which encodes the major haemolysin of *Vibrio vulnificus*, yet haemolytic activity and VvhA protein level were reduced in *fur* (Lee et al., 2013). Two metaldependent proteases are responsible for degradation of VvhA, VvpE, and VvpM and transcription of *vvpE* is under negative regulation by Fur. Through genetic and biochemical approaches, it was shown that VvpE and VvpM exhibited enhanced activity in *fur* resulting in reduction of the VvhA protein (Lee et al., 2013). Clearly, it can be appreciated from these two examples that the influence of Fur within the cell is global and typically involves multiple layers of regulation. Nevertheless, recent evidence indicates Fur may have a more direct role for activation of gene expression in bacteria (**Figure 2**).

#### **MECHANISMS OF ACTIVATION OF GENE EXPRESSION VIA DNA BINDING BY Fur: LOCATION, LOCATION, LOCATION**

Global gene expression studies have identified genes that require Fur for expression (Foster and Hall, 1992; D'Autreaux et al., 2002; Bjarnason et al., 2003; McHugh et al., 2003; Troxell et al., 2011a). Earlier work demonstrated a unique mechanism for Fur activation in *N. meningitidis* that involves Fur directly binding to *cis* regulatory elements upstream of a Fur-activated gene (Delany et al., 2004). Unlike Fur-repressed genes that possess a characteristic Fur-binding site overlapping the RNAP-binding site, Fur-activated genes [*norB*, *pan1* (*aniA*), and *nuoA*] contain Fur boxes located ≈100 bp upstream of the transcriptional start site, while the Fur-repressed *tbp* contains a Fur box that overlaps with the RNAP-binding site. The Fur box and activation of *norB*, which encodes a protein responsible for protection against NO (Anjum et al., 2002), is conserved in *N. gonorrhoeae* (Isabella et al., 2008). Moreover, in *Helicobacter pylori*, Fur activates expression of *oorB*, which encodes a 2-oxoglutarate:acceptor oxidoreductase (Hughes et al., 1998), by directly binding to a *cis* regulatory elements located 130 bp upstream of the transcriptional start site (Gilbreath et al., 2012). The importance of OorB in virulence is demonstrated by the significant reduction in colonization of the chicken gut by a *oorB* mutant strain of *Campylobacter jejuni* (Weerakoon et al., 2009). In *V. cholera*, Fur activates expression of the outer membrane porin, *ompT*, through binding a Fur box located 90 bp upstream of the transcriptional start site (Craig et al., 2011). In *S*. Typhimurium, transcription of the virulence factor *hilD* is activated by Fur through a Fur box located nearly 200 bp upstream of the transcriptional start site (Teixido et al., 2011). HilD is an AraC/XylS-type DNA-binding protein that regulates transcription of important virulence factors within *S*. Typhimurium and is required for infection (Ellermeier et al., 2005). Importantly, the sequence of the Fur box site for activated genes is virtually identical to the Fur box of repressed genes. Collectively, the molecular evidence suggests the location of the Fur box in proximity to the RNAP-binding site determines the ability of Fur to activate gene expression.

How does Fur activate gene expression? *In vitro* transcription experiments demonstrate that Fur can activate transcription of a target gene even though the Fur boxes are located ≈100 and 200 bp upstream of the transcriptional start site, respectively (Delany et al., 2004; Teixido et al., 2011). This example of Fur activation is rare, but may involve enhanced recruitment of RNAP to the promoter of target genes ("RNAP recruitment" activation model, **Figure 2B**). Surprisingly, addition of the Fur protein to the *in vitro* transcription assay stimulated the production of *hilD* mRNA, which suggests improved recruitment of RNAP to the promoter of *hilD* even though the Fur box is nearly 200 bp upstream of the transcriptional start site (Teixido et al., 2011). While deletion of *fur* reduces transcription of *hilD* (Teixido et al., 2011) overexpression of Fur results in little increased activation of the *hilD* promoter contrary to overexpression of a direct activator HilC, which increases *hilD*'s promoter activity by ≈5-fold (Ellermeier and Slauch, 2008). These results indicate the role of Fur in direct transcriptional activation of a target gene is complex.

Transcriptional activators that bind upstream of the RNAPbinding site have been shown to interact with the C-terminal domain of the α subunit (α-CTD) of RNAP, which promotes transcription of the target gene (Ishihama, 1992; Busby and Ebright, 1994; Ebright and Busby, 1995; Murakami et al., 1997; Hochschild and Dove, 1998). Contact between activators and α-CTD is inhibited when the upstream activator binding site is ≥100 bp upstream of the transcriptional start site (Murakami et al., 1997). Thus, transcription factor binding sites located further than 100 bp upstream of the transcriptional start site are unlikely to interact physically with the α-CTD of RNAP. However, oligomerization of the Fur protein at Fur boxes is known to occur (De Lorenzo et al., 1987; Tardat and Touati, 1993; Escolar et al., 2000; Nandal et al., 2010; Teixido et al., 2011), which suggests Fur proteins may extend to interact with other proteins nearby. Whether Fur contacts the RNAP is not known, but emerging *in vivo* evidence indicates there is another plausible molecular mechanism for Fur-dependent activation through binding DNA at a distal regulatory site.

#### *Roles of Fur and H-NS in the regulation of FtnA*

Fe2<sup>+</sup> activates expression of the Fe-storage gene *ftnA* in a Furdependent manner (Masse and Gottesman, 2002; Velayudhan et al., 2007). Overexpression of *ryhB* results in the down regulation of many Fe-cofactored proteins (i.e., SodB) and increases the intracellular Fe2<sup>+</sup> concentration resulting in enhanced Fur activation (Masse et al., 2005; Jacques et al., 2006). This is known as the "iron-sparing" response (Gaballa et al., 2008). Masse et al. theorized that Fur may negatively regulate a negative regulator of *ftnA*, which would manifest as a Fur activation. Evidence to support this theory was demonstrated by work from Simon C. Andrews' lab, which showed that Fur binds to a distal regulator site upstream of the RNAP-binding site in the promoter of *ftnA* to physically remove the histone-like protein, H-NS, which mediates repression of *ftnA* (Nandal et al., 2010). Unlike the activation of *norB* and *hilD*, Fur was not required for transcription of *ftnA* using *in vitro* transcription assays (Nandal et al., 2010). H-NS repressed transcription of *ftnA* and Fur was only required to relieve this repression. The role of Fur as an antirepressor in the activation of *ftnA* is supported with *in vivo* evidence: (1) *fur* is not required for *ftnA* expression in the absence of *hns*; and (2) *ftnA* expression is not reduced by Fe2+-chelation in *hns*(Nandal et al., 2010). Fur activation of gene expression by this mode represents a 3rd type of activation, the "antirepressor" activation model (**Figure 2C**). *In vivo* evidence supports the antirepressor model as a major mechanism for Fur-dependent activation of gene expression. Evidence for the antirepressor model is evident in *N. gonorrhoeae* because the Fur-binding site upstream of *norB* is not required for activation of expression when the *norB* repressor, ArsR, is deleted (Isabella et al., 2008). Thus, Fur antirepressor activity is an emerging model of Fur activation through DNA binding.

#### **Fur CONTROLS DEFENSES AGAINST ROS**

During bacterial infection the host responds to non-self molecules and initiates a potent antimicrobial response. However, bacterial pathogens are well-adapted to defending against the host antimicrobial response. In many bacterial pathogens the defense against ROS requires the Fur protein. Enzymatic defense against ROS occurs by the rapid enzymatic dismutation of superoxide (O2−) by superoxide dismutases (SODs) and detoxification of H2O2 by hydroperoxidases [i.e., the heme containing peroxidase/catalase (HPI), and the heme containing catalase (HPII)]. Unlike most pathogenic bacteria, *S*. Typhimurium contains 6 genes whose gene products are devoted toward degradation of H2O2. HPI (encoded by *katG*), HPII (encoded by *katE*), a Mndependent catalase (encoded by *katN*), an NADH-dependent alkyl peroxidase system (encoded by *ahpCF*), and two thiol specific peroxidases (encoded by *tsaA* and *tpx*). HPII and KatN are under positive regulation by the alternative σ factor RpoS, whereas HPI is induced by the redox sensing regulator OxyR during hydrogen peroxide stress (Tartaglia et al., 1989; Ivanova et al., 1994; Robbe-Saule et al., 2001; Vazquez-Torres, 2012). In addition, OxyR activates expression of *ahpC* (Storz et al., 1989; Tartaglia et al., 1989) and also *fur* (Zheng et al., 1999; Varghese et al., 2007). Regulation of *tsaA* appears Fur-independent (Delany et al., 2001) and there is a lack of evidence for whether Fe2<sup>+</sup> and perhaps Fur regulate *tpx*. Deletion of any single gene or in combinations does not influence virulence; only the combined deletion of 5 out of the 6 genes results in reduced virulence signifying the importance of redundant H2O2 scavengers to virulence (Hebrard et al., 2009; Horst et al., 2010). As evident from studies in other bacterial pathogens, there are profound redundancies that contribute to resistance to H2O2 and virulence *in vivo* (Cosgrove et al., 2007; Lindgren et al., 2007; Soler-Garcia and Jerse, 2007). Because SODs and H2O2-degrading enzymes require certain metals as cofactors for enzymatic function and because Fur is a redox sensing protein (Fleischhacker and Kiley, 2011), it is not surprising that Fur is involved in the regulation of defenses against ROS.

SODs and HPI/HPII require the appropriate cofactors; Fe2<sup>+</sup> is required for FeSOD (SodB) and Mn2<sup>+</sup> for MnSOD (SodA) whereas heme is required for HPI and HPII function (Keele et al., 1970; Yost and Fridovich, 1973; Hassan and Fridovich, 1978; Claiborne and Fridovich, 1979; Claiborne et al., 1979). Fur directly represses transcription of the gene encoding the MnSOD (*sodA*) and indirectly activates expression of the gene encoding the FeSOD (*sodB*; Niederhoffer et al., 1990; Tardat and Touati, 1991; Beaumont and Hassan, 1993). This indirect control of *sodB* requires the RNA chaperone Hfq or *ryhB* (Masse and Gottesman, 2002; Ellermeier and Slauch, 2008; Troxell et al., 2011a). In addition, Fur controls HPI/HPII activity in a complex manner that may depend on the ability of Fur to regulate biosynthesis of the heme cofactor (Hamza et al., 2000; Benov and Sequeira, 2003; Hoerter et al., 2005; Mey et al., 2005a; Gaballa et al., 2008) (R. Saah and H. M. Hassan, unpublished data). Surprisingly, despite the enhanced transcription of *sodA* in *fur*, a corresponding increase in MnSOD activity was not observed due to the increased [Fe2+] in the mutant. Indeed, increase in MnSOD activity in *fur* was only discernible upon supplementation of the growth medium with excess [Mn2+] in order to outcompete the available Fe2<sup>+</sup> for the active site of MnSOD (Hassan and Schrum, 1994; Schrum and Hassan, 1994; Troxell et al., 2011a). Thus, with respect to O− <sup>2</sup> defense *fur* behaves phenotypically like *sodAsodB* under Fe2<sup>+</sup> replete conditions. The Fur regulation of Mn2<sup>+</sup> transport is well-documented (Patzer and Hantke, 2001; Kehres et al., 2002; Guedon et al., 2003; Ikeda et al., 2005; Runyen-Janecky et al., 2006; Perry et al., 2012). Furthermore, because *katN* encodes a Mn-containing catalase and is activated by RpoS (Robbe-Saule et al., 2001) and repressed by H-NS (Beraud et al., 2010), it is likely that Fur is involved in *katN* expression in *S.* Typhimurium. Thus, the modulation of the intracellular Mn2<sup>+</sup> concentration will undoubtedly influence protection against ROS and likely virulence. In support of this, numerous studies have demonstrated the importance of Mn2<sup>+</sup> in the regulation of virulence and infectivity (Boyer et al., 2002; Corbin et al., 2008; Anderson et al., 2009; Ouyang et al., 2009; Ogunniyi et al., 2010; Wu et al., 2010; Champion et al., 2011; Kehl-Fie et al., 2011; Damo et al., 2013; Troxell et al., 2013). Likewise, additional members of the Fur family of metal-dependent transcription factors either bind Mn2<sup>+</sup> directly and/or regulate Mn2<sup>+</sup> transport.

#### **Fe2<sup>+</sup> SEQUESTRATION BY THE HOST**

Because Fur requires Fe2<sup>+</sup> as a corepressor the availability of this metal controls Fur activity. Moreover, the Fe2+-Fur complex is inactivated by ROS and reactive nitrogen species (RNS) (D'Autreaux et al., 2002; Varghese et al., 2007), both of which are generated by the host during infection. Humans and other higher eukaryotes produce numerous proteins that sequester free Fe2<sup>+</sup> and heme to deprive the pathogens of iron and meanwhile prevent the toxic formation of ROS. A potent antimicrobial response, including ROS production, produced by innate cells of the host's immune system is activated in response to detection of pathogen-associated molecular patterns (PAMPs) during bacterial infection. Innate cell activation by PAMPs initiates the synthesis of large amounts of Fe2<sup>+</sup> sequestering proteins to limit the available Fe pool for the pathogen, known as "nutritional immunity" (Kehl-Fie and Skaar, 2010; Hood and Skaar, 2012) and activates signaling pathways that causes the host to reduce dietary absorption of Fe that is known as "the anemia of inflammation." In addition, the host responds to infection by increasing the body temperature (the febrile response) as a means to inhibit bacterial growth. The antimicrobial host factors produced during activation of nutritional immunity can be inhibited by the addition of Fe (Weinberg, 1974). Furthermore, the febrile response to bacterial pathogens is antimicrobial, in part, due to the reduced ability of bacteria to acquire Fe2<sup>+</sup> at febrile temperatures (Kluger and Rothenburg, 1979).

Anemia of inflammation by the host in response to infection has been known for more than 60 years (Cartwright et al., 1946; Greenberg et al., 1947; Wintrobe et al., 1947) and the host protein, hepcidin, controls this response (Nicolas et al., 2002; Nemeth et al., 2004a,b). In addition, hepcidin is a host factor that strongly reduces the absorption of dietary Fe (Shayeghi et al., 2005; Drakesmith and Prentice, 2012; Prentice et al., 2012). Because Fe2<sup>+</sup> is required for cellular function within nearly all cells, limiting the availability of Fe2<sup>+</sup> starves pathogens for Fe2<sup>+</sup> and weakens the pathogens' ability to combat antimicrobial responses by the host. Not surprisingly, there is fierce competition for accessibility of Fe2<sup>+</sup> during infection. Phagocytosis of the intracellular pathogen *S.* Typhimurium by macrophages enhances expression of the Fe2<sup>+</sup> export protein ferroportin, which limits the available Fe2<sup>+</sup> during intracellular residence of *S*. Typhimurium (Nairz et al., 2007). Expression of ferroportin correlates directly with reduced bacterial burden of several intracellular pathogens (Paradkar et al., 2008). Thus, the host responds to infection by sequestering Fe2<sup>+</sup> from the local environment of pathogens, limits the absorption of dietary Fe resulting in a very Fe2<sup>+</sup> limiting host environment, and restricts available Fe2<sup>+</sup> within the phagosome.

An important host factor that controls bacterial infection is the natural resistance-associated macrophage protein 1 (NRAMP1, also known as *SLC11A1*) and several research groups determined the contribution of the *SLC11A1* locus to severity of infection within animal models (Plant and Glynn, 1976; Bradley, 1977; Skamene et al., 1982; Brown et al., 2013). *S*. Typhimurium lacking *fur* are avirulent within mice with a functional NRAMP1, whereas the isogenic parent is fully virulent. Mice lacking a functional NRAMP1 are partially resistant to infection with *fur* demonstrating that Fur function is important for virulence, in part, independent of the host NRAMP1 function (Troxell et al., 2011b). Evidence indicates that Fur is functional within an unstimulated macrophage cell-line expressing either a functional or mutated NRAMP1 (Taylor et al., 2009). The NRAMP1 protein is a highly conserved transporter of divalent cations and is expressed within phagocytic cells (Cellier et al., 1995; Canonne-Hergaux et al., 2002; Cellier, 2012); NRAMP1 functions as a transporter of manganese (Mn2+), Fe2+, or cobalt (Co) and is important for acidification of the phagosome (Hackam et al., 1998; Jabado et al., 2000; Forbes and Gros, 2003). Furthermore, NRAMP1 promotes additional host factors of the antimicrobial response including production of nitric oxide (NO) (Fritsche et al., 2003, 2008; Nairz et al., 2009) and production of lipocalin-2 (also called siderocalin), which binds to bacterial siderophores thereby sequestering bacterial Fe2<sup>+</sup> acquisition proteins (Fritsche et al., 2012). However, bacteria have evolved a counter defense mechanism by producing salmochelins, which are structurally distinct from enterochelin and therefore not susceptible to binding by lipocalin-2 (Smith, 2007). RNS and NO perturb Fur-Fe2<sup>+</sup> function within pathogens (Mukhopadhyay et al., 2004; Richardson et al., 2006; Bourret et al., 2008). NO is a crucial factor in the antimicrobial response and its production is regulated by Fe2<sup>+</sup> (Weiss et al., 1994; Melillo et al., 1997; Dlaska and Weiss, 1999). Consequently, the inability to generate NO increases the Fe2<sup>+</sup> content within macrophages, splenic cells, and hepatocytes thereby increasing disease severity in animal models of infection (Nairz et al., 2013). This signifies the importance of NRAMP1 in the ability to sequester Fe2<sup>+</sup> from pathogens and in general antimicrobial response.

#### **CONTROL OF VIRULENCE BY THE Fur FAMILY OF TRANSCRIPTIONAL REGULATORS**

The Fur protein contributes to virulence in animal models for numerous bacterial pathogens (**Table 1**). Although the precise mechanism for the observed attenuation of *fur* mutants is not clear, evidence indicates that a reduction in the activity of enzymes required for protection against ROS may be involved. Furthermore, virulence factors within the *fur* mutants exhibit altered expression or activity, which may additionally contribute to a decrease in virulence. Because Fur also controls expression or activity of enzymes within the TCA cycle, *fur* mutants are defective in the utilization of several carbon sources (i.e., succinate, etc.), which may contribute to the inability of *fur* mutants to cause disease within animal hosts.

There are additional transcription factors within the Fur family that require alternative metals to control gene regulation and virulence. First discovered by work in *B. subtilis* within the lab of John Helmann (Bsat et al., 1998; Mongkolsuk and Helmann, 2002), PerR is widespread in other bacteria and contributes to virulence within pathogens (Van Vliet et al., 1999; Horsburgh et al., 2001a; Rea et al., 2004, 2005; Gryllos et al., 2008).



The DNA-binding activity of PerR is sensitive to relevant concentrations of H2O2 and upon metal-dependent oxidation results in derepression of target genes (Lee and Helmann, 2006). PerR homodimers are detected as two forms, one which contains two ions of Zn2+/Fe2<sup>+</sup> per monomer and one which contains two ions of Zn2+/Mn2<sup>+</sup> per monomer. Only the Zn/Fe form is sensitive to H2O2-induced derepression and, as expected, PerR regulates genes whose protein products detoxify H2O2 (Herbig and Helmann, 2001; Lee and Helmann, 2006). Thus, the H2O2 sensing of PerR is directly influenced by the Mn2+:Fe2<sup>+</sup> ratio within the cell. Maintenance of the Mn2+:Fe2<sup>+</sup> ratio is an important aspect within bacterial pathogens (Veyrier et al., 2011).

Zinc (Zn2+) uptake regulator (Zur) is a Fur family regulator that responds to Zn2<sup>+</sup> and was discovered by two groups working with *E. coli* and *Bacillus subtilis* (*B. subtilis*) (Gaballa and Helmann, 1998; Patzer and Hantke, 1998). As expected for a Fur homolog, Zur represses transcription of Zn2<sup>+</sup> uptake when bound to the corepressor Zn2<sup>+</sup> (Patzer and Hantke, 2000; Gaballa and Helmann, 2002). Because ribosomal proteins utilize Zn2<sup>+</sup> for activity Zur also represses transcription of genes involved in mobilization of Zn2<sup>+</sup> by ribosomal protein paralogs, which may allow for protein synthesis under conditions of Zn2<sup>+</sup> limitation known as the "failsafe" model (Maciag et al., 2007; Natori et al., 2007; Gabriel and Helmann, 2009). The Zur protein or Zn2<sup>+</sup> uptake systems have an important role for bacterial pathogens, which demonstrate the importance of Zn2<sup>+</sup> acquisition during infection (Campoy et al., 2002; Ammendola et al., 2007; Sabri et al., 2009; Smith et al., 2009; Desrosiers et al., 2010; Pesciaroli et al., 2011; Corbett et al., 2012; Dowd et al., 2012; Gielda and Dirita, 2012). The ability to acquire Zn2<sup>+</sup> by bacterial pathogens is likely a broad requirement among bacterial pathogens during infection. More recently, a Fur-homolog was characterized as a Mn2+-dependent DNA-binding protein

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 24 July 2013; accepted: 18 September 2013; published online: 02 October 2013.*

*Citation: Troxell B and Hassan HM (2013) Transcriptional regulation by Ferric Uptake Regulator (Fur) in pathogenic bacteria. Front. Cell. Infect. Microbiol. 3:59. doi: 10.3389/fcimb. 2013.00059*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2013 Troxell and Hassan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Metal-dependent gene regulation in the causative agent of Lyme disease

## *Bryan Troxell\*† and X. Frank Yang*

*Department of Immunology and Microbiology, Indiana University School of Medicine, Indianapolis, IN, USA*

#### *Edited by:*

*Frédéric J. Veyrier, Institut Pasteur, France*

#### *Reviewed by:*

*Jon Skare, Texas A&M Health Science Center, USA Val Culotta, Johns Hopkins University, USA*

#### *\*Correspondence:*

*Bryan Troxell, Prestage Department of Poultry Science, North Carolina State University, 334E Scott Hall, Campus Box 7608, Raleigh, NC 27695-7608, USA e-mail: bryan\_troxell@ncsu.edu*

#### *†Present address:*

*Bryan Troxell, Prestage Department of Poultry Science, North Carolina State University, Raleigh, USA*

*Borrelia burgdorferi* (*Bb*) is the causative agent of Lyme disease transmitted to humans by ticks of the *Ixodes* spp. *Bb* is a unique bacterial pathogen because it does not require iron (Fe2+) for its metabolism. *Bb* encodes a ferritin-like Dps homolog called NapA (also called BicA), which can bind Fe or copper (Cu2+), and a manganese (Mn2+) transport protein, *Borrelia* metal transporter A (BmtA); both proteins are required for colonization of the tick vector, but BmtA is also required for the murine host. This demonstrates that *Bb*'s metal homeostasis is a critical facet of the complex enzootic life cycle between the arthropod and murine hosts. Although metals are known to influence the expression of virulence determinants during infection, it is unknown how or if metals regulate virulence in *Bb*. Recent evidence demonstrates that *Bb* modulates the intracellular Mn2<sup>+</sup> and zinc (Zn2+) content and, in turn, these metals regulate gene expression through influencing the Ferric Uptake Regulator (Fur) homolog *Borrelia* Oxidative Stress Regulator (BosR). This mini-review focuses on the burgeoning study of metal-dependent gene regulation within *Bb*.

**Keywords:** *Borrelia burgdorferi***, Lyme disease, copper, manganese, zinc, calprotectin**

## **INTRODUCTION**

*Borrelia burgdorferi* (*Bb*) is the causative agent of a multisystem disorder known as Lyme disease. *Bb* persists within an enzootic cycle that includes two diverse hosts, a tick vector and a warm-blooded host, typically small rodents. "Hard ticks" of the *Ixodes* genus are important arthropod hosts for colonization by *Bb*. *Ixodes* ticks are slow-feeding ticks (≈48 h for a bloodmeal) that have a 2-year life cycle including three distinct stages: larvae, nymph, and adult (**Figure 1**). At each stage, ticks will feed once on a warm-blooded host then undergo a molting process, which precedes a period of dormancy that may last months (**Figure 1**). Because *Bb* colonization of ticks does not appear to occur through transovarial transmittance, unfed larvae ticks are naïve and acquire *Bb* during feeding on an infected warmblooded host. Feeding ticks can acquire *Bb* at any stage of the usual 2-year life cycle and transmission of *Bb* can occur during feeding on an animal host at any subsequent stage of the life cycle. Small rodents (especially the white-footed mouse, *Peromyscus leucopus*) are the primary animal reservoirs for *Bb* within this enzootic cycle and are sources for the bloodmeal during the larval and nymphal stages (**Figure 1**). Unlike most bacterial pathogens, *Bb* lacks lipopolysaccharide (LPS), lipooligosaccharide (LOS), and capsule (Radolf and Samuels, 2010). *Bb* is highly motile due to the presence of flagella; however, *Bb*'s flagella are contained within the periplasmic space between the outer and inner membranes. Therefore, *Bb*'s flagella is not surface exposed and is called an endoflagella. The endoflagella are anchored at each end of the cell and provide *Bb* with a characteristic corkscrew movement. Despite *Bb*'s limited metabolism and fastidious nature *Bb* survives within two hosts, a tick vector and a small rodent host. Other animals, such as humans, are infected by *Bb*, but are not considered important for persistence of *Bb* within the enzootic cycle. Of significant interest, *Bb* is one of the few pathogens that does not require iron (Fe2+) to grow (Posey and Gherardini, 2000). Given the importance of Fe2<sup>+</sup> in the regulation of virulence within other bacteria, it is not clear which metals *Bb* utilize for regulating virulence factors. Recent work suggests that metals may play an important role in regulation of virulence within *Bb*.

Metal homeostasis is important to maintain the metabolism of bacterial pathogens. This is accomplished through the combined action of metal transporters, both importers and exporters, which control the abundance of specific metals and the ratio of the transition metals within the cell. Although some metal transporters are highly specific for a cognate metal, others are capable of importing several metals with different affinity of each metal. In addition to the importance of metals in bacterial physiology, metals play a critical role in the control of gene regulation within pathogens. The role of metals within *Bb* is not fully understood. Only a single protein, *Borrelia* metal transporter A (BmtA) is known to participate in metal transport. Analysis of the intracellular metal content with *in vitro* grown *Bb* suggests that BmtA transports Mn2<sup>+</sup> since this metal is nearly undetectable in *bmtA* strains (Ouyang et al., 2009a; Troxell et al., 2013). BmtA may also be involved in the import/export of other metals since deletion of *bmtA* alters the intracellular concentrations of Fe2+, Cu2+, and Zn2<sup>+</sup> (Wang et al., 2012). The mechanism of BmtA-dependent metal transport is still unknown, but recent evidence indicates that BmtA and Mn2<sup>+</sup> are involved in regulation of virulence through a Ferric uptake regulator (Fur) homolog named *Borrelia* Oxidative Stress Regulator (BosR). BosR is redox sensing DNA binding protein that utilizes Zn2<sup>+</sup> as a cofactor (Boylan et al., 2003; Katona et al., 2004). Discussed here is the role of metals in

**FIGURE 1 | The usual 2-year enzootic cycle of the Lyme disease spirochete.** A naïve *Ixodes scapularis* larvae will feed on a small rodent near the end of the Summer season or early Fall. The feeding larvae can acquire *Bb* at this feeding (1st feeding) and remain colonized throughout the molting process, which occurs during the Winter season. For the 2nd feeding, infected nymphs will feed late in the Spring season or early in the Summer season. The infected nymphs transmit *Bb* to either a small rodent host, which maintains the enzootic cycle in nature, or humans (accidental host). Infected humans develop Lyme disease and may develop erythema migrans (signified by a red bulls eye near the shoulder in the figure shown) shortly after an infected nymph feeds. Typically, if subject to late Lyme manifestations Lyme disease patients develop Lyme arthritis at one or both knee joints (signified by a red lightning bolt near the knee in the figure shown). For the final feeding (3rd feeding), nymphs will molt and emerge as adults to feed on large mammals, such as deer, during the Fall season. Deer are considered incompetent hosts for *Bb*, but the 3rd feeding is important in the enzootic cycle because female ticks will mate and lay eggs over the Winter season. Naïve larvae will emerge following hatching and the cycle begins anew.

*Bb* physiology and gene expression as it relates to virulence factors required *in vivo*.

## *Bb***: A NON-COMBATANT IN THE WAR FOR Fe2<sup>+</sup>**

Just as a siege limits the influx of food and supplies to an enemy's stronghold, during infection the host transports metals away from the locale of pathogens and synthesizes copious amounts of metal-chelating proteins to limit access of these essential micronutrients. The hosts' ability to produce metal-chelating proteins is important for defending against pathogens since deletion of the chelating protein calprotectin enhances virulence of *Acinetobacter baumannii*, *Staphylococcus aureus*, and the opportunistic yeast pathogen *Candida albicans* (Corbin et al., 2008; Kehl-Fie et al., 2011; Damo et al., 2013). Calprotectin can bind Mn2<sup>+</sup> and Zn2<sup>+</sup> and is an abundant protein present in neutrophils (Yui et al., 2003), which are an early host defender against invading pathogens. Some bacterial pathogens are capable of overcoming the growth inhibition exerted by calprotectin; *Salmonella enterica* serovar Typhimurium (*S*. Typhimurium) expresses a high affinity Zn2<sup>+</sup> ATP-binding cassette (ABC) transport system that outcompetes Zn2<sup>+</sup> chelation by calprotectin (Liu et al., 2012). Calprotectin is known to inhibit *in vitro* growth of *Bb* through Zn2<sup>+</sup> sequestration (Lusitani et al., 2003). The contribution of calprotectin to *Bb* growth *in vivo* is unknown, but *Bb* encodes several putative uncharacterized ABC transporters that could be involved in metal transport during infection. In addition, whether calprotectin inhibits *Bb* growth through Mn2<sup>+</sup> chelation is unknown. The fierce war between the pathogen and host for accessibility of Fe2<sup>+</sup> poses a problem to pathogens; however, *Bb* has evolved a novel solution by becoming a noncombatant in the war for Fe2+. *Bb* does not appear to transport Fe2+, lacks many biosynthetic and catabolic pathways that require Fe2+, and exhibits no defect in growth in the absence of detectable Fe2<sup>+</sup> (Posey and Gherardini, 2000). Although a recent study indicates there is detectable Fe2<sup>+</sup> within *Bb*, the physiological relevance of this finding remains uncertain (Wang et al., 2012). Another study did not detect intracellular Fe2<sup>+</sup> following *in vitro* cultivation of *Bb* (Aguirre et al., 2013). Therefore, additional experiments are required to address these discrepancies. At this point, how Fe2<sup>+</sup> is transported within *Bb* is unknown. Future work is required to determine the contribution of intracellular Fe2<sup>+</sup> to *Bb* gene regulation and metabolism. Instead, because calprotectin inhibits *Bb* growth by Zn2<sup>+</sup> sequestration, the existing data suggests that Zn2<sup>+</sup> is an important metal within the metabolism of *Bb*. This is supported by the Zn2+ dependent enzymatic activity of peptide deformylase (Nguyen et al., 2007) and the glycolytic enzyme fructose-1,6-bisphosphate aldolase (Bourret et al., 2011). Furthermore, peptide deformylase may be an essential enzyme (Jain et al., 2005) and, since glycolysis is the sole mechanism for the generation of ATP within *Bb*, Zn2<sup>+</sup> may be a critical metal for *Bb*.

## **BicA AND BmtA: TWO PROTEINS WITH NOVEL FUNCTION WITHIN** *Bb*

Bacteria encode metal binding proteins (ferritins or ferritinlike proteins) that store metals and serve as a facile source of essential metals when encountering a metal-depleted environment. *Bb* encodes a metal binding protein (NapA or BicA) that exhibits homology to the ferritin-like Dps present in other bacteria. Purified BicA is capable of binding Fe2<sup>+</sup> or Cu2+, but lacks either metal when isolated (Li et al., 2007; Wang et al., 2012). The majority of studies have focused on the role of Fe2<sup>+</sup> chelation by the host in nutritional immunity, but recent evidence demonstrates the importance of chelating Zn2<sup>+</sup> and Mn2<sup>+</sup> in thwarting bacterial infections (Kehl-Fie and Skaar, 2010). However, as part of the antimicrobial defense present within ticks, an antimicrobial peptide, known as microplusin, inhibits bacterial growth by Cu2<sup>+</sup> chelation (Silva et al., 2009, 2011). Microplusin is expressed within the hemocele of ticks (Esteves et al., 2009), implying that this locale is a Cu2<sup>+</sup> limited environment. *Bb* does appear to regulate its intracellular Cu2+, but the relevance or need for Cu2<sup>+</sup> is unknown (Wang et al., 2012). The importance of BicA to the enzootic cycle is restricted to residence within the tick vector (Li et al., 2007), implying that Zn2<sup>+</sup> and Cu2<sup>+</sup> are limiting within this host.

The role of Mn2<sup>+</sup> in *Bb* metabolism is not understood. The gene *bmtA*, encoding a Mn2<sup>+</sup> transport protein BmtA, is not essential for *in vitro* growth within virulent *Bb* strains from the B31 (tick isolated) and 297 (human isolated) lineages despite reducing cellular Mn2<sup>+</sup> to near undetectable concentrations (Ouyang et al., 2009a; Troxell et al., 2013). *Bb* cultivation *in vitro* requires a complex growth medium called BSK (Barbour, 1984). Treatment of BSK medium with a chelating resin, called Chelex, results in significant changes of the concentrations of metals. Chelex treatment of BSK reduces Zn2+, but Mn2<sup>+</sup> becomes undetectable in the medium. Despite the undetectable Mn2<sup>+</sup> in Chelex-treated BSK growth medium, no growth defects are observed for wild-type or *bmtA* strains during cultivation in this medium (Troxell et al., 2013). BmtA has homology to the GufA family of metal transporters (Guerinot, 2000). BmtA has 8 membrane spanning domains and is predicted to transport cations through a novel mechanism (Ouyang et al., 2009a). To date, only a single protein within *Bb* is characterized as being Mn2+-dependent; specifically, the superoxide dismutase (SOD) encoded by *sodA* (Troxell et al., 2012; Aguirre et al., 2013). The expression of *bmtA* and the intracellular concentration of Mn2<sup>+</sup> are enhanced during cultivation at 25◦C, suggesting there may be a requirement for Mn2<sup>+</sup> at cooler temperatures (Ojaimi et al., 2003; Troxell et al., 2013). The physiological need for more Mn2<sup>+</sup> at 25◦C is unknown, but this may be due to the need for defense against reactive oxygen species (ROS) because *Bb* encodes a Mn-dependent SOD and lower temperatures contain increased concentrations of dissolved O2 that could lead to enhanced formation of superoxide radical (O− <sup>2</sup> ) (Troxell et al., 2012; Aguirre et al., 2013). However, *Bb* may encode additional proteins that require Mn2+.

Mn2<sup>+</sup> is considered an essential trace element within biology. In bacteria, Mn2<sup>+</sup> is critical for defense against several stresses such as oxidative stress, bile stress, and resistance to antibiotics (Anjem et al., 2009; Srinivasan et al., 2012). In addition, Mn2<sup>+</sup> is involved in gene regulation through indirect mechanisms. For instance, the alarmone guanosine tetraphosphate (ppGpp) is synthesized and degraded by SpoT/RelA homolog proteins. During conditions of nutrient deprivation, ppGpp is synthesized and binds to the RNA polymerase (RNAP) in order to enhance transcription of genes important for survival or virulence while reducing transcription of genes involved in growth and cell division (Magnusson et al., 2005). SpoT/RelA homologs contain a highly conserved Mn2<sup>+</sup> binding site and require Mn2<sup>+</sup> as a cofactor for the enzymatic degradation of ppGpp (Sy, 1977; Sun et al., 2010). *Bb* encodes a SpoT/RelA homolog, *bb0198*, that is induced during serum starvation and is responsible for both synthesis and degradation of ppGpp (Concepcion and Nelson, 2003; Bugrysheva et al., 2005). This suggests that *Bb* may require Mn2<sup>+</sup> in order to initiate cell growth. Recently, *Bb*'s peptide deformylase was isolated with bound Mn2<sup>+</sup> (Aguirre et al., 2013); however, an enzymatic assay of the Mn-bound enzyme was not conducted. Whether peptide deformylase functions with Mn2<sup>+</sup> is unknown, but this enzyme is active with Zn2<sup>+</sup> as a cofactor (Nguyen et al., 2007). Future work is needed to determine the metal specificity of BB0198 and the peptide deformylase and to identify *Bb* proteins that require Mn2+.

Surprisingly, some enhancement in the intracellular concentration of Zn2<sup>+</sup> for *bmtA* has been noted (Ouyang et al., 2009a; Wang et al., 2012). It has been hypothesized that within *bmtA* there may be compensation for the reduction of Mn2<sup>+</sup> by enhancing the transport of Zn2<sup>+</sup> and thereby replacing the requirement of Mn2<sup>+</sup> with Zn2+. Although future work is required to fully test this hypothesis, the replacement of Mn2<sup>+</sup> for Zn2<sup>+</sup> in Mn2+-dependent enzymes causes a pronounced reduction in catalytic efficiency or abrogates enzymatic activity altogether (Ose and Fridovich, 1976; Sobota and Imlay, 2011; Gu and Imlay, 2013). Metal-dependent transcription factors can utilize a variety of metals for function, i.e., Mn2<sup>+</sup> or Fe2<sup>+</sup> in the case of Fur (Privalle and Fridovich, 1993), and host metalsequestering proteins exhibit promiscuity in metal binding, which is demonstrated by the Mn2<sup>+</sup> or Zn2<sup>+</sup> binding site (S1 site) in calprotectin (Damo et al., 2013). This is in contrast to metaldependent enzymes, which exhibit stringent metal specificity for activity, as is the case for SpoT/RelA homologs and *Bb*'s SodA (Sy, 1977; Troxell et al., 2012; Aguirre et al., 2013). However, because many of *Bb*'s putative metalloenzymes are uncharacterized, the possibility exists that a significant number of these proteins can utilize either Mn2<sup>+</sup> or Zn2<sup>+</sup> within the cell.

## *Bb***'S METAL REQUIREMENT WITHIN THE TICK**

The unfed tick is presumed to be a nutrient deprived environment for *Bb*. Starvation conditions may mimic oxidative stress conditions and factors responsible for defense against ROS are also important for survival during starvation (Jenkins et al., 1988; Nystrom et al., 1996). *Bb* may require Mn2<sup>+</sup> in order to defend against ROS that occurs during onset of the bloodmeal. Although Mn2<sup>+</sup> complexed with other biological compounds, such as bicarbonate, are capable of degrading ROS, this requires large concentrations of intracellular Mn2<sup>+</sup> that occurs within *Lactobacillus plantarum* (Archibald and Fridovich, 1981, 1982; Stadtman et al., 1990). In the only report to compare directly the intracellular Mn2<sup>+</sup> content of *L. plantarum* with *Bb*, it was observed that *Bb* contains 20 to 100-fold lower intracellular Mn2<sup>+</sup> compared to *L. plantarum*, indicating this is an unlikely mechanism for ROS defense within *Bb* (Posey and Gherardini, 2000). However, *Bb*'s intracellular Mn2<sup>+</sup> can fluctuate during *in vitro* growth conditions (Troxell et al., 2013), suggesting that environmental conditions within the tick-mouse life cycle may exist whereby *Bb* could contain sufficient intracellular Mn2<sup>+</sup> to degrade ROS in a manner similar to *L. plantarum*. Although *sodA* is required for infection of the murine host (Esteve-Gassent et al., 2009), the contribution of *sodA* within the tick vector is unknown. It is currently unclear if *Bb* contains a high intracellular Mn2<sup>+</sup> within the unfed tick or is starved for metals. Because of the involvement of BicA in Cu2<sup>+</sup> and Zn2<sup>+</sup> homeostasis and since *bicA* exhibits a defect within the unfed tick (Li et al., 2007), the results support the notion that these two metals are limiting. In addition, the contribution of BmtA to the unfed tick is unknown.

## **REGULATION OF σ<sup>S</sup> BY Zn2<sup>+</sup> AND Mn2<sup>+</sup> WITHIN** *Bb*

*Bb* is capable of surviving within two diverse hosts through changes in gene expression, specifically outer surface lipoproteins that modulate adaptation within each host. Outer Surface Proteins A (OspA) and C (OspC) are a lipoproteins produced by *Bb* within the tick and animal host, respectively. *Bb* contains a limited genome that contains a relatively small number of transcription factors and sigma factors: *Bb* encodes only three sigma factors the housekeeping σ70, and two alternative sigma factors, RpoN (σ54) and RpoS (σS) (Fraser et al., 1997; Samuels, 2011; Radolf et al., 2012). In addition, *Bb* genome encodes only one bacterial enhancer binding protein (bEBP), known as Rrp2, which is involved in σ<sup>54</sup> activation. The requirement of the Rrp2- RpoN-RpoS pathway (or Rrp2-σ54-σ<sup>S</sup> sigma factor cascade) in the regulation of *ospA* and *ospC* demonstrates the importance of this regulatory network (Hubner et al., 2001; Yang et al., 2003; Caimano et al., 2004; Fisher et al., 2005; Gilbert et al., 2007). Rrp2 and σ<sup>54</sup> directly activates transcription of *rpoS* (Smith et al., 2007; Blevins et al., 2009). σ*<sup>S</sup>* then activates transcription of *ospC* by direct binding to the promoter of *ospC* (Yang et al., 2005) and also represses expression of *ospA* (Caimano et al., 2007). In addition, BosR, a Fur/PerR-like family transcription factor and a Zn2+-dependent DNA binding protein, has been shown to be essential for transcription of *rpoS* (Ouyang et al., 2009b, 2011). More recently, Wang et al. demonstrated that BosR may also directly repress *ospA* (Wang et al., 2013). Because RpoS regulates many genes important for *Bb* transmission and mammalian infection such as *ospC*, this pathway is essential for the enzootic cycle of *Bb* (Caimano et al., 2004; Grimm et al., 2004; Pal et al., 2004; Boardman et al., 2008; Ouyang et al., 2008). Moreover, *bosR* is required for transmission from the tick vector and infection of the mammalian host (Hyde et al., 2009; Ouyang et al., 2009b). Thus, *Bb* has evolved to utilize the transcription factor BosR for virulence.

*Bb* is a highly fastidious pathogen. The cultivation of *Bb* requires a complex medium that is analogous to cell culture media for eukaryotic cells (Barbour, 1984). Comparisons of *Bb* replication within a feeding tick and during *in vitro* growth at 35–37◦C demonstrate that both conditions support growth with a generation time of ≈8–10 h (De Silva and Fikrig, 1995). Metal analysis of the cultivation medium for *Bb* indicates there is <sup>≈</sup>5μM Zn2+, <sup>≈</sup>4μM Cu2+, and <sup>≈</sup>0.1μM Mn2<sup>+</sup> (Wang et al., 2012; Troxell et al., 2013). Besides Fe2+, other transition metals, such as Zn2<sup>+</sup> and Mn2<sup>+</sup> are known to influence gene regulation within bacterial pathogens (Corbin et al., 2008). Based on the Zn2+-dependent nature of BosR (Boylan et al., 2003; Katona et al., 2004), and because BosR regulates *rpoS*, Zn2<sup>+</sup> could regulate *rpoS* within *Bb*.

Metal analysis indicates that while intracellular Zn2<sup>+</sup> remained relatively constant under different conditions, Mn2<sup>+</sup> was subject to temperature-dependent regulation within *Bb* (Troxell et al., 2013). Moreover, the intracellular Mn2<sup>+</sup> can fluctuate 20-fold during *in vitro* growth conditions and the temperaturedependent inverse concentration of intracellular of Mn2<sup>+</sup> is reminiscent of the inverse regulation of *ospA* and *ospC* within *Bb* (Stevenson et al., 1995; Obonyo et al., 1999; Yang et al., 2000; Alverson et al., 2003). To test if Mn2<sup>+</sup> could suppress regulation by σS, MnCl2 was added to cultures growing under conditions of σ<sup>S</sup> activation. The addition of MnCl2 increases intracellular Mn2<sup>+</sup> and reduces the expression of *rpoS* and σS-activated *ospC* (Troxell et al., 2013). The addition of excess ZnSO4 increases the intracellular Zn2+, increases the level of BosR protein, and abrogates the repression of *rpoS* by Mn2+. Surprisingly, MnCl2 did

**FIGURE 2 | Known and putative roles of Mn2+, Cu2+, and Zn2<sup>+</sup> in gene regulation and metabolism of** *Bb***.** A schematic of the importance of transition metals within *Bb* is shown with a magnification of a section from a single *Bb* cell. Extracellular Mn2<sup>+</sup> is transported through BmtA and supplies the appropriate cofactor for the Mn-SOD and possibly the SpoT/RelA homolog BB0198 (designated by a pink arrow). In addition, Mn2<sup>+</sup> reduces the level of BosR protein (designated by a pink blunted line), which controls transcription of the alternative sigma factor, *rpoS* (not shown). The putative role of Mn2<sup>+</sup> as a cofactor for additional unknown enzymes is shown with a pink box. Zn2<sup>+</sup> transport is uncharacterized in *Bb*, but is presumed to be transported by a membrane bound protein. The requirement for Zn2<sup>+</sup> within

*Bb* is likely to include enzymes within glycolysis, such as fructose 1,6-bisphosphatase (BB0445), and the peptide deformylase (BB0065) shown in the white box. Zn2<sup>+</sup> is a known cofactor for the DNA binding protein BosR. Therefore, the intracellular Mn2+:Zn2<sup>+</sup> can modulate the level of BosR protein. The transporter for Cu2<sup>+</sup> and the role of Cu2<sup>+</sup> within *Bb* is unknown, but BicA may be involved in transport and homeostasis (blue box). Moreover, the contribution of Cu2<sup>+</sup> to gene regulation within *Bb* is unknown, but is predicted to involve redox sensing transcription factors (Changela et al., 2003; Gomez-Santos et al., 2011). Future work is required to elucidate the complete role of these metals in gene regulation and physiology of this important vector borne pathogen.

not influence transcription of *bosR*, but reduced the level of the BosR protein (Troxell et al., 2013). In addition, deletion of *bmtA* in two infectious strains does not alter *bosR* transcription, but enhances temperature-dependent activation in the level of BosR, which results in increased transcription of *rpoS* and *ospC*. As an earlier study shows, the BosR protein level is increased by CO2 despite the inability of dissolved CO2 to regulate transcription of *bosR* (Hyde et al., 2007). These combined results suggest that either metals or CO2 may control the level of the BosR protein, which activates transcription of *rpoS*. Correlation of the intracellular Mn2+:Zn2<sup>+</sup> indicates that the ratio between these two metals play an important role in the level of BosR protein and *rpoS* regulation. Collectively, these results support the hypothesis that a combined reduction in intracellular Mn2<sup>+</sup> while increasing Zn2<sup>+</sup> regulates σ<sup>S</sup> by dramatically enhancing the level of BosR protein.

Why does *Bb* require *bmtA* for the enzootic cycle? The presence of excess Mn2<sup>+</sup> suggests there is a collection of unknown targets that require Mn2<sup>+</sup> for activity. One function may be to control σ<sup>S</sup> activation during the enzootic cycle. Precise regulation of *ospC* and other outer surface proteins, such as *vlsE*, is required for infection of the murine host; constitutive activation of either surface protein results in rapid elimination of *Bb* by either innate cells or the humoral response of the host (Liang et al., 2002; Xu et al., 2006, 2008a,b). In the absence of any defined metabolic requirement for Mn2<sup>+</sup> within *Bb*, the importance of Mn2<sup>+</sup> to the enzootic cycle could be to control regulation of highly immunogenic outer surface proteins. Nevertheless, the limited genome of *Bb* encodes several homologs that may require Mn2<sup>+</sup> for activity. Furthermore, BmtA appears to influence not only the intracellular Mn2<sup>+</sup> concentration, but also the concentrations of Cu2<sup>+</sup> and Zn2+. How Zn2<sup>+</sup> and Cu2<sup>+</sup> are transported within *Bb* is unknown, but it is likely that these metals are required for proper regulation of virulence genes and unknown metabolic genes. Future work will no doubt shed light on the importance of these metals as cofactors and their influence on gene regulation. This is summarized in **Figure 2**, which depicts the known and putative roles of Mn2+, Cu2+, and Zn2<sup>+</sup> within *Bb*.

#### **CONCLUSIONS**

Unlike most bacterial pathogens, *Bb* does not require Fe2<sup>+</sup> for growth, which presents a unique model system to study metaldependent gene regulation and stress responses. The bloodmeal is rich in Zn2+/Cu2<sup>+</sup> and relatively poor in Mn2+, which suggests that *Bb*'s intracellular Zn2+/Cu2<sup>+</sup> content may increase through unidentified transporters (**Figure 2**). Because Mn2<sup>+</sup> regulates the BosR protein level, but not *bosR* transcription (Troxell et al., 2013), the low Mn2<sup>+</sup> content in blood may further enhance expression *rpoS*, which is required for *Bb* to exit the tick midgut and reach the salivary glands during tick feeding (Fisher et al., 2005; Dunham-Ems et al., 2012). How does *Bb* coordinate the regulation of transport of Mn2+, Cu2+, and Zn2<sup>+</sup> during the enzootic cycle? What is apparent from *in vitro* work is that the intracellular Mn2+:Zn2<sup>+</sup> ratio regulates transcription of the alternative sigma factor, *rpoS*, which controls activation of genes required for infection of mammals. A caveat to these studies is the heavy reliance on *in vitro* experiments due to the difficulties of measuring intracellular metal content while detecting changes in gene expression during *in vivo* studies. Infection studies with *bicA* and *bmtA* demonstrate the importance of these genes within the enzootic cycle, but the mechanism for why *Bb* requires them is unknown. Although the contribution to the unfed tick is known for *bicA*, the contribution of *bmtA* to survival within the dormant tick is unknown. How BicA and BmtA control metal homeostasis or gene expression *in vivo* would greatly improve our understanding of their importance in infection. Moreover, the identification of a dedicated Zn transport system and Cu transport system within *Bb* would provide additional and much needed clarity. It is clear that we are only beginning to understand the importance of metals in the metabolism and gene regulation within the Lyme disease spirochete

#### **ACKNOWLEDGMENTS**

Bryan Troxell was supported by NIH T32 AI060519. Funding for this work was partially provided by NIH grants AI083640 and AI085242, Indiana INGEN and METACyt grants of Indiana University, funded by the Lilly Endowment, Inc (to X. Frank Yang).

## **REFERENCES**


lipoproteins primarily expressed in the tick during mammalian infection. *Mol. Microbiol.* 89, 1140–1153. doi: 10.1111/mmi.12337


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 30 July 2013; accepted: 29 October 2013; published online: 15 November 2013.*

*Citation: Troxell B and Yang XF (2013) Metal-dependent gene regulation in the causative agent of Lyme disease. Front. Cell. Infect. Microbiol. 3:79. doi: 10.3389/ fcimb.2013.00079*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2013 Troxell and Yang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*