AUTHOR=Huang Yan , Zheng Xiuping , Zhang Hongli , Ding Haojie , Guo Xiaolu , Yang Yi , Chen Xueqiu , Zhou Qianjin , Du Aifang 

TITLE=Site-Directed Mutagenesis Study Revealed Three Important Residues in Hc-DAF-22, a Key Enzyme Regulating Diapause of Haemonchus contortus

JOURNAL=Frontiers in Microbiology

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2017.02176

DOI=10.3389/fmicb.2017.02176

ISSN=1664-302X

ABSTRACT=<p><italic>Haemonchus contortus</italic> (<italic>H. contortus</italic>) is one of the most important parasites of small ruminants, especially goats and sheep. The complex life cycle of this nematode is a main obstacle for the control and prevention of haemonchosis. So far, a special form of arrested development called diapause different from the dauer stage in <italic>Caenorhabditis elegans</italic> (<italic>C. elegans</italic>) has been found in many parasitic nematodes. In our previous study, we have characterized a novel gene <italic>Hc-daf-22</italic> from <italic>H. contortus</italic> sharing high homology with <italic>Ce-daf-22</italic> and functional analysis showed this gene has similar biological function with <italic>Ce-daf-22</italic>. In this study, <italic>Hc-daf-22</italic> mutants were constructed using site-directed mutagenesis, and carried out rescue experiments, RNA interference (RNAi) experiments and <italic>in vitro</italic> enzyme activity analysis with the mutants to further explore the precise function site of Hc-DAF-22. The results showed that <italic>Hc-daf-22</italic> mutants could be expressed in the rescued <italic>ok693</italic> worms and the expression positions were mainly in the intestine which was identical with that of <italic>Hc-daf-22</italic> rescued worms. Through lipid staining we found that <italic>Hc-daf-22</italic> could rescue <italic>daf-22</italic> mutant (<italic>ok693</italic>) from the fatty acid metabolism deficiency while <italic>Hc-daf-22</italic> mutants failed. Brood size and body length analyses in rescue experiment along with body length and life span analyses in RNAi experiment elucidated that <italic>Hc-daf-22</italic> resembled <italic>Ce-daf-22</italic> in effecting the development and capacity of <italic>C. elegans</italic> and mutants impaired the function of <italic>Hc-daf-22</italic>. Together with the protease activity assay, this research revealed three important active resides 84C/299H/349H in Hc-DAF-22 by site-directed mutagenesis.</p>