Objectives: Nitric oxide (NO) works as an
antimicrobial agent of many pathogens due to its reactivity as S-nitrosylating
agent. The aim of this study was to determine whether S-nitrosylation
of proteins of the parasite, Entamoeba histolytica regulates their
function.
Methodology & Results: We used the resin-assisted
capture method to identify S-nitrosylated proteins. Some of the S-nitrosylated
proteins as peroxiredoxin and superoxide dismutase may be involved in the
resistance to NO. However, very little information is available on the
mechanism of resistance to NO in this parasite. We observed that exposure of
the parasites to NO strongly reduces their viability and protein synthesis;
however, the deleterious effects of NO were significantly reduced in
trophozoites overexpressing the cytosine-5 methyltransferases of the Dnmt2
family (Ehmeth). In addition, these trophozoites exhibit high levels of tRNAAsp
methylation suggesting that Ehmeth –mediated- tRNAAsp
methylation is part of the resistance mechanism to NO.
We previously reported that enolase, a glycolytic
enzyme, binds to Ehmeth and inhibits its activity. We observed that the amount
of Ehmeth-enolase complex is significantly reduced in NO-treated E.
histolytica which explains the aforementioned increase of tRNA methylation.
Indeed, we demonstrated via
site-directed mutagenesis that cysteine residues 228
and 229 in Ehmeth are susceptible to S-nitrosylation and are crucial for Ehmeth
binding to enolase.
Conclusion: Our work provides the first global analysis of S-nitrosylated
proteins in E.histolytica and indicates that Ehmeth plays a central role
in the response of the parasite to NO.
