Nitric oxide (NO) works as an antimicrobial agent of many
pathogens due to its reactivity as S-nitrosylating agent. In
order to determine whether S-nitrosylation of proteins of the parasite, Entamoeba
histolytica regulates their function, we used the resin-assisted capture
method to identify S-nitrosylated proteins. We observed that S-nitrosylated
proteins of E. histolytica are mainly involved in glycolysis/
gluconeogenesis, pyruvate metabolism, and ribosome synthesis. Indeed, NO
inhibits many of the key enzymes involved in the metabolism and virulence of
the parasite E. histolytica via their S-nitrosylation.
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, another 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.
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 and it
contributes to the growing evidence towards NO as an emerging
regulator of epigenetic mechanisms.