In the human large intestine, nitrosative stress, which is generated by innate immune cells, is one of the various environmental challenges that Entamoeba histolytica encounters during its life cycle. Why nitric oxide (NO) kills this parasite and can the parasite develop resistance to NO are open questions that have not been thoroughly resolved. In order to fill this knowledge gap, we identified S-nitrosylated (SNO) proteins in trophozoites exposed to NO, using resin-assisted capture (RAC). We found that many of the key enzymes that are involved in the metabolism and virulence of E.histolytica are nitrosylated which lead to their inhibition and consequently to the parasite’s death. We also observed that protein synthesis is inhibited by NO; however, the deleterious effects of NO were significantly reduced in trophozoites overexpressing the cytosine-5 methyltransferases of the Dnmt2 family (Ehmeth). These trophozoites exhibit high levels of tRNAAsp methylation and they selectively express proteins that are involved in the resistance to nitrosative stress like peroredoxin. To gain insight into the mechanism of resistance to NO, trophozoites were made resistant to lethal amount of NO by stepwise exposures to increasing amounts of this molecule. The outcome of several physiological and omics experiments (RNA seq & SNO RAC) to characterize the NO-resistant strain will be discussed.
In summary, this work provides the first global analysis of SNO proteins in E.histolytica and reveals an unexpected link between Dnmt2-mediated tRNA methylation and resistance to nitrosative stress. We also demonstrated that E.histolytica can develop resistance to NO.
