The Entamoeba histolytica Dnmt2 Homolog (Ehmeth) Confers Resistance to Nitrosative Stress

Abstract

c Nitric oxide (NO) has antimicrobial properties against many pathogens due to its reactivity as an S-nitrosylating agent. It inhibits many of the key enzymes that are involved in the metabolism and virulence of the parasite Entamoeba histolytica through S-nitrosylation of essential cysteine residues. Very little information is available on the mechanism of resistance to NO by pathogens in general and by this parasite in particular. Here, we report that exposure of the parasites to S-nitrosoglutathione (GSNO), an NO donor molecule, strongly reduces their viability and protein synthesis. However, the deleterious effects of NO were significantly reduced in trophozoites overexpressing Ehmeth, the cytosine-5 methyltransferase of the Dnmt2 family. Since these trophozoites also exhibited high levels of tRNA Asp methylation, the high levels suggested that Ehmeth-mediated tRNA Asp 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 was significantly reduced in GSNOtreated E. histolytica, which explains the aforementioned increase of tRNA methylation. Specifically, we demonstrated via sitedirected mutagenesis that cysteine residues 228 and 229 of Ehmeth are susceptible to S-nitrosylation and are crucial for Ehmeth binding to enolase and for Ehmeth-mediated resistance to NO. These results indicate that Ehmeth has a central role in the response of the parasite to NO, and they contribute to the growing evidence that NO is a regulator of epigenetic mechanisms.

N-acetyl ornithine deacetylase is a moonlighting protein and is involved in the adaptation of Entamoeba histolytica to nitrosative stress

“…The QRT-PCR values were normalized to the level of rDNA74. Data are expressed as the mean ± SD of three independent experiments.…”

Downregulation of methyltransferase Dnmt2 results in condition‐dependent telomere shortening and senescence or apoptosis in mouse fibroblasts

“…Overexpression of dDnmt2 promoted longevity that was accompanied by resistance to oxidative stress (paraquat treatment) and upregulation of small heat shock proteins, namely Hsp22, Hsp23, and Hsp26 (Lin et al, ). Overexpression of another Dnmt2, namely Ehmeth, also conferred resistance to HP treatment and nitrosative stress ( S ‐nitrosoglutathione stimulation) and caused upregulation of heat shock protein 70 in the protozoan parasite E. histolytica (Fisher et al, ; Hertz et al, ). Moreover, oxidative stress‐induced Dnmt2 upregulation was observed in different human and mouse cell lines with unmodified physiological levels of Dnmt2 (Lewinska, Wnuk et al, ; Mytych et al, ; Mytych, Zebrowski, Lewinska, & Wnuk, ; Thiagarajan et al, ).…”

“…HP‐treated Dnmt2‐silenced fibroblasts were also more prone to apoptosis than control cells (this study) and Dnmt2 may be considered as a pro‐survival signal during stress conditions. Dnmt2‐mediated resistance to stress conditions is associated with high levels of tRNA Asp methylation in Drosophila and E. histolytica (Hertz et al, ; Schaefer et al, ). It has been shown that Dnmt2‐mediated tRNA methylation protected tRNAs against stress‐induced cleavage in Drosophila (Schaefer et al, ).…”

“…Overexpression of Dnmt2 homologs resulted in upregulation of heat shock proteins and resistance to oxidative stress in D. melanogaster and E. histolytica (Fisher, Siman‐Tov, & Ankri, ; Lin, Tang, Reddy, & Shen, ). Overexpression of Ehmeth also conferred resistance to nitrosative stress that was associated with elevated levels of tRNA Asp methylation (Hertz et al, ).…”

Deciphering the epitranscriptome: A green perspective

“…Recently, the tRNA modification 2’‐0‐cytosine methylation (Cm) was shown to be increased in response to pathogen infection in Arabidopsis (Ramirez et al ). In other organisms, several tRNA modifications were shown to be induced under stress conditions such as oxidative stress (Chan et al ; Chan et al ), nutrient starvation (Preston et al ) and toxins (Hertz et al ).…”