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The Entamoeba histolytica Dnmt2 homolog (Ehmeth) confers resistance to nitrosative stress.

ABSTRACT: 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 GSNO-treated E. histolytica, which explains the aforementioned increase of tRNA methylation. Specifically, we demonstrated via site-directed 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.

SUBMITTER: Hertz R

PROVIDER: S-EPMC4000097 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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The Entamoeba histolytica Dnmt2 homolog (Ehmeth) confers resistance to nitrosative stress.

Hertz Rivi R Tovy Ayala A Kirschenbaum Michael M Geffen Meirav M Nozaki Tomoyoshi T Adir Noam N Ankri Serge S

Eukaryotic cell 20140221 4

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-nitrosoglutat ...[more]

PMID: 24562908

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Project description:Entamoeba histolytica is an intestinal parasite infecting over 50 million people worldwide and is the causative agent of amebic dysentery and amoebic liver abscess. In the human host, E. histolytica experiences stress brought on by nutrient deprivation and the host immune response. To be a successful parasite, E. histolytica must counter the stress; therefore, understanding the stress response may uncover new drug targets. In many systems, the stress response includes down-regulation of protein translation, which is regulated by phosphorylation of eukaryotic initiation factor (eIF-2?). Previous work has demonstrated that phosphorylation of the E. histolytica eIF-2? (EheIF-2?) increases significantly when exposed to long-term serum starvation, oxidative stress, and long-term heat shock. However, the effects of reagents that are known to induce nitrosative or endoplasmic reticulum (ER) stresses, on EheIF-2? have yet to be evaluated. Nitrosative stress is part of the host's immune response and ER stress can be caused by several physiological or pathological factors. We treated E. histolytica cells with various reagents known to induce nitrosative stress (DPTA-NONOate and SNP) or ER stress (BFA and DTT). We examined the morphology of the ER, tracked phosphorylation of EheIF-2?, and assessed protein translation in control and stressed cells. While all four stress-inducing reagents caused a global reduction in protein translation, only DTT was capable of also inducing changes in the morphology of the ER (consistent with ER stress) and phosphorylation of EheIF-2?. This suggests that DTT authentically induces ER stress in E. histolytica and that this stress is managed by the eIF-2?-based system. This was supported by the observation that cells expressing a non-phosphorylatable version of eIF-2? were also highly sensitive to DTT-stress. Since protein translation decreased in the absence of phosphorylation of eIF-2? (after treatment with DPTA-NONOate, SNP or BFA), the data also indicate that there are alternative protein-translational control pathways in E. histolytica. Overall, our study further illuminates the stress response to nitrosative stress and ER stress in E. histolytica.

Project description:The protozoan parasite, Entamoeba histolytica, invades the host colon causing significant tissue destruction and inflammation. Upon host infection, the parasite is confronted with reactive oxygen and nitrogen species (ROS/RNS) that cause large-scale changes in gene expression profiles, which likely support the parasite's adaptation to the host environment. We have previously identified oxidative and nitrosative stress responsive genes using whole-genome expression profiling. Functional studies on two such genes are now reported and demonstrate that they have roles in parasite virulence. EHI_056680 encodes a small hypothetical protein named E. histolytica stress-induced adhesion factor (EhSIAF); EHI_188210 encodes a putative phospholipid transporting P-type ATPase/flippase (EhPTPA). Over-expression of each protein in E. histolytica trophozoites enhanced parasite survival in response to oxidative stress. Exposure to oxidative and nitrosative stress did not affect the localization of EhSIAF or EhPTPA but markedly increased EhPTPA protein levels. Interestingly, over-expression of each gene resulted in parasites with increased adherence to healthy mammalian cells, but increased adherence to apoptotic cells was noted only in EhSIAF over-expressing parasites. However, despite having increased adherence to both healthy and apoptotic host cells, EhSIAF-over-expressing parasites were reduced in their ability to destroy mammalian cell monolayers, raising the intriguing possibility that EhSIAF over-expression caused signaling defects or resulted in a dominant negative phenotype. Over-expression of EhSIAF and EhPTPA also resulted in decreased motility in a transwell motility assay. Thus, we have confirmed that two genes that are upregulated by ROS confer increased resistance to oxidative stress and have identified an unexpected role of EhSIAF and EhPTPA in host cell adherence and a role of EhSIAF in parasite virulence. Our data imply that stress response genes may play multi-factorial roles in amoebic pathogenesis.

Dataset Information

The Entamoeba histolytica Dnmt2 homolog (Ehmeth) confers resistance to nitrosative stress.

Publications

The Entamoeba histolytica Dnmt2 homolog (Ehmeth) confers resistance to nitrosative stress.

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