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

Preeti Shahi,Mohit Mazumder,Sharon Alterzon-Baumel,Karen Methling,Gourinath Samudrala,Rivka Hertz,Meirav Trebicz-Geffen,Serge Ankri,Shruti Nagaraja,Michael Lalk

doi:10.1038/srep36323

Abstract

Adaptation of the Entamoeba histolytica parasite to toxic levels of nitric oxide (NO) that are produced by phagocytes may be essential for the establishment of chronic amebiasis and the parasite’s survival in its host. In order to obtain insight into the mechanism of E. histolytica’s adaptation to NO, E. histolytica trophozoites were progressively adapted to increasing concentrations of the NO donor drug, S-nitrosoglutathione (GSNO) up to a concentration of 110 μM. The transcriptome of NO adapted trophozoites (NAT) was investigated by RNA sequencing (RNA-seq). N-acetyl ornithine deacetylase (NAOD) was among the 208 genes that were upregulated in NAT. NAOD catalyzes the deacetylation of N-acetyl-L-ornithine to yield ornithine and acetate. Here, we report that NAOD contributes to the better adaptation of the parasite to nitrosative stress (NS) and that this function does not depend on NAOD catalytic activity. We also demonstrated that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is detrimental to E. histolytica exposed to NS and that this detrimental effect is neutralized by NAOD or by a catalytically inactive NAOD (mNAOD). These results establish NAOD as a moonlighting protein, and highlight the unexpected role of this metabolic enzyme in the adaptation of the parasite to NS.

Highlights

Cytotoxic for E. histolytica, and this cytotoxicity is implemented by S-nitrosylation of key metabolic enzymes and by fragmenting the endoplasmic reticulum (ER)[5]
We revealed that N-acetyl-L-ornithine deacetylase (NAOD) is a moonlighting protein which has been recruited by E. histolytica for its adaptation to nitric oxide (NO)
In order to determine whether E. histolytica can be adapted to survive in micromolar concentrations of NO, E. histolytica trophozoites were exposed to increasing GSNO concentrations by stepwise additions of GSNO to the culture medium to a final concentration of 110 μM over one month

Summary

Introduction

Cytotoxic for E. histolytica, and this cytotoxicity is implemented by S-nitrosylation of key metabolic enzymes and by fragmenting the endoplasmic reticulum (ER)[5]. We completed a high-throughput proteomic analysis of S-nitrosylated (SNO) proteins in NO-exposed E. histolytica using resin-assisted capture of SNO proteins (SNO-RAC)[3], and found that SNO proteins are involved in glycolysis, translation, protein transport, and virulence. In order to obtain insight into the mechanism of NO resistance in E. histolytica, NO adapted trophozoites (NAT) were produced in vitro by stepwise exposure to increasing amounts of S-nitrosoglutathione (GSNO), an NO donor, which is the S-nitrosated derivative of glutathione. Phenotypic analysis of NAT show that they are more resistant to activated macrophages and their binding to target cells is substantially stronger that of trophozoites which were exposed to an acute nitrosative stress (TEANS)[15]. We present the results of a comparative transcriptome analysis of control untreated trophozoites and NAT. We revealed that N-acetyl-L-ornithine deacetylase (NAOD) is a moonlighting protein which has been recruited by E. histolytica for its adaptation to NO

Methods

Results

Conclusion