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

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.

Formation of oxidised (OX) proteins inEntamoeba histolyticaexposed to auranofin and consequences on the parasite virulence

“…We have previously reported that OS induced by H 2 O 2 inhibits protein synthesis in E. histolytica (Shahi et al, ; Trebicz‐Geffen et al, ). The presence of proteins that are involved in translation among OXs, such as the elongation factor‐1 alpha and elongation factor 2, suggests that AF regulates the translation of proteins in the AF‐treated trophozoites.…”

“…The culture was continued for an additional 24 hr. The viability of the trophozoites untreated or treated with AF was determined by the eosin dye exclusion method (Shahi, Trebicz‐Geffen, Nagaraja, Hertz, et al, ). The experiment was performed in duplicates and repeated three times.…”

Escherichia coli mediated resistance of Entamoeba histolytica to oxidative stress is triggered by oxaloacetate

“…For this purpose, we exposed E . histolytica trophozoites to the nitric oxide (NO) donor, S-nitrosoglutathione (350 μM for 120 minutes)[ 41 ]. We found that E .…”

“…The expression of HA-tagged EcMDH in the trophozoites was confirmed by western blotting ( Fig 3A ) and detecting MDH activity in their whole lysates ( Table 2 ). When we compared the resistance of HA-tagged EcMDH trophozoites to H 2 O 2 -induced oxidative stress ( Fig 3B ) and trophozoites transformed with the pcontrol plasmid[ 41 ], we found that the sensitivity of the HA-tagged EcMDH trophozoites and the pcontrol trophozoites to H 2 O 2 -induced oxidative stress was identical. Incubation of the HA-tagged EcMDH trophozoites in presence of malate (2 mM) prior to their exposure to H 2 O 2 -induced oxidative stress did not increase their resistance to H 2 O 2 -induced oxidative stress ( Fig 3B ).…”

“…Several different explanations may account for this experimental observation: (i) EhMDHs are very sensitive to environmental conditions and are denatured once the parasite is lysed, (ii) the parasite has lost an active MDH during the course of evolution because it can relies on malate and oxaloacetate produced by the gut microbiota and (iii) EhMDHs may have a different enzymatic activity unrelated to their metabolic function as reported for a number of E . histolytica moonlighting enzymes [ 41 , 70 ]. This third hypothesis is supported by the unexpected presence of two MDHs (EHI_030810 and EHI_165350) on the parasite’s surface [ 71 ].…”