Identification of S-Nitrosylated (SNO) Proteins in Entamoeba histolytica Adapted to Nitrosative Stress: Insights into the Role of SNO Actin and In vitro Virulence

Abstract

We have recently reported that Entamoeba histolytica trophozoites can adapt to toxic levels of the nitric oxide (NO) donor, S-nitrosoglutathione (GSNO). Even if the consequences of this adaptation on the modulation of gene expression in NO-adapted trophozoites (NAT) have been previously explored, insight on S-nitrosylated (SNO) proteins in NAT is missing. Our study aims to fill this knowledge gap by performing a screening of SNO proteins in NAT. Employing SNO resin-assisted capture (RAC), we identified 242 putative SNO proteins with key functions in calcium binding, enzyme modulation, redox homeostasis, and actin cytoskeleton. Of the SNO proteins in NAT, proteins that are associated with actin family cytoskeleton protein are significantly enriched. Here we report that the formation of actin filaments (F-actin) is impaired in NAT. Consequently, the ability of NAT to ingest erythrocytes and their motility and their cytopathic activity are impaired. These phenotypes can be imitated by treating control parasite with cytochalasin D (CytD), a drug that binds to F-actin polymer and prevent polymerization of actin monomers. Removal of GSNO from the culture medium of NAT restored the sensitivity of the parasite to nitrosative stress (NS) and its ability to form F-actin formation and its virulence. These results establish the central role of NO in shaping the virulence of the parasite through its effect on F-actin formation and highlight the impressive ability of this parasite to adapt 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.…”

“…Lane 1: Untreated control trophozoites, Lane 2: trophozoites treated with 10‐μg ml −1 puromycin for 20 min, Lane 3: Trophozoites treated with 1.5‐μM AF for 24 hr and then labelled with 10‐μg ml −1 puromycin for 20 min, Lane 4: Trophozoites treated with 2.5 μM H 2 O 2 for 30 min and then treated with puromycin for 20 min, Lane 4: Trophozoites treated with cycloheximide (100 μg ml −1 ) for 5 min and then treated with puromycin for 20 min. The protein extracts were separated by denaturing electrophoresis and analysed by immunoblotting with a monoclonal puromycin antibody 12D10 clone as previously described (Trebicz‐Geffen et al, )…”

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

“…The cytopathic activity of E . histolytica trophozoites was determined using a previously described protocol [ 84 ].…”

Entamoeba histolytica under Oxidative Stress: What Countermeasure Mechanisms Are in Place?

“…Motility is an essential function for the survival of E. histolytica [ 67 , 68 ] not only for displacement and phagocytosis of host cells, but also for the intracellular trafficking of virulence factors [ 69 ]. Another project correlating the transcriptomic data of NO-exposed parasites to the proteomic data of NO-adapted trophozoites [ 70 ] showed an up-regulation in the gene expression of cytoskeleton proteins that were previously identified by proteomics. Mammalian actin S -nitrosylation can decrease actin polymerization and rearrange its cytoskeleton.…”

“…Mass spectrometry analysis of NO-adapted proteins showed that the E. histolytica actin residue Cys286 was S -nitrosylated, suggesting that it could indeed impair the cytoskeleton functions in E. histolytica . These NO-adapted trophozoites had reduced virulence functions (i.e., erythrophagocytosis, cytophatic activity, and motility), similar to when actin is inhibited by Cytochalasin D, suggesting that NO exposure not only affects the endomembrane network [ 25 ], but also the cytoskeleton functions [ 70 ] by increasing its gene expression profile and modifying the proteins. Interestingly, once the NO pressure is removed, the trophozoites regained the same levels of virulence functions as the control parasites [ 70 ], demonstrating once again the ability of the amoeba to quickly adapt to different environments.…”