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.
Highlights
Amebiasis is caused by the single-celled protozoan, Entamoeba histolytica
In order to confirm the consistency of our SNORAC analysis, actin was selected and the presence of SNO actin was confirmed by western blotting (Figure 1C)
E. histolytica has a remarkable ability to adapt to various stresses, such as glucose starvation (Baumel-Alterzon and Ankri, 2014), serum starvation (Ahamad et al, 2015), treatment with metronidazole (Penuliar et al, 2015), or nitrosative stress (NS) (Shahi et al, 2016b) and comprehensive transcriptome analyses have been conducted in these stressed parasites
Summary
Amebiasis is caused by the single-celled protozoan, Entamoeba histolytica. The disease is mostly prevalent in developing countries, and is one of the three most common causes of death from parasitic diseases (WHO, 1997). Transcriptomic analyses of NO-adapted trophozoites (NAT) have revealed an unexpected function of N-acetyl ornithine deacetylase (NAOD) in the adaptation of the parasite to NO. This function does not depend on NAOD’s catalytic activity but is mediated by blunting the detrimental effect of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) on E. histolytica exposed to NS (Shahi et al, 2016b). Motility and actin cytoskeletal dynamics functions are directly linked to the pathogenicity of the parasite (Aguilar-Rojas et al, 2016) Despite these instructive data on the transcriptomics of NAT, information on the identity of nitrosylated proteins in NAT is lacking. We report that (a) some of the parasite’s functions, such as erythrophagocytosis, motility, and virulence, become impaired in NAT during its adaptation to NO and (b) these functions are restored when NAT are no longer exposed to NO
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