Proteomic Identification of Oxidized Proteins in Entamoeba histolytica by Resin-Assisted Capture: Insights into the Role of Arginase in Resistance to Oxidative Stress

Abstract

Entamoeba histolytica is an obligate protozoan parasite of humans, and amebiasis, an infectious disease which targets the intestine and/or liver, is the second most common cause of human death due to a protozoan after malaria. Although amebiasis is usually asymptomatic, E. histolytica has potent pathogenic potential. During host infection, the parasite is exposed to reactive oxygen species that are produced and released by cells of the innate immune system at the site of infection. The ability of the parasite to survive oxidative stress (OS) is essential for a successful invasion of the host. Although the effects of OS on the regulation of gene expression in E. histolytica and the characterization of some proteins whose function in the parasite's defense against OS have been previously studied, our knowledge of oxidized proteins in E. histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale identification and quantification of the oxidized proteins in oxidatively stressed E. histolytica trophozoites using resin-assisted capture coupled to mass spectrometry. We detected 154 oxidized proteins (OXs) and the functions of some of these proteins were associated with antioxidant activity, maintaining the parasite's cytoskeleton, translation, catalysis, and transport. We also found that oxidation of the Gal/GalNAc impairs its function and contributes to the inhibition of E. histolytica adherence to host cells. We also provide evidence that arginase, an enzyme which converts L-arginine into L-ornithine and urea, is involved in the protection of the parasite against OS. Collectively, these results emphasize the importance of OS as a critical regulator of E. histolytica's functions and indicate a new role for arginase in E. histolytica's resistance to OS.

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

“…According to previous reports in mammalian cells, the cysteines in actin are some of the most susceptible targets of oxidation and nitrosylation and their modification is the likely cause of reduced polymerization activity and altered binding of actin with actin binding proteins45. We have previously reported that a number of cysteine residues in E. histolytica actin are susceptible to oxidation in oxidatively stressed trophozoites20 and to S-nitrosylation in NAT (manuscript in preparation). Based on this information, it is tempting to speculate that the upregulation of cytoskeletal proteins in NAT is a mechanism used by the parasite to replace SNO cytoskeletal proteins.…”

“…We have recently showed that the accumulation of the polyamine putrescine and E. histolytica ’s resistance to OS are linked20. The synthesis of polyamines has been also linked to NS resistance in bacteria2122.…”

“…The measurement of intracellular putrescine and acetate concentrations was done using a previously described protocol20.…”

Proteome analysis of excretory-secretory proteins of Entamoeba histolytica HM1:IMSS via LC–ESI–MS/MS and LC–MALDI–TOF/TOF

“…They are strongly acidic proteins that are commonly found on the surface of the ribosome in all organisms [40]. Eukaryotic 60 s acidic ribosomal proteins are called ribosomal P proteins because they can be phosphorylated [41]. P protein is a structural constituent of the ribosome and is involved in nucleic acid binding and translation.…”

Phosphorylation of Eukaryotic Initiation Factor-2α during Stress and Encystation in Entamoeba Species

“…Our findings are also consistent with the observation that protein biosynthesis is inhibited during oxidative stress in E . histolytica [25]. It has been suggested that oxidation of components of the parasite’s translation machinery (e.g., ribosomal proteins, elongation factors) [25], and enzymatic down-regulation of almost all tRNA species [31] are responsible for reduced protein biosynthesis during H 2 O 2 exposure.…”