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Proteomic identification of S-nitrosylated proteins in the parasite Entamoeba histolytica by resin-assisted capture: insights into the regulation of the Gal/GalNAc lectin by nitric oxide.

ABSTRACT: Entamoeba histolytica is a gastrointestinal protozoan parasite that causes amebiasis, a disease which has a worldwide distribution with substantial morbidity and mortality. Nitrosative stress, which is generated by innate immune cells, is one of the various environmental challenges that E. histolytica encounters during its life cycle. Although the effects of nitric oxide (NO) on the regulation of gene expression in this parasite have been previously investigated, our knowledge on S-nitrosylated proteins in E.histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale detection of S-nitrosylated (SNO) proteins in E.histolytica trophozoites that were treated with the NO donor, S-nitrosocysteine by resin-assisted capture (RAC). We found that proteins involved in glycolysis, gluconeogenesis, translation, protein transport, and adherence to target cells such as the heavy subunit of Gal/GalNac lectin are among the S-nitrosylated proteins that were enriched by SNO-RAC. We also found that the S-nitrosylated cysteine residues in the carbohydrate recognition domain (CRD) of Gal/GalNAc lectin impairs its function and contributes to the inhibition of E.histolytica adherence to host cells. Collectively, these results advance our understanding of the mechanism of reduced E.histolytica adherence to mammalian cells by NO and emphasize the importance of NO as a regulator of key physiological functions in E.histolytica.

SUBMITTER: Hertz R

PROVIDER: S-EPMC3953491 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Proteomic identification of S-nitrosylated proteins in the parasite Entamoeba histolytica by resin-assisted capture: insights into the regulation of the Gal/GalNAc lectin by nitric oxide.

Hertz Rivka R Ben Lulu Shani S Shahi Preeti P Trebicz-Geffen Meirav M Benhar Moran M Ankri Serge S

PloS one 20140313 3

Entamoeba histolytica is a gastrointestinal protozoan parasite that causes amebiasis, a disease which has a worldwide distribution with substantial morbidity and mortality. Nitrosative stress, which is generated by innate immune cells, is one of the various environmental challenges that E. histolytica encounters during its life cycle. Although the effects of nitric oxide (NO) on the regulation of gene expression in this parasite have been previously investigated, our knowledge on S-nitrosylated ...[more]

PMID: 24626316

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Project description:BackgroundEntamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis, including amebic dysentery and liver abscesses. E. histolytica invades host tissues by adhering onto cells and phagocytosing them depending on the adaptation and expression of pathogenic factors, including Gal/GalNAc lectin. We have previously reported that E. histolytica possesses multiple CXXC sequence motifs, with the intermediate subunit of Gal/GalNAc lectin (i.e., Igl) as a key factor affecting the amoeba's pathogenicity. The present work showed the effect of immunization with recombinant Igl on amebic liver abscess formation and the corresponding immunological properties.Methodology/principal findingsA prokaryotic expression system was used to prepare the full-length Igl and the N-terminal, middle, and C-terminal fragments (C-Igl) of Igl. Vaccine efficacy was assessed by challenging hamsters with an intrahepatic injection of E. histolytica trophozoites. Hamsters intramuscularly immunized with full-length Igl and C-Igl were found to be 92% and 96% immune to liver abscess formation, respectively. Immune-response evaluation revealed that C-Igl can generate significant humoral immune responses, with high levels of antibodies in sera from immunized hamsters inhibiting 80% of trophozoites adherence to mammalian cells and inducing 80% more complement-mediated lysis of trophozoites compared with the control. C-Igl was further assessed for its cellular response by cytokine-gene qPCR analysis. The productions of IL-4 (8.4-fold) and IL-10 (2-fold) in the spleen cells of immunized hamsters were enhanced after in vitro stimulation. IL-4 expression was also supported by increased programmed cell death 1 ligand 1 gene.Conclusions/significanceImmunobiochemical characterization strongly suggests the potential of recombinant Igl, especially the C-terminal fragment, as a vaccine candidate against amoebiasis. Moreover, protection through Th2-cell participation enabled effective humoral immunity against amebic liver abscesses.

Project description:Entamoeba histolytica is a food- and waterborne parasite that causes amebic dysentery and amoebic liver abscesses. Adhesion is one of the most important virulence functions as it facilitates motility, colonization of host, destruction of host tissue, and uptake of nutrients by the parasite. The parasite cell surface adhesin, the Gal/GalNAc lectin, facilitates parasite-host interaction by binding to galactose or N-acetylgalactosamine residues on host components. It is composed of heavy (Hgl), intermediate (Igl), and light (Lgl) subunits. Igl is constitutively localized to lipid rafts (cholesterol-rich membrane domains), whereas Hgl and Lgl transiently associate with rafts. When all three subunits are localized to rafts, galactose-sensitive adhesion is enhanced. Thus, submembrane location may regulate the function of this adhesion. Rhomboid proteases are a conserved family of intramembrane proteases that also participate in the regulation of parasite-host interactions. In E. histolytica, one rhomboid protease, EhROM1, cleaves Hgl as a substrate, and knockdown of its expression inhibits parasite-host interactions. Since rhomboid proteases are found within membranes, it is not surprising that lipid composition regulates their activity and enzyme-substrate binding. Given the importance of the lipid environment for both rhomboid proteases and the Gal/GalNAc lectin, we sought to gain insight into the relationship between rhomboid proteases and submembrane location of the lectin in E. histolytica. We demonstrated that EhROM1, itself, is enriched in highly buoyant triton-insoluble membranes reminiscent of rafts. Reducing rhomboid protease activity, either pharmacologically or genetically, correlated with an enrichment of Hgl and Lgl in rafts. In a mutant cell line with reduced EhROM1 expression, there was also a significant augmentation of the level of all three Gal/GalNAc subunits on the cell surface and an increase in the molecular weight of Hgl and Lgl. Overall, the study provides insight into the molecular mechanisms governing parasite-host adhesion for this pathogen.

Dataset Information

Proteomic identification of S-nitrosylated proteins in the parasite Entamoeba histolytica by resin-assisted capture: insights into the regulation of the Gal/GalNAc lectin by nitric oxide.

Publications

Proteomic identification of S-nitrosylated proteins in the parasite Entamoeba histolytica by resin-assisted capture: insights into the regulation of the Gal/GalNAc lectin by nitric oxide.

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