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.
Highlights
Amebiasis is a parasitic infection of the human intestine and is caused by the single-celled protozoa, Entamoeba histolytica
The heavy subunit of Gal/GalNAc lectin, which is essential for the adherence of the ameba to its target cells [9], was among the S-nitrosylated proteins that we identified in NOexposed E. histolytica trophozoites by resinassisted capture (RAC)
In order to assess the reliability of MS-based identification of the S-nitrosylated proteins, three proteins, namely enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the heavy subunit of Gal/GalNac lectin, were selected and their S-nitrosylation was confirmed by SNO-RAC and western blotting (Fig. 1D)
Summary
Amebiasis is a parasitic infection of the human intestine and is caused by the single-celled protozoa, Entamoeba histolytica. The parasite has two stages in its life cycle in the host: the infective cyst and the invasive trophozoite. The parasite must adapt to various environmental stresses during infection of their human hosts, and the host-parasite relationship is dependent upon the host’s immune system. Nitric oxide (NO) is a potent cytotoxin, which is released by activated macrophages, natural killer cells, and other phagocytic cells, and has been reported to be the major cytotoxin for killing E.histolytica trophozoites [2]. It has been reported that NO triggers stress responses in E.histolytica and that NO directly inhibits glycolysis and stimulates cysteine synthase activity [7]
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