A first evidence of a glutaredoxin-like protein in entamoeba histolytica

Abstract

Entamoeba histolytica, an intestinal parasitic protozoan, is the causative agent of amoebiasis. The parasite usually lives and multiplies within the human gut, an environment of reduced oxygen pressure. During tissue invasion, E. histolytica is exposed to elevated amounts of exogenous reactive oxygen species (ROS), which are highly toxic for the parasite. The metabolic pathway for ROS detoxification in this organism is a matter of controversy. Because neither glutathione nor its associated enzymes were found to occur, it has been proposed the cysteine as a main intracellular thiol and one of the compounds responsible for maintaining the intracellular redox balance. In this work, we present the functional characterization of a glutaredoxin-like protein from E. histolytica (EhGrx1). Biochemical assays showed that EhGrx1 was able to catalyze the in vitro reduction of GSH-derivate low molecular mass disulfides and cystine. The protein obtained by recombinant expression in Escherichia coli presented an apo- monomeric structure; however, a holo-protein form was obtained from supplemented culture media with ferric citrate and cysteine. The ability to ligate iron-sulfur centers (ISCs) was evaluated by UV-Vis spectroscopy and gel filtration chromatography, showing evidence that EhGrx1 could bind ISCs. The Grx activity was not detected in holo-EhGrx1, suggesting that its catalytic cysteine residue would be linked to ISC. We also evaluated by western blot the relative abundance of EhGrx1 in E. histolytica cells exposed to exogenous oxidative species and metronidazole (the preferred drug for amoebiasis treatment). The results showed that the protein level increases respect to no-treated cells. Similar behavior was observed in the subcellular localization analysis, carried out for different oxidative conditions by confocal immunofluorescence microscopy. Altogether, the results suggest that EhGrx1 could be involved in oxidative and nitrosative stress protection in the parasite. To the best of our knowledge, this is the first characterization of this type of protein in E. histolytica.