Overexpression of a Mutant Form of EhRabA, a Unique Rab GTPase of Entamoeba histolytica , Alters Endoplasmic Reticulum Morphology and Localization of the Gal/GalNAc Adherence Lectin

Abstract

Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. Vesicle trafficking events, such as phagocytosis and delivery of plasma membrane proteins, have been implicated in pathogenicity. Rab GTPases are proteins whose primary function is to regulate vesicle trafficking; therefore, understanding the function of Rabs in this organism may provide insight into virulence. E. histolytica possesses a number of unique Rabs that exhibit limited homology to host Rabs. In this study we examined the function of one such Rab, EhRabA, by characterizing a mutant overexpressing a constitutively GTP-bound version of the protein. Overexpression of mutant EhRabA resulted in decreased adhesion to and phagocytosis of human red blood cells and in the appearance of large tubular organelles that could be stained with endoplasmic reticulum (ER)-specific but not Golgi complex-specific antibodies. Consistent with the adhesion defect, two subunits of a cell surface adhesin, the galactose/N-acetylgalactosamine lectin, were mislocalized to the novel organelle. A cysteine protease, EhCP2, was also localized to the ER-like compartment in the mutant; however, the localization of two additional cell surface proteins, Igl and SREHP, remained unchanged in the mutant. The phenotype of the mutant could be recapitulated by treatment with brefeldin A, a cellular toxin that disrupts ER-to-Golgi apparatus vesicle traffic. This suggests that EhRabA influences vesicle trafficking pathways that are also sensitive to brefeldin A. Together, the data indicate that EhRabA directly or indirectly influences the morphology of secretory organelles and regulates trafficking of a subset of secretory proteins in E. histolytica.Entamoeba histolytica, a protozoan parasite, is the causative agent of amoebic dysentery and causes liver abscess. It is prevalent in developing countries that cannot prevent its fecal-oral spread and is responsible for considerable global morbidity and mortality (reviewed in reference 12). Infection is acquired by ingestion of the cyst form of the parasite. Excystation occurs in the small intestine, and released amoeboid trophozoites move to, and colonize, the bowel lumen.

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

“…histolytica expression vector pGIR209, which confers G418 (neomycin) resistance and allows for tetracycline-inducible expression of exogenous genes [20]. A standard electroporation protocol [21] was utilized to introduce the expression vector into trophozoites that had been previously transfected with another plasmid, pGIR308. This partner plasmid encodes the tetracycline repressor protein, which is necessary for tetracycline inducibility.…”

A Genomewide Overexpression Screen Identifies Genes Involved in the Phosphatidylinositol 3-Kinase Pathway in the Human Protozoan Parasite Entamoeba histolytica

“…Expression from this plasmid is driven by upstream ferredoxin regulatory sequences. The cDNA library in pAH-DEST was transfected into HM1: IMSS trophozoites using standard protocols (10,29). Transfectants were maintained by the addition of 23 g/ml hygromycin to the medium.…”

“…histolytica trophozoites were transfected with expression plasmids encoding SREHP or EhCoactosin using standard transfection protocols (10,29). These 2nd-generation SREHP and EhCoactosin overexpressors were maintained in a medium supplemented with 23 g/ml hygromycin, and the drug concentration was increased to 46 g/ml for 1 week prior to conducting experiments.…”

Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania

“…Although the secretory/endocytic pathway is not well characterized in Entamoeba histolytica, it has been shown that fibronectin receptor and Gal/GalNAc lectin trafficking to plasma membrane is brefeldin-A-dependent, whereas thiolproteinase secretion is brefeldin-A-independent in this parasite (33). Further characterization reveals that overexpression of a GTP-locked mutant of EhRabA alters ER morphology and mislocalizes Gal/GalNAc lectin without affecting the localization of other cell surface proteins, indicating the specific role of EhRabA in early secretory pathway (34). Bioinformatic analyses have shown the presence of a Rab1 homologue in Giardia lamblia (35), Plasmodium falciparum (36), and Leishmania major (37); however, the functional role of Rab1 in the secretory pathway of these parasites is not yet elucidated.…”