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

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. Here, the pathogen acquires nutrients via phagocytosis of colonic bacteria, host cells, and host cell debris. The ability of the pathogen to carry out phagocytosis has been correlated to its virulence potential. For example, phagocytosis-deficient mutants of E. histolytica exhibit reduced pathogenicity in vitro and in vivo (24, 33), and a noninvasive Entamoeba species, E. dispar, exhibits low rates of phagocytosis (26).Given the importance of phagocytosis to virulence, the identification of proteins that directly or indirectly regulate this process has been the focus of a considerable research effort (reviewed in references 16 and 23). Proteomic analyses of purified phagosomes have identified numerous proteins that may regulate phagocytosis in E. histolytica (2, 17, 22). Functional studies have identified several cell surface proteins as possible phagocytic receptors. One such receptor, the galactose/N-acetylgalactosamine-inhibitable lectin (Gal/GalNAc lectin) (6, 31), consists of a transmembrane heavy subunit (Hgl) which is covalently associated with a glycosylphosphatidylinositol (GPI)-linked light subunit (Lgl). This heterodimer is noncovalently associated with a GPI-anchored intermediate subunit (Igl). Other cell surface receptors include SREHP, a serine-rich protein that is proposed to be lipid anchored (42), and PATMK, a transmembrane kinase (2).The complement of receptors found on the cell surface is influenced both by anterograde and retrograde vesicle trafficking pathways. For example, newly synthesized proteins are incorporated into the membrane of the endoplasmic reticulum (ER), transported to the Golgi apparatus, and finally delivered to the plasma membrane via anterograde transport vesicles. On the other hand, cell surface receptors may be internalized and delivered to intracellular compartments or back to the plasma membrane via retrograde transport vesicles (44). While E. histolytica trophozoites have been shown to possess an ER and Golgi apparatus (8, 19, 41), insight into the molecular mechanisms regulating the morphological and functional integrity of these organelles is limited. Such insight would contribute to our understanding of phagocytosis, which relies on the proper functioning of these organelles for localization of cell surface receptors.In other systems, secretory vesicle trafficking events are regulated by Rab GTPases, a family of GTP-binding proteins involved in the docking and fusion of transport vesicles with target membranes. Substantial evidence suggests that Rab proteins carry out their function by cycling between a GDP-bound cytosolic form and a GTP-bound membrane form (reviewed in reference 39). In silico genome mining has shown that E. histolytica possesses greater than 90 Rab GTPases (34). A number of these exhibit limited homology to other known Rabs and thus are considered unique to E. histolytica (34). We previously reported that one of these unique Rabs, EhRabA, may be involved in the regulation of polarization, motility, and actin cytoskeletal dynamics (47, 48). In the current study, we demonstrate that overexpression of the putatively GTP-bound form of EhRabA results in the alteration of ER morphology, mislocalization of two subunits of the Gal/GalNAc lectin, and reduced phagocytosis, suggesting that this Rab plays a direct or indirect role in cellular functions that contribute to virulence.