Characterization of the Rab11-like GTPase, EhRab11, of the protozoan parasite Entamoeba histolytica

The parasite, Entamoeba histolytica, infects 10% of the world's population leading to 50 million cases of invasive amoebiasis and causing 100,000 deaths annually. Infection is acquired by ingestion of the quadranucleate cyst form of the organism. Excystation occurs in the intestines giving rise to mature trophozoites capable of lysing host epithelial cells leading to invasive colitis and extra-intestinal infections. Vesicle trafficking pathways have been implicated in pathogenicity as the uptake of nutrients, the secretion of hydrolases and pore forming peptides, and the trafficking of cyst wall components are all virulence associated processes. In other eukaryotes the Ras-related Rab GTPases are proposed to be key regulators of vesicle trafficking. To date, 16 Entamoeba Rabs have been identified---the largest family being the Rab11-like family of GTPases. To further the understanding of the role of Rab11-like GTPases in Entamoeba virulence, specific antibodies were generated for one member of the Rab11-like family, EhRab11. Immunofluorescence confocal microscopy using EhRab11-specific antibodies revealed no colocalization with several organelle-specific markers. In addition, EhRab11 was not found to be associated with markers for various endocytic compartments. However, under conditions of iron and serum starvation, EhRab11 was translocated to the periphery of the cell. The altered cellular localization was accompanied by multinucleation of the cells as well as the acquisition of detergent resistance by the cells, features that are characteristic of Entamoeba cysts. To gain further insight into the role the EhRab11 in Entamoeba, a mutant cell line expressing a dominant negative version of the protein was generated and characterized. Mutants over-expressing the dominant negative version of EhRab111 were defective in their ability to phagocytose latex beads, secrete cysteine proteases and to lyse target host epithelial cells, though there was no decrease in their ability to adhere to host cells. Together, these data suggest that EhRab11 participates in virulence by regulating phagocytic and secretory events and encystation in E. histolytica. This is the first characterization of a Rab GTPase of E. histolytica by subcellular localization and mutational analysis. This is also the first report of a Rab-GTPase playing a role in encystation in any organism.