Proteomics approach to identify novel proteins on the surface of Trichomonas vaginalis involved in pathogenesis

DE MIGUEL N; RIESTRA A; JOHNSON PJ

Congreso; IX Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2011

Sociedad Argentina de Parasitologia

The unicellular parasitic Trichomonas vaginalis is the causative agent of trichomoniasis, the most prevalent non-viral sexually transmitted disease in humans. Because T. vaginalis is an obligate extracellular pathogen, proteins on the surface may play a critical role host-parasite interaction and the consequent pathogenesis. In this study, we used cell surface biotinylation and multidimensional protein identification technology (MudPIT) to identify the surface proteome of six strains of Trichomonas vaginalis with differing adherence capacities to vaginal epithelial cells (1). A combined total of 411 proteins were identified, and of these, 11 were found to be more abundant in adherent strains relative to less adherent parasites. As proof of principle and to investigate a possible role in pathogenesis, we report the identification and functional characterization of two surface proteins detected in the proteome: Tetraspanin-6 (TSP6) and Tetraspanin-1 (TSP1). These proteins are members of the tetraspanin family, which comprises a large superfamily of cell surface-associated membrane proteins that has been implicated in fundamental biological processes, including cell adhesion, migration, fusion and proliferation (2). Our studies showed that TSP6 targets to both the plasma membrane and the flagella of free living parasites (Fig 1). The protein relocalizes during exposure to vaginal epithelial cells (VECs). The complex localization and expression pattern of TSP6 suggests that its intracellular trafficking and distribution must be tightly regulated during parasite pathogenesis. In order to evaluate TSP6 function, cell adhesion and migration assays were performed using parasites that over-express full length and C-terminal truncated version of TSP6. Although no effect was observed on parasite that overexpress full length TSP6, when the C-terminal tail is removed (TSP6DCt), exogenous TSP6 exerted a dominant negative effect, as it significantly reduced migration through matrigel invasion chambers compared with parasites transfected with empty plasmid. In addition, TSP6DCt does not relocalize upon exposure to VECs as observed for the full length protein. In conclusion, TSP6 appears to influence cellular migration, with the TSP6 tail being of particular importance in determining the “outside-in” signals that follow ligand engagement. The elucidation of determinants involved in the process of migration may reveal virulence factors and novel therapeutic targets to combat disease. In addition, a related protein, TSP1, has been shown to reside in exosomes as well as large multivesicular bodies that accumulate upon contact of the parasite with host cells. Exosomes are small membrane vesicles (50- to 100-nm) released into the extracellular environment upon exocytic fusion of multivesicular bodies with the cell surface. These vesicles are increasingly recognized as significant vehicles for intercellular communication (3). Our results indicate that TSP1 is present in vesicles with morphological and biochemical characteristics of exosomes. Interestingly, these vesicles appear to modulate host-parasite communication.