Characterization of ectosomes-like vesicles released by Trichomonas vaginalis

NIEVAS Y. R; COCERES V.M; BENCHIMOL M.; DE MIGUEL N

Congreso; X Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2014

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Here, we report the identification and further characterization of ectosome-like microvesicles (MVs) released by T. vaginalis. "Ectosomes" or "shedding vesicles" are derived from budding or shedding from the plasma membrane and are known to be heterogeneous in size. As exosomes, ectosomes are considered an universal transport vehicle for intercellular communication. Both types of MVs can incorporate peptides, proteins, lipids, miRNA, and mRNA, all of which can be transferred to target cells through receptor-ligand interactions, fusion with the cell membrane, and delivery of a functional cargo to the cytoplasm of the target cell. In this report, using parasites transfected with a Tetraspanin (TSP1) fused to an haemaglutinin tag, we identify structures that shed from the plasma membrane of the parasite; reminiscent to ectosomes released from other types of cells. Hence, we set up a protocol based on filtration with different pore sizes and ultracentrifugation to differentially isolate ectosomes and exosomes from parasite conditioned media. Examination of the preparation by electron microscopy (EM) revealed vesicles with size ranging 100 nm-1 μm in diameter. Using flow cytometry, isolated MVs were phosphatidil-serine (PS) positive with a size range between 0.1?0.9 μm diameter, similar as described for ectosomes in other cells. As mammalian ectosomes, these structures are enriched in three Tetraspanin proteins (TSP1, TSP3 and TSP8). Moreover, PCR analysis on isolated MVs of transfected parasites indicated that ectosomes from T. vaginalis can cargo DNA plasmid. Finally, we demonstrated that plasmid contained in isolated MVs could be transferred between parasites; suggesting a role in intercellular communication.