Coceres, V. M.; Miranda-Magalhaes, A.; de Figueiredo, R. C. B. Q.; de Miguel, N.; Pereira-Neves, A.

Tritrichomonas foetus is an extracellular flagellated protist responsible for bovine and feline tritrichomonosis, yet several aspects of its cell biology and pathogenicity remain poorly understood. Here, we evaluated the presence of extracellular vesicles (EVs) and membrane protrusions in T. foetus using complementary electron microscopy and immunolabeling approaches. EV-like structures of different sizes were found protruding from the parasite cell body and anterior flagella, and ultrathin sections showed continuity between the membranes of these protrusions and the plasma or flagellar membrane. Multivesicular bodies containing intraluminal vesicles were also observed, and an EV-enriched fraction isolated from conditioned medium contained vesicles with a broad size distribution, consistent with heterogeneous EV populations. Immunofluorescence further showed tetraspanin-1 in intracellular vesicles, flagella, and vesicular structures protruding from the parasite surface. Exposure to bovine preputial epithelial cells resulted in a ninefold increase in the percentage of parasites displaying flagellar cytoneme-like structures, suggesting a possible role for these projections in host-parasite interactions. VPS32, a component of the ESCRT-III machinery, was found in intraluminal vesicles, vesicles protruding from the cell body and flagella, isolated EVs, and flagellar membrane protrusions. In parasites overexpressing VPS32, the number of vesicles per flagellum increased, and the percentage of cells with flagellar cytoneme-like projections was fivefold higher than in control parasites. Pearling-like protrusions containing VPS32 were also observed. Together, these findings provide ultrastructural evidence for heterogeneous EV populations associated with the cell body and flagella of T. foetus and suggest that VPS32 could contribute to flagellar EV and membrane protrusion formation.