Trichomonas vaginalis: Monolayer and Cluster Formation-Ultrastructural Aspects Using High-Resolution Scanning Electron Microscopy.

Trichomonas vaginalis is an extracellular protozoan parasite that causes human trichomoniasis, a sexually transmitted infection (STI) that affects approximately 270 million people worldwide. The phenomenon of T. vaginalis adhesion to inert substrates has been described in several reports. Still, very few studies on cluster formation have been conducted, and more detailed analyses of the contact regions between the parasites' membranes in these aggregate formations have not been carried out. The present study aims to show that T. vaginalis forms a tight monolayer, similar to an epithelium, with parasites firmly adhered to the culture flask bottom by interdigitations and in the absence of host cells. In addition, we analyzed and compared the formation of the clusters, focusing on parasite aggregates that float in the culture flasks. We employed various imaging techniques, including high-resolution scanning electron microscopy, transmission electron microscopy, cytochemistry, TEM tomography, and dye injection. We analyzed whether the monolayer behaves as an epithelium, analyzing cell junctions, cell communication, and ultrastructural aspects, and concluded that monolayer formation differs from cluster formation in many aspects. The monolayers form strong adhesion, whereas the clusters have fragile attachments. We did not find fusion or the passage of molecules between neighbor-attached cells; there is no need for different strains to form filopodia, cytonemes, and extracellular vesicles during cluster and monolayer formation.

Expansion Microscopy of trichomonads.

Light microscopy has significantly advanced in recent decades, especially concerning the increased resolution obtained in fluorescence images. Here we present the Expansion Microscopy (ExM) technique in two parasites, Trichomonas vaginalis and Tritrichomonas foetus, which significantly improved the localization of distinct proteins closely associated with cytoskeleton by immunofluorescence microscopy. The ExM techniques have been used in various cell types, tissues and other protist parasites. It requires the embedment of the samples in a swellable gel that is highly hydrophilic. As a result, cells are expanded 4.5 times in an isotropic manner, offering a spatial resolution of ∼70 nm. We used this new methodology not only to observe the structural organization of protozoa in more detail but also to increase the resolution by immunofluorescence microscopy of two major proteins such as tubulin, found in structures formed by microtubules, and costain 1, the only protein identified until now in the T. foetus's costa, a unique rod-shaped like structure. The individualized microtubules of the axostyle were seen for the first time in fluorescence microscopy and several other details are presented after this technique.