Omics Analyses of Trichomonas vaginalis Actin and Tubulin and Their Participation in Intercellular Interactions and Cytokinesis.

Actin and tubulin proteins from Trichomonas vaginalis are crucial for morphogenesis and mitosis. This parasite has 10 and 11 genes coding bonafide actin and tubulin proteins, respectively. Hence, the goal of this work was to analyze these actin and tubulin genes, their expression at the mRNA and protein levels, and their parasite localization in intercellular interaction and cytokinesis. Representative bonafide actin (tvact1) and tubulin (tvtubα1) genes were cloned into and expressed in Escherichia coli. The recombinant proteins TvACT1r and TvTUBα1r were affinity purified and used as antigens to produce polyclonal antibodies. These antibodies were used in 1DE and 2DE WB and indirect immunofluorescence assays (IFA). By IFA, actin was detected as a ring on the periphery of ameboid, ovoid, and cold-induced cyst-like parasites, on pseudopods of amoeboid parasites, and in cytoplasmic extensions (filopodia) in cell-cell interactions. Tubulin was detected in the axostyle, flagellum, undulating membrane, and paradesmose during mitosis. Paradesmose was observed by IFA mainly during cytokinesis. By scanning electron microscopy, a tubulin-containing nanotubular structure similar to the tunneling nanotubes (TNTs) was also detected in the last stage of cytokinesis. In conclusion, actin and tubulin are multigene families differentially expressed that play important roles in intercellular interactions and cytokinesis.

Trichomonas vaginalis cathepsin D-like aspartic proteinase (Tv-CatD) is positively regulated by glucose and degrades human hemoglobin.

Trichomonas vaginalis genome encodes ∼440 proteases, six of which are aspartic proteases (APs). However, only one belongs to a clan AA (EC 3.4.23.5), family A1 (pepsin A), cathepsin D-like protease. This AP is encoded by an 1113-bp gene (tv-catd), which translates into a 370-aa residues zymogen of 40.7-kDa and a theoretical pI of 4.6, generating a ∼35 kDa active enzyme after maturation (Tv-CatD). The goal of this study was to identify and analyze the effect of glucose on the expression of Tv-CatD at the transcript and protein levels, subcellular localization, and proteolytic activity. The qRT-PCR assays showed a ∼2-fold increase in tv-catd mRNA under high-glucose (HG) conditions compared to glucose-restriction (GR) conditions. We amplified, cloned, and expressed the tv-catd gene, and purified the recombinant precursor enzyme (Tv-CatDr) to generate a polyclonal antibody (anti-Tv-CatDr). Western blot (WB) and immunolocalization assays showed that glucose increases the amount of Tv-CatD in different subcellular localizations and in in vitro secretions. Additionally, Tv-CatD proteolytic activity was detected in protease-resistant extracts (PREs) using a synthetic fluorogenic peptide specific for cathepsin D/E APs at different pHs and in the presence of AP inhibitors. In a two-dimensional (2-DE) WB analysis of a PRE from parasites grown under GR and HG conditions, an anti-Tv-CatDr antibody detected a 35-kDa protein spot at pI 5.0 identified as the mature Tv-CatD form by mass spectrometry that showed proteolytic activity in 2-DE zymograms copolymerized with hemoglobin under both glucose conditions. Thus, Tv-CatD could be involved in trichomonal hemolysis.