Trichomonas vaginalis is the causative agent of trichomoniasis, the most prevalent nonviral, neglected sexually transmitted disease worldwide. T. vaginalis has one of the largest degradomes among unicellular parasites. Cysteine peptidases (CPs) are the most abundant peptidases, constituting 50% of the degradome. Some CPs are virulence factors recognized by antibodies in trichomoniasis patient sera, and a few are found in vaginal secretions that show fluctuations in glucose concentrations during infection. The CPs of clan CD in T. vaginalis include 10 genes encoding legumain-like peptidases of the C13 family. TvLEGU-2 is one of them and has been identified in multiple proteomes, including the immunoproteome obtained with Tv (+) patient sera. Thus, our goals were to assess the effect of glucose on TvLEGU-2 expression, localization, and in vitro secretion and determine whether TvLEGU-2 is expressed during trichomonal infection. We performed qRT-PCR assays using parasites grown under different glucose conditions. We also generated a specific anti-TvLEGU-2 antibody against a synthetic peptide of the most divergent region of this CP and used it in Western blot (WB) and immunolocalization assays. Additionally, we cloned and expressed the tvlegu-2 gene (TVAG_385340), purified the recombinant TvLEGU-2 protein, and used it as an antigen for immunogenicity assays to test human sera from patients with vaginitis. Our results show that glucose does not affect tvlegu-2 expression but does affect localization in different parasite organelles, such as the plasma membrane, Golgi complex, hydrogenosomes, lysosomes, and secretion vesicles. TvLEGU-2 is secreted in vitro, is present in vaginal secretions, and is immunogenic in sera from Tv (+) patients, suggesting its relevance during trichomonal infection.

Autophagy is a conserved cellular degradation process involving ATG proteins, with ATG4 proteases essential for processing ATG8 family proteins during autophagosome formation. In Trichomonas vaginalis, the role of autophagin proteases in processing autophagy markers TvAtg8a and TvAtg8b has not been fully characterized. In this study, we expressed and purified recombinant TvAtg4.4 and demonstrated its cysteine protease activity in vitro. TvAtg4.4 rapidly processed TvAtg8aGST and, to a lesser extent, TvAtg8bGST. Enzymatic assays confirmed substrate specificity and inhibition by cysteine protease inhibitors. TvAtg4.4 mRNA expression increased under glucose restriction, and immunolocalization showed its presence in autophagic vesicles, cytoplasm, endoplasmic reticulum, Golgi, lysosomes, hydrogenosomes, and nucleus. Colocalization with TvAtg8a and TvAtg8b supports its functional role in autophagy. The localization of TvAtg4.4 in T. vaginalis autophagosomes and ER suggests its involvement in the cleavage of TvAtg8a and TvAtg8b after synthesis and in the delipidation or deconjugation of these proteins from the autophagosome outer membrane before autophagosome-lysosome fusion. These findings clarify the enzymatic function and cellular localization of TvAtg4.4, provide insight into autophagy mechanisms in T. vaginalis, and suggest potential novel roles for this protease in parasite biology.