Identification of autophagy-related protein 3 in the ancient protist Trichomonas vaginalis

Background: Autophagy has been suggested to be involved in the pathogenesis of protists. While the molecular mechanisms of autophagy are mainly studied in model organisms, functional characterization of autophagy-related (Atg) proteins is poorly understood in deep-branching protists. Trichomoniasis is the most common nonviral sexually transmitted infection caused by Trichomonas vaginalis. Bioinformatics analysis of the T. vaginalis genome reveals that the parasite possesses the genes encoding proteins of the Atg8 conjugation system. Herein, we sought to characterize whether the T. vaginalis Atg3 ortholog (TVAG_447140), a putative component of the TvAtg8 conjugation system, regulates autophagy in this parasite. Methods: The recombinant protein of T. vaginalis Atg3 ortholog (TvAtg3) (rTvAtg3) and the polyclonal antibody against rTvAtg3 were generated. The expression and localization was monitored upon autophagy induction by glucose restriction (GR) compared with glucose-rich cultivation. The role of TvAtg3 in autophagy was clarified using small interfering RNA targeting TvAtg3 gene. Results: Phylogenic analysis of Atg3 proteins from different organisms showed that T. vaginalis was not in a close evolutionary relationship with any other protozoan. The expression of TvAtg3 was upregulated in the late-stationary phase of GR culture, implying its involvement in autophagy. Immunofluorescence analysis revealed a much higher TvAtg3 fluorescent intensity located on the round and/or linear structures close to the nucleus. Silencing Tvatg3 expression suppressed GR-induced TvAtg8 expression and autophagic vacuoles formation. Conclusions: These findings suggest the potential role of TvAtg3 in T. vaginalis autophagy and enhance our understanding of the autophagy regulatory network in the deep-branching eukaryotes.