tRNA-derived stress-induced RNAs, exhibiting translation-inhibitory 5’-terminal oligoguanine motifs, are produced by Trichomonas vaginalis and packaged into extracellular vesicles

Abstract

Trichomoniasis, caused by the pathogenic protozoan parasite Trichomonas vaginalis, is a widespread sexually transmitted disease causing mostly vaginitis and urethritis but also significant morbidities. Despite its high global prevalence, research into T. vaginalis struggles to gain adequate attention from the scientific community leaving substantial gaps of knowledge on the pathobiology of this medically important pathogen. A key avenue of investigation comprises understanding the impact that the RNA cargo of extracellular vesicles (EVs) produced by T. vaginalis may have on parasite-to-parasite and parasite-to-host communication. T. vaginalis EVs have a subpopulation of tRNA-derived small RNAs (tsRNAs) alike to tRNA-derived stress-induced RNAs (tiRNAs). In mammals, 5’-tiRNAAla and 5’-tiRNACys harbouring a 5’ terminal oligoguanine (TOG) motif are known for promoting translation inhibition through dissociation of the eukaryotic initiation factor 4F (eIF4F) complex as an endogenous response to nutritional stress. To date, this pathway has not been formally described in protozoan parasites; even less is known about these tsRNA populations in protozoans and how they are produced in cells and packaged into EVs. In this study, we aimed to identify TOG-containing 5’-tiRNAAla and 5’-tiRNACys in T. vaginalis, and determine their relative abundances upon nutritional stress in cells and EVs via quantitative PCR. Secondly, we wanted to test methodologies that could allow us to determine translational inhibitory properties of these 5’-tiRNAs for future research. These include testing the performance of: antibodies to detect individual components of the human eIF4F and a luciferase reporter assay to quantify translation activity in human cells. This research demonstrated that T. vaginalis TOG-containing 5’-tiRNAAla and 5’-tiRNACys can be detected qualitatively with primers. Quantitative analysis further revealed that when nutritionally stressed, T. vaginalis cells upregulate TOG-containing 5’-tiRNAs, and this relationship is transferrable to EVs. While we could not ascertain the translational inhibitory properties of TOG-containing 5’-tiRNAs so far due to technical errors, we are one step closer to displaying this mechanism clearly. Collectively, the findings in this research broaden our knowledge of the packaging of tsRNAs into EVs and strengthen the hypothesis that T. vaginalis TOG-containing 5’-tiRNAs play a crucial role in parasite-to-host communication.