Conference Agenda

Trichomonas vaginalis (TV), the most common non-viral sexually transmitted human pathogen, feeds on the vaginal microbiota including protective Lactobacillus spp. (LB) through phagocytosis. Subsequent destruction occurs in lysosomes through degradative hydrolases. Secretion of protein factors by TV may play a fundamental role in TV-LB interaction prior to phagocytosis. However, a comprehensive profile of the secretome upon interaction remains elusive.

Here we co-cultivated TV and LB for multiple time frames to investigate their interactions. Phagocytosis was observed and quantified by microscopy and Fluorescence-activated cell sorting. The dynamics of protein secretion during TV-LB co-incubation was analyzed in the cell-free supernatant by label-free quantitative mass spectrometry. Gene expression levels of differentially secreted proteins were examined through real-time quantitative PCR. LB significantly stimulated the secretion of 24 TV proteins including a variety of proteases and surface proteins such as Lysozyme-like, Cathepsin-L like, and GP63-like peptidases, and Trichomonas beta-sandwich repeat proteins, a group of heterogenous surface transmembrane proteins of unknown function. Overexpression of selected proteins in TV confirmed their effect on LB viability.

Our analysis provides a comprehensive set of secreted proteins that may be involved in parasite-LB interactions and potentially contribute to the decolonization of LB during trichomoniasis and the development of vaginal dysbiosis.

Infection with Trichomonas vaginalis is associated with an imbalance in the bacteria that normally reside in the female reproductive tract. T. vaginalis is increasingly detected in women that have reduced amounts or a complete absence of Lactobacillus species. This is alarming, as Lactobacillus crispatus promotes better pregnancy outcomes and protects against urogenital infection. We hypothesize that T. vaginalis has antibacterial activity against L. crispatus. Using confocal microscopy, we observed that T. vaginalis adheres to L. crispatus as soon as both microbes are exposed to one another. Furthermore, T. vaginalis exerts an antibacterial effect against L. crispatus leading to a statistically significant reduction in L. crispatus colony forming units as early as 30 minutes, and even greater microbicidal activity is observed at 3 and 6 hours of co-incubation. Additionally, T. vaginalis displayed this antibacterial activity in nutrient-rich and nutrient-limiting conditions, whereas the viability of T. vaginalis was unaffected by exposure to L. crispatus. The molecular mechanisms contributing to this microbe-microbe interaction are under study. Our work will yield critical insight as to how T. vaginalis infection leads to changes in the cervicovaginal microbiome and potentially reveal a therapeutic need to ameliorate the microbiological insults inflicted by T. vaginalis.

Lactobacillus iners is the most common bacteria found in the cervicovaginal microbiome. This bacterial community is most prone to shift towards a microbial community found in women with bacterial vaginosis. Infection with Trichomonas vaginalis is also associated with bacterial vaginosis. To our knowledge, nothing is known about T. vaginalis-L. iners interactions. We hypothesize that T. vaginalis exerts antibacterial effects on L. iners. We first optimized the conditions to maximally quantify L. iners colony formation on agar plates. Upon co-incubation with T. vaginalis, we found that at a ratio of 155 L. iners to 1 T. vaginalis, there was a statistically significant 96% reduction in L. iners colony formation. At a ratio of 0.02 bacteria to 1 parasite, L. iners growth was promoted by 1.7 fold in the presence of T. vaginalis. Interestingly, in 12 biologically independent experiments, T. vaginalis viability was unaffected by the presence of L.iners. Lastly, using transmission electron microscopy, we have observed L. iners uptake by T. vaginalis. Delineating the factors that influence the balance between coexistence vs. antibacterial killing of L. iners by T. vaginalis can potentially lead to the identification of novel therapeutic targets to ease the burden of T. vaginalis infections.

The human vaginal microbiome is a complex ecosystem primarily governed by lactobacilli, which create an acidic environment and defend against pathogens. However, when lactobacilli decrease, anaerobic bacteria such as, Gardnerella vaginalis among others may flourish as a polymicrobial condition known as bacterial vaginosis (BV). Coincidently, the vaginal infection by the protozoan Trichomonas vaginalis (i.e., trichomoniasis) is accompanied by this dysbiotic BV-like microbiome including G. vaginalis. Therefore, the interactions between this bacterium and the protozoan, despite poorly understood, might be crucial for disease development. Because G. vaginalis is the initiator of a pathogenic biofilm, here we aimed to understand how these two microorganisms may help disperse each other. We employed complementary approaches to evaluate biofilm growth and dispersion, and the adhesion of T. vaginalis to this biofilm. We also examined whether extracellular vesicles (EVs) produced by lactobacilli and G. vaginalis would influence the dispersion of T. vaginalis on agar-based assays. We show that T. vaginalis avidly adheres to the G. vaginalis biofilm and enhances bacterial dispersion in a strain-independent manner while bacterial EVs affect T. vaginalis dispersion. Further experiments are necessary to quantify these phenotypes and to reveal the molecular mechanisms underlying the synergistic interactions between G. vaginalis and T. vaginalis.

Vaginal trichomoniasis, resulting from Trichomonas vaginalis infection, is characterized by a dysbiotic microbiome including anaerobic bacterial species associated with bacterial vaginosis (BV). Recent research by us and others highlights important disease-related synergism between BV-bacteria and T. vaginalis. BV-bacteria are known to metabolically collaborate among themselves. Therefore, our study aimed to investigate whether BV-bacteria and T. vaginalis would also engage in metabolic cooperation. Here we demonstrate that, unlike host-protective lactobacilli, BV-bacteria and T. vaginalis mutually support each other's growth, even in depleted conditions where neither organism can grow alone. Furthermore, metabolomics analyses using gas-chromatography coupled with mass-spectrometry revealed alterations in metabolite profiles, indicating a metabolic shunt towards amino acid catabolism, notably through upregulation of the arginine dihydrolase (ADH) pathway. This metabolic pattern mirrors previous reports of cooperation between T. vaginalis and its mycoplasma endosymbionts. Gene expression analysis further confirmed upregulation of ADH enzymes, particularly carbamate kinase. In conclusion, our findings unveil a previously unrecognized metabolic cooperation between T. vaginalis and the associated dysbiotic microbiome, providing insights into the synergistic relationships underlying vaginal trichomoniasis.

Trichomonas species live at various mucosal surfaces characterised by complex microbiota amongst a range of animal hosts. Trichomonas infections associate with significant changes in microbiota taxonomic composition, commonly referred to as dysbiosis. Trichomonas vaginalis and Trichomonas gallinae (a bird parasite) infections result in the depletion of the mutualist bacteria Lactobacillus spp., known to play important roles in female urogenital defence against pathogens. However, the molecular and cellular basis of interactions between Trichomonas and the microbiota are poorly understood.

We exploited Trichomonas gallinae - Escherichia coli co-cultures as a model system alongside comparative genomics and transcriptomics (RNAseq) to identify candidate enzymes and peptides targeting bacteria. A number of genes encoding homologues of bacterial cell wall targeting enzymes, including candidate lysozymes, and anti-microbial peptides, which are conserved throughout Trichomonas spp., were identified and with a number of them being significantly upregulated within the co-culture system. We also observed changes in bacterial and parasite behaviour and morphology.

Integrating comparative genomics between species, transcriptomics within our model and known interactions between Trichomonas and bacteria, for example the peptidoglycan targeting NlpC/P60 endopeptidases, illustrates a number of mechanisms for the parasites to potentially out-compete bacteria, with a likely important role of deconstructing bacterial cell wall peptidoglycans.