Postdoctoral Fellow University of Calgary, Canada Calgary, Alberta, Canada
Background: Bacterial vaginosis (BV) is a prevalent dysbiotic vaginal condition that increases the risk of sexually transmitted infection (STI). Elevated cervicovaginal proteolysis occurs during BV and increased human matrix metalloproteinase (MMP) expression is linked to HIV acquisition. However, the role of BV-associated bacterial proteases in initiating proteolysis is poorly defined and their human protein targets remain unknown. We hypothesize that BV-associated bacteria secrete proteases that disrupt the endocervical barrier and promote pathogen invasion.
Methods: BV-associated bacteria were screened for proteolytic activity using zymography or fluorophore-conjugated substrates. Casein was used to assess general protease activity, while structural (collagen I/IV and elastin) and regulatory (MMP-8) cervical protein substrates were used to assess effects on endocervical tissue integrity. Protease inhibitors were used to confirm the enzyme types. To evaluate bacterial protease-mediated barrier disruption, a polarized endocervical epithelial model was exposed to BV-associated bacteria or cell-free supernatants and then subjected to a solute flux assay. Results: Proteolytic activity was detected across diverse BV-associated bacteria, but the highest activity was observed in Porphyromonas and Prevotella species. These BV-associated bacteria were found to produce diverse and redundant enzymes that degrade key structural barrier proteins, including collagens and elastin. Next, we evaluated whether BV-associated bacteria proteolytically process MMP-8, a human protease that targets collagen and immune substrates. Vaginal Prevotella species were found to target MMP-8 for activation and degradation, producing several unique proteolyzed breakdown products over time. Prevotella bivia exhibited the highest levels of protease activity and the most rapid MMP-8 processing. Furthermore, P. bivia secreted proteases disrupted endocervical barrier function in a polarized epithelial model.
Conclusions: Vaginal Porphyromonas and Prevotella species are high prevalence/low abundance BV-associated bacteria linked to HIV acquisition. These BV-associated bacteria exhibit high levels of secreted proteolytic activity targeting structural proteins involved in maintaining endocervical barrier function. Prevotella species can further exacerbate endocervical barrier disruption by proteolytically activating the human cervical-remodelling protease, MMP-8. Taken together, these results demonstrate that proteases secreted by BV-associated bacteria contribute to endocervical barrier disruption, which could promote pathogen invasion and STI acquisition. Further characterization of secreted proteases and their targets could unveil new therapeutics for BV and STIs.
