Protease activities of vaginal Porphyromonas species disrupt coagulation and extracellular matrix in the cervicovaginal niche

Abstract

Porphyromonas asaccahrolytica and Porphyromonas uenonis are frequently isolated from the human vagina and are linked to bacterial vaginosis and preterm labour. However, little is known about the pathogenesis mechanisms of these bacteria. The related oral opportunistic pathogen, Porphyromonas gingivalis, is comparatively well-studied and known to secrete numerous extracellular matrix-targeting proteases. Among these are the gingipain family of cysteine proteases that drive periodontal disease progression and hematogenic transmission to the placenta. Given their phylogenetic relatedness, we hypothesized that vaginal Porphyromonas species possess gingipain-like protease activity targeting host extracellular matrix in the female reproductive tract. In this study, we demonstrate that vaginal Porphyromonas species degrade type I collagen (cervix), type IV collagen (chorioamnion/placenta), and fibrinogen, but not through the activity of gingipain orthologs. Bioinformatic queries identified 5 candidate collagenases in each species, including serine, cysteine and metalloproteases, with signal peptides directing them to the extracellular environment. Inhibition assays revealed both species secrete metalloproteases that degrade collagen and casein, while P. asaccharolytica also secretes a metalloprotease that degrades fibrinogen. Phylogenetic analysis of the predicted collagen-degrading metalloprotease revealed an orthologous relationship with the P. gingivalis endopeptidase PepO. Cloning and expression of P. asaccharolytica PepO confirmed this proteins collagenase and caseinase activities, which have not previously been attributed to PepO homologs in other bacteria. Altogether, this description of the first known virulence factor in Porphyromonas species colonizing the human vagina sheds light on their potential to alter the structural integrity and homeostasis of reproductive tissues. ImportancePorphyromonas species are common inhabitants of the vaginal microbiome, but their presence has been liked to adverse health outcomes for women, including bacterial vaginosis and preterm birth. We determined that P. asaccharolytica and P. uenonis secrete broad-acting proteases capable of freely diffusing within the cervicovaginal niche and degrading important components of host tissues, namely the extracellular matrix. We show that secreted Porphyromonas proteases degrade collagens that are enriched within the cervix (type I) and chorioamniotic membranes (type IV). Furthermore, these Porphyromonas proteases can also degrade fibrinogen and inhibit clot formation. These activities can be partially attributed to a metalloprotease that exhibits broad-acting protease activity and is distantly related to the P. gingivalis endopeptidase PepO. This initial characterization of virulence activities in vaginal Porphyromonas species highlights their potential to harm human pregnancy through clotting disruption, fetal membrane weakening, and premature cervical remodeling.