Porphyromonads: high prevalence, low abundance troublemakers emerging from the human microbiome
Anaerobes from the genus Porphyromonas are commonly found within the lung, oral, gastric, cutaneous and vaginal microbiomes. However, these low-abundance bacteria are emerging as frequent offenders linked to adverse health outcomes such as cancers and periodontitis1. Vaginal Porphyromonas species are common members of the vaginal microbiome that increase a woman’s risk for bacterial vaginosis, pelvic inflammatory disease, HIV acquisition, gynecological cancer and preterm birth2-9. Given our limited knowledge about these species, we sought to functionally characterize two vaginal Porphyromonas species to better understand their contributions to adverse sexual and reproductive health outcomes.
Urogenital cousins of gingipain-encoding Porphyromonas gingivalis
The most recognizable Porphyromonad is P. gingivalis, a periodontal pathogen best known for secreting the broad-acting cysteine proteases known as gingipains. Gingipains (RgA, RgB, Kgp) contribute to local tissue destruction by degrading extracellular matrix proteins and immune factors. They also activate host collagen-degrading enzymes10-12. Intriguingly, the gingipains have even been implicated in preterm birth13. Given the association between vaginal Porphyromonas species and preterm birth, we were curious to explore whether gingipain-like enzymes may be encoded in vaginal Porphyromonas species.
Vaginal Porphyromonas species carve their own path of destruction
In our recent study, we show that the vaginal Porphyromonas species, Porphyromonas asaccharolytica and Porphyromonas uenonis, secrete broad-acting proteases that degrade type I collagen, type IV collagen as well as the universal protease substrate casein. Collagens are key structural proteins found within pregnancy tissues and significantly remodeled during labour. We also found that these bacteria impaired fibrin clotting by degrading fibrinogen. Despite these gingipain-like activities, we found no evidence that the vaginal Porphyromonas species encode gingipains, confirming the results from a previous comparative genomics study14.
Given these results, we anticipated that the vaginal Porphyromonas species encoded novel proteases, so we set out to identify the enzymes responsible for the observed collagen, casein and fibrinogen degradation. We created a list of all predicted peptidases in P. asaccharolytica and P. uenonis and predicted which classes were candidate collagen degrading enzymes. Inhibitors specific for these enzyme classes were incorporated into degradation assays with Porphyromonas cell-free supernatants, revealing that a metalloprotease inhibitor blocked degradation of secreted collagenase (type I, type IV) and caseinase activities in both P. asaccharolytica and P. uenonis. This led us to conclude that a candidate M13 metalloprotease found in P. asaccharolytica and P. uenonis was conferring the observed proteolytic activity.
PepO: ubiquitous Porphyromonas metalloproteases
In order to validate the activity of our candidate protease, the M13 metalloprotease from P. asaccharolytica was cloned and expressed. We found that this recombinant protein degraded type I collagen and casein, but not type IV collagen. These results confirm that the P. asaccharolytica M13 metalloprotease is a host-targeting collagenase, but also highlight the existence of at least one additional unidentified Porphyromonas metalloprotease that degrades type IV collagen. Comparative genomics revealed that the M13 metalloprotease is ubiquitous amongst Porphyromonas species isolated from the urogenital tract and oral niche. In fact, the P. gingivalis M13 metalloprotease has been previously characterized as PepO, a secreted endopeptidase with roles in host attachment/invasion and proteolytic activation of the vasoconstrictor endothelin-115-17.
Host-targeting Porphyromonas metalloproteases: implications for pregnancy complications
Our interest in the vaginal Porphyromonas species originally stemmed from their association with adverse health outcomes for women, particularly preterm birth. Preterm birth (<37 weeks gestation) is a major cause of infant and child mortality affecting 1 in 10 pregnancies worldwide18,19. Although a significant proportion of preterm labour cases are the result of intrauterine infection, antibiotic therapies do not prevent preterm birth, nor improve outcomes20-22. Our work represents the first functional characterization of preterm labour-associated vaginal Porphyromonas species and uncovers possible mechanisms underlying their association with pregnancy complications. Our observations that the vaginal Porphyromonas species degrade fibrinogen and impair clot formation could leave women at elevated risk for postpartum haemorrhage. Furthermore, our findings that vaginal Porphyromonas species degrade collagens that are enriched in pregnancy tissues has important implications for obstetrical outcomes. Remodeling of collagens in the cervix contributes to cervical softening that precedes dilation and delivery23,24. Furthermore, collagen degradation in chorioamniotic (fetal/maternal) membranes contributes to membrane rupture (water breaking)25,26. Our work suggests that secreted host-targeted proteases from vaginal Porphyromonas species could initiate key events leading to preterm birth, as inappropriately timed collagenase activity could prematurely trigger cervical dilation, chorioamniotic membrane rupture and ultimately preterm labour. Any way you cut it, the secreted proteases of vaginal Porphyromonas species are likely to be key modulators of mucosal environment in the female genital tract.
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