Vaginal Porphyromonas species carve their own path of destruction

A tale of secreted proteolytic activities from vaginal Porphyromonas species that contributes to structural remodeling within the female genital tract.

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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

Model prepared in BioRender

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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References

1            Guilloux, C. A., Lamoureux, C., Beauruelle, C. & Hery-Arnaud, G. Porphyromonas: A neglected potential key genus in human microbiomes. Anaerobe 68, 102230, doi:10.1016/j.anaerobe.2020.102230 (2021).

2            Elovitz, M. A. et al. Cervicovaginal microbiota and local immune response modulate the risk of spontaneous preterm delivery. Nat Commun 10, 1305, doi:10.1038/s41467-019-09285-9 (2019).

3            Holst, E., Goffeng, A. R. & Andersch, B. Bacterial vaginosis and vaginal microorganisms in idiopathic premature labor and association with pregnancy outcome. J Clin Microbiol 32, 176-186, doi:10.1128/JCM.32.1.176-186.1994 (1994).

4            Petrina, M. A. B., Cosentino, L. A., Wiesenfeld, H. C., Darville, T. & Hillier, S. L. Susceptibility of endometrial isolates recovered from women with clinical pelvic inflammatory disease or histological endometritis to antimicrobial agents. Anaerobe 56, 61-65, doi:10.1016/j.anaerobe.2019.02.005 (2019).

5            Haggerty, C. L., Hillier, S. L., Bass, D. C. & Ness, R. B. Bacterial vaginosis and anaerobic bacteria are associated with endometritis. Clin Infect Dis 39, 990-995, doi:CID33509 [pii]

10.1086/423963 (2004).

6            Chao, X. et al. Research of the potential biomarkers in vaginal microbiome for persistent high-risk human papillomavirus infection. Ann Transl Med 8, 100, doi:10.21037/atm.2019.12.115 (2020).

7            Chen, Y. et al. Human papillomavirus infection and cervical intraepithelial neoplasia progression are associated with increased vaginal microbiome diversity in a Chinese cohort. BMC Infect Dis 20, 629, doi:10.1186/s12879-020-05324-9 (2020).

8            Walther-Antonio, M. R. et al. Potential contribution of the uterine microbiome in the development of endometrial cancer. Genome Med 8, 122, doi:10.1186/s13073-016-0368-y (2016).

9            Summanen, P. H. et al. Porphyromonas somerae sp. nov., a pathogen isolated from humans and distinct from porphyromonas levii. J Clin Microbiol 43, 4455-4459, doi:10.1128/JCM.43.9.4455-4459.2005 (2005).

10          Imamura, T., Travis, J. & Potempa, J. The biphasic virulence activities of gingipains: activation and inactivation of host proteins. Curr Protein Pept Sci 4, 443-450, doi:10.2174/1389203033487027 (2003).

11          Bedi, G. S. & Williams, T. Purification and characterization of a collagen-degrading protease from Porphyromonas gingivalis. J Biol Chem 269, 599-606 (1994).

12          Tada, H. et al. Proteolysis of ICAM-1 on human oral epithelial cells by gingipains. J Dent Res 82, 796-801, doi:10.1177/154405910308201007 (2003).

13          Takii, R., Kadowaki, T., Tsukuba, T. & Yamamoto, K. Inhibition of gingipains prevents Porphyromonas gingivalis-induced preterm birth and fetal death in pregnant mice. Eur J Pharmacol 824, 48-56, doi:10.1016/j.ejphar.2018.01.028 (2018).

14          O'Flynn, C. et al. Comparative Genomics of the Genus Porphyromonas Identifies Adaptations for Heme Synthesis within the Prevalent Canine Oral Species Porphyromonas cangingivalis. Genome Biol Evol 7, 3397-3413, doi:10.1093/gbe/evv220 (2015).

15          Park, Y., Yilmaz, O., Jung, I. Y. & Lamont, R. J. Identification of Porphyromonas gingivalis genes specifically expressed in human gingival epithelial cells by using differential display reverse transcription-PCR. Infect Immun 72, 3752-3758, doi:10.1128/IAI.72.7.3752-3758.2004 (2004).

16          Ansai, T., Yu, W., Urnowey, S., Barik, S. & Takehara, T. Construction of a pepO gene-deficient mutant of Porphyromonas gingivalis: potential role of endopeptidase O in the invasion of host cells. Oral Microbiol Immunol 18, 398-400, doi:10.1046/j.0902-0055.2003.00080.x (2003).

17          Awano, S. et al. Sequencing, expression and biochemical characterization of the Porphyromonas gingivalis pepO gene encoding a protein homologous to human endothelin-converting enzyme. FEBS Lett 460, 139-144, doi:10.1016/s0014-5793(99)01326-5 (1999).

18          Liu, L. et al. Global, regional, and national causes of under-5 mortality in 2000-15: an updated systematic analysis with implications for the Sustainable Development Goals. Lancet 388, 3027-3035, doi:10.1016/S0140-6736(16)31593-8 (2016).

19          Blencowe, H. et al. Born too soon: the global epidemiology of 15 million preterm births. Reprod Health 10 Suppl 1, S2, doi:10.1186/1742-4755-10-S1-S2 (2013).

20          Lettieri, L., Vintzileos, A. M., Rodis, J. F., Albini, S. M. & Salafia, C. M. Does "idiopathic" preterm labor resulting in preterm birth exist? Am J Obstet Gynecol 168, 1480-1485, doi:10.1016/s0002-9378(11)90785-6 (1993).

21          Muglia, L. J. & Katz, M. The enigma of spontaneous preterm birth. N Engl J Med 362, 529-535, doi:10.1056/NEJMra0904308 (2010).

22          Iams, J. D., Romero, R., Culhane, J. F. & Goldenberg, R. L. Primary, secondary, and tertiary interventions to reduce the morbidity and mortality of preterm birth. Lancet 371, 164-175, doi:10.1016/S0140-6736(08)60108-7 (2008).

23          Osmers, R. et al. Collagenase activity in the cervix of non-pregnant and pregnant women. Arch Gynecol Obstet 248, 75-80, doi:10.1007/BF02389578 (1990).

24          Rath, W., Adelmann-Grill, B. C., Pieper, U. & Kuhn, W. Collagen degradation in the pregnant human cervix at term and after prostaglandin-induced cervical ripening. Arch Gynecol 240, 177-184, doi:10.1007/BF00207713 (1987).

25          Wang, H. et al. Genetic and epigenetic mechanisms combine to control MMP1 expression and its association with preterm premature rupture of membranes. Hum Mol Genet 17, 1087-1096, doi:10.1093/hmg/ddm381 (2008).

26          McLaren, J., Taylor, D. J. & Bell, S. C. Increased concentration of pro-matrix metalloproteinase 9 in term fetal membranes overlying the cervix before labor: implications for membrane remodeling and rupture. Am J Obstet Gynecol 182, 409-416, doi:10.1016/s0002-9378(00)70232-8 (2000).

Karen Lithgow

Postdoctoral Fellow, University of Calgary