Parvimonas micra stimulates expression of gingipains from Porphyromonas gingivalis in multi-species communities

Dental biofilms are complex ecosystems containing many bacterial species that live in mutualistic relationships. These interactions can profoundly affect the virulence properties of the community. In this study we investigated how the production of gingipains, virulence factors from Porphyromonas gingivalis important in periodontal disease, was affected by other commonly found members of the sub-gingival microbiome. To mimic the subgingival microbiome, multispecies consortia (P. gingivalis, Fusobacterium nucleatum, Actinomyces naeslundii, Streptococus oralis, Streptococcus mitis, Streptococcus gordonii and Streptococcus cristatus, with or without Parvimonas micra) as well as dual species consortia (P. gingivalis with P. micra, S. oralis or F. nucleatum) were constructed and maintained anaerobically in 10% serum for up to seven days. The number of P. gingivalis was determined by plating on Brucella agar and the gingipain specific fluorogenic substrate BikKam-10 was used to investigate gingipain activity. The effect of secreted products from P. micra on gingipain activity was investigated by adding supernatants from P. micra to P. gingivalis cultures. The most prominent secreted proteins in the supernatant were identified using mass spectrometry. P. gingivalis was unable to grow in serum, either alone or in the presence of S. oralis or F. nucleatum. In contrast, with P. micra growth was significantly enhanced and this was associated with an increase in gingipain activity. In the multi-species consortia, the presence of P. micra caused a 13-fold increase in gingipain activity. Exposure of P. gingivalis to supernatants from P. micra for 24 h caused a 3-fold increase in gingipain activity. This effect was reduced by 43% after heat-treatment of the supernatant. Two dimensional gel electrophoresis revealed that several of the most prominent proteins in the P. micra supernatant were glycolytic enzymes. The results from this study suggests that gingipains are produced in response to a P. micra derived signalling molecule that is most likely a protein. This is the first time it has been shown that P. micra can affect P. gingivalis virulence properties. This is likely to be of significance for the development of be of periodontitis since these two microorganisms are often found together in the subgingival biofilm