Polymicrobial Synergy Stimulates Porphyromonas Gingivalis Survival And Gingipain Expression In A Multispecies Subgingival Community

Abstract

Background Dysbiosis in subgingival microbial communities, resulting from increased inflammatory transudate from the gingival tissues, is an important factor in initiation and development of periodontitis. Dysbiotic communities are characterized by increased numbers of bacteria that exploit the serum-like transudate for nutrients, giving rise to a proteolytic community phenotype. Here we investigate the contribution of interactions between members of a sub-gingival community to survival and development of virulence in a serum environment - modelling that in the subgingival pocket. Methods Growth and proteolytic activity of three P. gingivalis strains in nutrient-rich broth or a serum environment were assessed using A600 and a fluorescent protease substrate, respectively. Adherence of P. gingivalis strains to serum-coated surfaces was studied with confocal microscopy and 2D-gel electrophoresis of bacterial supernatants used to investigate extracellular proteins. A model multi-species sub-gingival community containing Fusobacterium nucleatum, Streptococcus constellatus, Parvimonas micra with wild type or isogenic mutants was then created and growth and proteolytic activity in serum assessed as above. Community composition over time was monitored using culture techniques and qPCR. Results The P. gingivalis strains showed different growth rates in nutrient-rich broth related to the level of proteolytic activity (largely gingipains) in the cultures. Despite being able to adhere to serum-coated surfaces, none of the strains was able to grow alone in a serum environment. In the subgingival consortium however, all the included species were able to grow in the serum environment and the community adopted a proteolytic phenotype. Inclusion of P. gingivalis strains lacking gingipains in the consortium revealed that the ability of the community to grow was largely due to Rgp gingipain. Conclusions In the multi-species consortium, growth was facilitated by the wild-type and Rgp-expressing strains of P. gingivalis, suggesting that Rgp is involved in delivery of nutrients to the whole community through degradation of complex serum substrates. Whereas they are constitutively expressed by P. gingivalis in nutrient-rich broth, gingipain expression in the model periodontal pocket environment (serum) appears to be orchestrated through signaling to P. gingivalis from other members of the community, a phenomenon which can then promote growth of the whole community.