Ginigipains Determine the Clinical Outcome of Mixed Infection with P.gingivalis&F.nucleatum

Background: P.gingivalis and F.nucleatum are considered as important pathogens in pathogenesis of periodontal disease. We have showed that mixed infection with P.gingivalis and F.nucleatum induces a stronger inflammatory response than mono-infection. Gingipains, a group of cysteine proteases produced by P.gingivalis, were proposed as an interaction factor with other bacterial species, and may have a role in co-aggregation between P.gingivalis and other bacteria. Objectives: To determine the role of P.gingivalis proteases in inducing the synergistic effect observed in mixed infection with F.nucleatum. Methods: Two models were used in this study - the subcutaneous model used to investigate the local inflammatory evoked by bacterial infection, and the alveolar bone loss model used to investigate the effect of bacterial infection on the periodontium. Mixed infection of F.nucleatum with mutant strains of P.gingivalis lacking RgpA or kgp, were compared with wild-type(WT) P.gingivalis. Co-aggregation assay was preformed to evaluate effect of Gingipains on co-aggregation with F.nucleatum in vitro. Results: Mixed infection with P.gingivalis lacking RgpA or Kgp exhibited less co-aggregation in vitro and induced less leukocyte migration in vivo compared to WT P.gingivalis. Mixed infection with P.gingivalis mutants also induced less TNFα, IL-1β and IL-10, but not IFNγ, compared to mixed infection with the WT bacteria. Mice infected orally with P.gingivalis RgpA^-/- and F.nucleatum were more resistant to alveolar bone loss than mice infected with F.nucleatum and the WT or Kgp mutant of P.gingivalis. Conclusions: Gingipains are responsible, in part, for the co-aggregation between P.gingivalis and F.nucleatum. P.gingivalis lacking RgpA or Kgp induces less leukocyte migration and reduced inflammatory response using our mixed infection model. The reduced IL-10 level indicates a shift toward Th1 dominance in mixed infection with P.gingivalis lacking Gingipains. RgpA, but not Kgp, has a critical role in inducing bone loss both in P.gingivalis mono-infection and in mixed infection with F.nucleatum.