In vivo expression of proteases and protease inhibitor, a serpin, by periodontal pathogens at teeth and implants.

Porphyromonas gingivalis and Tannerella forsythia secrete proteases, gingipains and KLIKK-proteases. In addition, T. forsythia produces a serpin (miropin) with broad inhibitory spectrum. The aim of this pilot study was to determine the level of expression of miropin and individual proteases in vivo in periodontal and peri-implant health and disease conditions. Biofilm and gingival crevicular fluid (GCF)/ peri-implant sulcular fluid (PISF) samples were taken from healthy tooth and implant sites (n = 10), gingivitis and mucositis sites (n = 12), and periodontitis and peri-implantitis sites (n = 10). Concentration of interleukin-8 (IL-8), IL-1ß and IL-10 in GCF was determined by enzyme-linked immunosorbent assay. Loads of P. gingivalis and T. forsythia and the presence of proteases and miropin genes were assessed in biofilm by quantitative PCR, whereas gene expression was estimated by quantitative RT-PCR. The presence of P. gingivalis and T. forsythia, as well as the level of IL-8 and IL-1ß, were associated with disease severity in the periodontal and peri-implant tissues. In biofilm samples harboring T. forsythia, genes encoding proteases were found to be present at 72.4% for karilysin and 100% for other KLIKK-protease genes and miropin. At the same time, detectable mRNA expression of individual genes ranged from 20.7% to 58.6% of samples (for forsylisin and miropsin-1, respectively). In comparison with the T. forsythia proteases, miropin and the gingipains were highly expressed. The level of expression of gingipains was associated with those of miropin and certain T. forsythia proteases around teeth but not implants. Cumulatively, KLIKK-proteases and especially miropin, might play a role in pathogenesis of both periodontal and peri-implant diseases.

Peptidylarginine deiminase from Porphyromonas gingivalis contributes to infection of gingival fibroblasts and induction of prostaglandin E2 -signaling pathway.

Porphyromonas gingivalis (P. gingivalis) expres-ses the enzyme peptidylarginine deiminase (PPAD), which has a strong preference for C-terminal arginines. Due to the combined activity of PPAD and Arg-specific gingipains, P. gingivalis on the cell surface is highly citrullinated. To investigate the contribution of PPAD to the interaction of P. gingivalis with primary human gingival fibroblasts (PHGF) and P. gingivalis-induced synthesis of prostaglandin E2 (PGE2 ), PHGF were infected with wild-type P. gingivalis ATCC 33277, an isogenic PPAD-knockout strain (∆ppad) or a mutated strain (C351A) expressing an inactive enzyme in which the catalytic cysteine has been mutated to alanine (PPAD(C351A) ). Cells were infected in medium containing the mutants alone or in medium supplemented with purified, active PPAD. PHGF infection was assessed by colony-forming assay, microscopic analysis and flow cytometry. Expression of cyclo-oxygenase 2 (COX-2) and microsomal PGE synthase-1 (mPGES-1), key factors in the prostaglandin synthesis pathway, was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), while PGE2 synthesis was evaluated by enzyme immunoassay. PHGF were infected more efficiently by wild-type P. gingivalis than by the ∆ppad strain, which correlated with strong induction of COX-2 and mPGES-1 expression by wild-type P. gingivalis, but not by the PPAD activity-null mutant strains (Δppad and C351A). The impaired ability of the Δppad strain to adhere to and/or invade PHGF and both Δppad and C351A to stimulate the PGE2 -synthesis pathway was fully restored by the addition of purified PPAD. The latter effect was strongly inhibited by aspirin. Collectively, our results implicate PPAD activity, but not PPAD itself, as an important factor for gingival fibroblast infection and activation of PGE2 synthesis, the latter of which may strongly contribute to bone resorption and eventual tooth loss.