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 E-2 (PGE(2)), 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 PGE(2) 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 (ppad and C351A). The impaired ability of the ppad strain to adhere to and/or invade PHGF and both ppad and C351A to stimulate the PGE(2)-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 PGE(2) synthesis, the latter of which may strongly contribute to bone resorption and eventual tooth loss.