Periodontitis (PD), an oral inflammatory disease, is primarily caused by P. gingivalis. Peptidylarginine deiminase (PPAD) is considered an attractive virulence factor because, due to protein citrullination, it may have deleterious effects on host tissues. In this study, the ppad gene sequences from P. gingivalis were analyzed in the context of its impact on bacterial virulence and potential targets for PD therapy. Analyses of ppad sequences from 58 patients with various clinical stages of PD, 20 controls, and 60 sequences from public databases were conducted. Overall, 55 substitutions assigned as polymorphic variants (4), missense mutations (10), or synonymous variants (35) were identified in PD, and 22 synonymous variants were identified in controls. Among them, the G231N, E232T, N235D variant was found in ~25% of P. gingivalis strains from PD samples. It was located close to the catalytic triad and had two-fold higher activity in comparison with reference P. gingivalis, upregulated expression of key inflammatory mediators, and contributed to worsening periodontium conditions in advanced PD, suggesting their unambiguous impact on P. gingivalis virulence. Our results indicate the G231N, E232T, N235D variant of the ppad gene as a potential candidate, opening a path to searching for novel targets for supportive therapy of PD. Further validation of the identified mutations is needed in future studies.

This study compared immune activation of gingival fibroblasts (GFs) by Porphyromonas gingivalis strains carrying different variants of peptidylarginine deiminase (PPAD). To this aim, GFs from donors with periodontitis (PD) (n=4) and healthy controls (CTRL) (n=2) were infected with P. gingivalis strains expressing either ppad with missense mutations (A390T + T421I) (n=2, moderate PD) or clusters of specific polymorphic variants (S203P + G231N, E232T, N235D) and (S203P + G231N, E232T, N235D + N291D + A515V + S528G) (n=2, advanced PD). Enzymatic activity of PPAD from these strains was determined using a colorimetric assay. Immune activation of infected GFs was analysed using qRT-PCR. P. gingivalis strains carrying clusters of specific polymorphic variants of ppad induced ~two-fold increase of PPAD activity and upregulated TNF-α, IL-6, and COX-2 expression by infected GFs compared to the reference ATCC 33277 strain. Contrary, P. gingivalis strains containing missense mutations showed decreased PPAD activity and elicited inflammatory response comparable to that presented by the ATCC 33277 strain. This study demonstrated that the inflammatory outcome of P. gingivalis infection results from the interplay between bacterial virulence driven by PPAD sequence diversity and enzymatic activity, and the intrinsic hyperreactivity of host GFs associated with PD.