Pathogenicity of P. gingivalis strains carrying clusters of specific polymorphic variants of peptidylarginine deiminase gene.

INTRODUCTION

Periodontitis (PD) is one of the most prevalent human inflammatory diseases of bacterial etiology, affecting over 50% of adult population worldwide. It is initiated by bacterial dysbiosis and bacteria-elicited inflammation. Among key periopathogens, the anaerobic bacterium, P. gingivalis has attracted a special interest. It secretes peptidylarginine deiminase (PPAD), an enzymatic protein, which due to the conversion of arginine to citrulline residue may alter protein structure and antigenicity. Despite increasing evidence supporting involvement of PPAD in pathogenesis of PD, functional and clinical significance of ppad gene variability remains poorly understood. The goal of this work was analysis of genetic variability of ppad gene from recently collected PD cohort in the context of P. gingivalis virulence and host-pathogen interactions.

METHODS

Clinical isolates from gingival crevicular fluid (GCF) of 35 PD patients and 20 healthy donors were analyzed for the presence, sequence variability, expression, and enzymatic activity of the ppad gene in P. gingivalis using PCR, BLAST, qRT-PCR, and colorimetric assays. Virulence was assessed in murine osteoblasts (MC3T3-E1) and primary human gingival fibroblasts (PHGFs) infected with P. gingivalis clinical strains, followed by analysis of proinflammatory cytokines, PGE2-dependent pathway enzymes, and the bone resorption marker OPG.

RESULTS

The ppad gene of P. gingivalis strains revealed pronounced variability in PD patients, affecting gene expression and enzymatic activity. Three predominant clusters of specific polymorphic variants were identified: i) S191F + N291D, ii) S191F + N291D + S528G, iii) S203P + G231N, E232T, N235D + N291D + A515V + S528G. Infection with strains harboring the S203P + G231N, E232T, N235D + N291D + A515V + S528G resulted in elevated PPAD activity, which correlated with clinical indicators of disease severity, induced COX-2 (~6-fold) and IL-1b (~7-fold) expression in osteoblasts and led to 94% reduction in OPG. Similarly, these strains triggered upregulation of TNF-α, IL-6, COX-1, and COX-2 in PHGFs compared to the P. gingivalis ATCC 33277.

DISCUSSION: Collectively, these findings underscore that clusters of specific polymorphic variants of ppad are critical determinants of enzymatic function, host immune modulation and periodontal tissue destruction in PD.