Background Periodontitis (PD) is the most common chronic inflammatory disease caused by bacterial infection resulting in alveolar bone resorption and tooth loss.1,2 A main causative agent of PD is Porphyromonas gingivalis (Pg). 3,4 This periodontopathogen produces peptidylarginine deiminase (PPAD) catalysing conversion of arginine to citrulline.5,6 PD shares common mechanism and risk factors with rheumatoid arthritis (RA). Due to the presence of pathogenic autoantibodies reactive with citrullinated proteins in RA, citrullination of host and bacterial proteins by PPAD was proposed as a mechanistic link between PD and RA.7–9 The aim of this study was to investigate the impact of a novel polymorphism identified in the PPAD gene on bacterial virulence and PD clinical status.
Materials and methods Gingival crevicular fluid (GCF) from 20 patients with PD was plated on blood agar and Pg colonies re-cultured to isolate individual strains of the bacterium. From each strain the PPAD gene was cloned, sequenced and analysed. The native PPAD gene in the reference Pg ATCC33277 strain was substituted with the polymorphic gene. The phenotype of clinical isolates harbouring polymorphism, the mutated and native ATCC33277 strains was examined. Further, periodontal clinical parameters were compared amongst patients infected by Pg expressing PPAD with and w/o polymorphism.
Results A three amino acid polymorphism (G231N, E232T, N235D) was identified in the vicinity of the PPAD catalytic His residue in Pg isolates obtained from 30% of PD patients. The PPAD activity of clinical strains with polymorphism and the ATCC33277 mutant was 2-fold higher in comparison to the reference strain. Gingival fibroblasts infection with strains carrying polymorphic PPAD caused significantly higher upregulation of cyclooxygenase 2 (COX-2) than infection with native ATCC33277. Probing pocket depth (PPD) and clinical attachment level (CAL) assessment revealed that patients infected with Pg expressing polymorphic PPAD suffered from more severe disease than those carrying ‘classical’ Pg w/o polymorphism.
Conclusions A three amino acid polymorphism of PPAD augments the virulence of Pg and severity of periodontitis apparently due to higher PPAD activity. The increased citrullination of bacterial and/or host proteins by Pg with the characterised PPAD genotype can trigger autoimmune response in RA.
Acknowledgments National Science Centre, Poland (2012/07/B/NZ6/03524, K.G.), Foundation for Polish Science (TEAM, DPS/424-329/10, J.P.).
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