Generation and characterisation of Porphyromonas gingivalis mutant lacking peptidylarginine deiminase activity

Abstract

Porphyromonas gingivalis peptidylarginine deiminase (PPAD) is the focus of several studies due to its ability to citrullinate in vitro human proteins, which have been linked to the aetiopathogenesis of rheumatoid arthritis (RA). The aim of this work was the generation by homologous recombination and characterisation of a P. gingivalis W50 mutant lacking pad gene (PG1424) to study the role of PPAD in RA. To confirm the absence of PPAD activity in P. gingivalis PG1424, cells were incubated with arginine-containing substrates and citrullination of L-arginine measured using a colorimetric assay and thin-layer chromatography. Furthermore, arginine and lysine protease (gingipain) activities were assessed and immunoblotting was performed using monoclonal antibody 1B5 (mAb1B5) and a commercial anti-modified citrulline antibody (AMC) to detect differences in virulence factor expression. The deletion of pad gene in P. gingivalis PG1424 completely abolished the ability to autocitrullinate P. gingivalis proteins in the mutant strain and also the citrullination of used substrates but not free L-arginine. Moreover, the wild-type and mutant strains had similar total gingipain activities and reactivity with mAb1B5. In conclusion, this work has produced a well-characterised PPAD-deleted P. gingivalis strain, which can be used to help determine the role of citrullination by this microorganism in RA.