An investigation of Porphyromonas gingivalis peptidylarginine deiminase: a putative virulence factor in an animal model of inflammation.

Abstract

Porphyromonas gingivalis, an oral periodontopathogen linked to chronic periodontitis expresses peptidylarginine deiminase (PAD), an enzyme that converts peptide-bound arginine to citrulline. A relationship between human PADs and chronic inflammatory diseases has been proposed. Citrullinated a-enolase is a candidate auto-antigen in rheumatoid arthritis. Vimentin and fibrin are also likely target proteins in disease development. This study partially characterised the enzyme and the ability of P. gingivalis cells to citrullinate peptides and these rheumatoid arthritis relevant proteins. In addition, the influence of gingipains, key P. gingivalis virulence factors, on PgPAD activity was investigated. A limited histological survey was performed on selected tissues to investigate the effect of P. gingivalis in an animal model of adjuvant arthritis. A colourimetric assay to quantify citrulline was developed and used to determine the effect of environmental pH and temperature on enzyme activity. Enzyme localization was investigated by comparing reaction rates of whole cells to cell sonicates. Enzyme specificity was determined by incubation of cells with a range of arginine analogues and arginine-containing peptides. The rates of citrullination of enolase, vimentin and fibrin by P. gingivalis cells were calculated. The influence of the gingipains on citrullination was measured by comparing the rate of citrullination of albumin in the presence and absence of the proteolytic inhibitors tosyl phenylalanyl chloromethyl ketone and leupeptin. Tissue sections from three regions of the animal heads were stained for polymorphonuclear cells and osteoclasts. In addition sponge samples were surveyed for polymorphonuclear cells and citrullinated proteins detected using immunohistochemical technique. PgPAD activity was heat stable, predominantly cell-surface expressed and exhibited optimal activity between pH 7.5 and 8. The enzyme was highly specific for arginine and citrullinated arginine residues in all positions in the peptides tested. PgPAD was able to citrullinate all rheumatoid arthritis relevant proteins, at rates slower than peptides. Inhibition of the gingipains failed to influence the rate of citrullination of albumin. In the adjuvant arthritis animal study, pre-treatment with P. gingivalis produced increased inflammatory cellular infiltrate at the site of exposure but no similar affect in the head tissue. There was a significant increase numbers of polymorphonuclear cells in the bone marrow from the head region and in the implanted sponge infiltrate from rats with prior exposure to P. gingivalis. Although citrullinated proteins were detected in sponge sections from both adjuvant arthritis-induced rat groups, no difference between them was observed. A similar result was seen with osteoclasts, as both groups exhibited increased numbers over the control group. This study has shown that P. gingivalis peptidylarginine deiminase has potential to influence the inflammatory process by citrullinating arginine containing peptides and rheumatoid arthritis relevant proteins. An examination of rats exposed to the bacterium in an animal model of rheumatoid arthritis did not appear to exacerbate inflammation in selected tissues.