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Title: Protein citrullination by Porphyromonas gingivalis and its implications for autoimmunity in rheumatoid arthritis
Author: Wegner, Natalia
ISNI:       0000 0004 2705 0534
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2011
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Autoantibodies to citrullinated proteins are characteristic for rheumatoid arthritis (RA). RA has been associated with periodontitis in epidemiologic studies. Chronic periodontal infection with Porphyromonas gingivalis (P. gingivalis) is a possible risk factor for developing RA, as this bacterium expresses a peptidylarginine deiminase enzyme (PPAD) which could generate antigenic citrullinated proteins within an infectious context. The aim of this project was to investigate protein citrullination by P. gingivalis and its potential for breaking tolerance to citrullinated proteins in RA. Endogenous citrullinated proteins were abundant in cell extracts of P. gingivalis but lacking in other tested oral bacteria. Deletion of the PPAD gene resulted in abrogation of protein citrullination. Inactivation of arginine-gingipains but not lysine-gingipains, the major proteolytic virulence factors, led to decreased citrullination. Incubation of wildtype P. gingivalis with fibrinogen or α-enolase caused degradation of the proteins by bacterial proteinases and peptidases and citrullination of the resulting peptides at carboxy-terminal arginine residues. No statistically significant antibody reactivity was found in RA serum with two of these peptides tested. The PPAD enzyme was cloned and expressed in E. coli in an enzymatically active form and used for in vitro enzymatic assays to confirm substrate specificity for carboxy-terminal arginine residues and demonstrate inhibition by 2- chloroacetamidine, but not by conventional RA drugs. Site-directed mutagenesis identified five amino acids crucial for catalysis. A polyclonal antibody was developed and demonstrated that PPAD is localised in the bacterial outer membrane. The results demonstrate that P. gingivalis generates citrullinated endogenous and host peptides by proteolytic cleavage at arginine-X peptide bonds by arginine-gingipains followed by citrullination of carboxy-terminal arginines by PPAD. Mutagenesis and inhibition studies point to a critical cysteine (Cys-351) in the catalytic centre. The work provides the basis for future studies of the role of PPAD in bacterial virulence and tolerance breakdown in RA.
Supervisor: Venables, Patrick Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral