Protein citrullination by Porphyromonas gingivalis and its implications for autoimmunity in rheumatoid arthritis
Author(s)
Wegner, Natalia
Type
Thesis
Abstract
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.
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.
Date Issued
2011-03
Date Awarded
2011-07
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Venables, Patrick
Creator
Wegner, Natalia
Publisher Department
Medicine: Kennedy Institute of Rheumatology
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)