In the present study we provide evidence that the periodontal pathogen P. gingivalis
is an alternative source in the human host for generating citrullinated proteins and peptides. The underlying mechanism - proteolytic cleavage and subsequent citrullination at carboxy-terminal arginine residues - differs from that of the human PAD enzymes, which citrullinate internal arginine residues in whole proteins most efficiently. This finding suggests that protein citrullination by PPAD has the potential to generate epitopes to which immunological tolerance does not exist, not only due to the presence of foreign citrullinated proteins from the bacterium, but also through a foreign mode of proteolytic processing and post-translational modification of host antigens. It also indicates that citrullination of bacterial proteins at internal arginines, as a potential mechanism for triggering autoantibodies via molecular mimicry (5
), is more likely to be due to the action of human PAD enzymes present at the site of inflammation.
Endogenous citrullinated proteins were detected exclusively in P. gingivalis
, out of eleven oral bacterial species tested, indicating that a bacterial PAD gene is expressed or active only in this bacterium amongst those tested. To substantiate this finding, we performed similarity searches using BLAST and Psi Blast (41
). This revealed numerous orthologues distantly related to PPAD among prokaryotes, including several of the oral organisms we tested. Most share the predicted conserved catalytic residues of PPAD and other members of the guanidino-group modifying enzymes superfamily, although they most likely possess agmatine iminohydrolase or arginine deiminase rather than peptidylarginine deiminase activity (42
Using fibrinogen as a model antigen, we show that P. gingivalis
rapidly generates small fibrinogen peptides with carboxy-terminal citrulline residues. Fibrinopeptide A, which normally results from thrombin cleavage of the fibrinogen α-chain after arginine-16, was also detected in its citrullinated form (1
) in samples incubated with P. gingivalis
wild-type, but only in the native, arginine-containing form in the Δppad
samples. It is known that P. gingivalis
gingipain-mediated degradation of human fibrinogen inhibits fibrinogen polymerization and results in the localised bleeding tendency which is typical for chronic periodontitis (35
). A recent study has shown that, in intact fibrinogen, internal citrullination at arginine-16 by mammalian PAD impairs thrombin-catalyzed cleavage and fibrin polymerization (43
), indicating at least two possible pathogenic roles for citrullinated fibrinogen in RA, serving as an autoantigen and disturbing the coagulation cascade and linked pathways. The pathophysiological role of fibrinogen peptides with carboxy-terminal citrulline residues, generated by the concerted action of gingipain and PPAD, is yet unknown, opening up a novel area for future investigations. Similarly, it is known that P. gingivalis
arginine-gingipains cleave a number of other human proteins, releasing biologically active peptides with important roles in immunity and inflammation, such as C5a (44
) and bradykinin (45
), and simultaneous citrullination of these peptides by P. gingivalis
PAD might have a previously unappreciated role in human disease.
The lower levels of detectable citrullination of α-enolase peptides with the P. gingivalis wild-type are likely to be the result of a combination of physiological and technical factors. Enolase has a lower percentage of arginine residues (3.9%) compared to fibrinogen (5.2%), and a higher percentage of lysine residues (enolase 8.8% versus fibrinogen 6.9%). Further, it appears to be more extensively cleaved by non-arg/lys peptidases. Combined, this would result in fewer suitable PPAD substrates, and overall very small peptides, which would not be detected using the methods employed. Thus, lower levels of detectable citrullination may simply be due to a relative paucity of the substrate, and technical shortcomings with the detection of short peptides.
Here, we have demonstrated that P. gingivalis
efficiently citrullinates its own proteins and peptides from host fibrinogen, and, to a lesser extent, α-enolase. The two major findings of this study, that proteolytic processing is required for citrullination by P. gingivalis
and that host peptides with exclusively carboxy-terminal citrulline residues are generated, provides a strong basis for future in vivo
studies, aimed at identifying citrullinated peptides at the site of gingival inflammation and exploring their potency for triggering a T- and/or B -cell response. Citrullinated host peptides, generated by P. gingivalis
, are likely to expose epitopes previously hidden from immune surveillance, which, in the context of bacterial infection in a genetically susceptible host, may trigger an immune response. The slightly increased prevalence of anti-citrullinated protein antibodies reported in patients with periodontitis compared to healthy controls (47
) supports this concept, but the lower frequency and titre than that found in RA suggest that P. gingivalis
infection is not sufficient on its own for the mature autoimmune response. However, once tolerance is breached, we predict that exposure to host proteins in the inflamed joint, citrullinated by human PADs (31
), leads to intra- and inter-molecular epitope spreading to additional peptides from the initiating porteins and other autoantigens. Thus we propose a “two-hit” model of RA, based on (i) tolerance breakdown to specific citrullinated peptides generated by P. gingivalis
at the site of gingival inflammation, followed by (ii) epitope-spreading to other host citrullinated proteins in the inflamed joint. This self-sustaining immune response would then result in the chronic and destructive inflammation that typifies RA. The unique nature of the bacterial deiminase, along with its location on the cell-surface of the bacterium (26
), provides a target for treatment designed to prevent this otherwise incurable disease.