Abstract
Evasion of killing by the complement system, a crucial part of innate immunity, is a key evolutionary strategy of many human pathogens. A major etiological agent of chronic periodontitis, the Gram-negative bacterium Porphyromonas gingivalis, produces a vast arsenal of virulence factors that compromise human defense mechanisms. One of these is peptidylarginine deiminase (PPAD), an enzyme unique to P. gingivalis among bacteria, which converts Arg residues in polypeptide chains into citrulline. Here, we report that PPAD citrullination of a critical C-terminal arginine of the anaphylatoxin C5a disabled the protein function. Treatment of C5a with PPAD in vitro resulted in decreased chemotaxis of human neutrophils and diminished calcium signaling in monocytic cell line U937 transfected with the C5a receptor (C5aR) and loaded with a fluorescent intracellular calcium probe: Fura-2 AM. Moreover, a low degree of citrullination of internal arginine residues by PPAD was also detected using mass spectrometry. Further, after treatment of C5 with outer membrane vesicles naturally shed by P. gingivalis, we observed generation of C5a totally citrullinated at the C-terminal Arg-74 residue (Arg74Cit). In stark contrast, only native C5a was detected after treatment with PPAD-null outer membrane vesicles. Our study suggests reduced antibacterial and proinflammatory capacity of citrullinated C5a, achieved via lower level of chemotactic potential of the modified molecule, and weaker cell activation. In the context of previous studies, which showed crosstalk between C5aR and Toll-like receptors, as well as enhanced arthritis development in mice infected with PPAD-expressing P. gingivalis, our findings support a crucial role of PPAD in the virulence of P. gingivalis.
Highlights
Pathogenic bacteria avoid killing by phagocytes through inhibition of C5a chemotactic activity
C5a in the native form eluted in two peaks from the C-18 column, whereas PPADtreated C5a eluted in four peaks, at retention times different from those of the native peptide
Among tryptic peptides derived from peptidylarginine deiminase (PPAD)-incubated C5a, one abundant peptide showed a clear shift in retention time as compared with the control C5a-derived peptides
Summary
Pathogenic bacteria avoid killing by phagocytes through inhibition of C5a chemotactic activity. Porphyromonas gingivalis is a major causative agent of periodontitis, a chronic inflammatory disease of tooth-supporting structures that affects up to 30% of the world’s population [1] This Gram-negative, anaerobic bacterium uses a large arsenal of virulence factors such as hemagglutinins/adhesins, fimbriae, and proteolytic enzymes to facilitate colonization of the gingival sulcus, to generate nutrients and growth factors, and to provide protection from the host immune system [2]. The latter is achieved by sophisticated manipulation of the host inflammatory response through activation of coagulation factors and contact activation systems, corrupting complement functions, shedding receptors, and modifying cytokines and intracellular signaling [3]. This modification decreased the ability of C5a to induce calcium influx in a monocytic cell line expressing C5aR and strongly reduced its chemotactic potential for neutrophils
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