Peptidyl arginine deiminase from Porphyromonas gingivalis abolishes anaphylatoxin C5a activity

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.

Keywords: Bacteria; Chemotaxis; Complement System; Inflammation; Periodontal Disease.

FIGURE 1.

Citrullination of C5a by P. gingivalis PPAD. A, Laemmli SDS-PAGE followed by silver staining of PPAD purified from genetically modified P. gingivalis strain W83 culture medium. LMW, low molecular weight marker. B, C5a (10 μg), incubated in the reaction buffer alone or with 3.22 μm PPAD for 3 h at 37 °C, was analyzed with HPLC on a C18 column. C, samples (the same as for B digested with trypsin/clostripain) were analyzed with nLC-MS/MS. Results indicated significant citrullination of the C-terminal Arg in C5a treated with PPAD. D, C5 (10 μg), incubated with OMVs from P. gingivalis W83 wild-type strain and Δppad mutant strain at 25:1 molar ratio to Rgp for 30 min at 37 °C, was analyzed with nLC-MS/MS after trypsin digestion. Results demonstrate significant citrullination of the Arg-74 residue in C5a formed from C5 by Rgp and modified by PPAD present in OMVs from W83 strain. C5a was not citrullinated in the sample treated with OMVs from the Δppad mutant strain. mAU, milliabsorbance units.

FIGURE 2.

PPAD citrullinates C5a, decreasing its chemotactic ability against neutrophils and calcium influx into U937 C5aR cells. A, C5a (12.5 nm) was incubated with serial dilutions of PPAD for 1 h at 37 °C. Neutrophil migration was measured after 1 h of incubation as fluorescence intensity of carboxyfluorescein succinimidyl ester-labeled cells in the lower chamber of the transmigration assay plate. Buffer with KYT-1 and l-cysteine, PPAD alone, and C5a-desArg (125 nm) incubated with 0.1 μm PPAD or without were used as negative controls, and human C5a (12.5 nm) was used as the positive control. Results are the average of three independent experiments with S.D. Statistical significance was calculated using a one-way analysis of variance with Dunnett's post test. **, p < 0.01; ***, p < 0.001 B, C5a (10 nm) was incubated with increasing PPAD concentrations for 1 h at 37 °C. Changes in [Ca²⁺] were monitored in U937 C5aR cells loaded with Fura-2 AM in the presence of 1.3 mm calcium. FI ratio, fluorescence intensity ratio. C, C5a-desArg (100 nm) was incubated with PPAD or alone in buffer supplemented with KYT-1 (used as negative control). Depicted are the results of one representative experiment.