title: 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.
authors.affiliation: From the Department of Laboratory Medicine, Medical Protein Chemistry, Lund University, SE-205 02 Malmö, Sweden, the Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krakow, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Bielecka
authors.affiliation: the Department of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center, Aarhus University, DK-8000 Aarhus, Denmark.
authors.forename: Carsten
authors.initials: C
authors.lastname: Scavenius
authors.affiliation: the Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krakow, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Kantyka
authors.affiliation: From the Department of Laboratory Medicine, Medical Protein Chemistry, Lund University, SE-205 02 Malmö, Sweden.
authors.forename: Monika
authors.initials: M
authors.lastname: Jusko
authors.affiliation: the Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krakow, Poland.
authors.forename: Danuta
authors.initials: D
authors.lastname: Mizgalska
authors.affiliation: the Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krakow, Poland.
authors.forename: Borys
authors.initials: B
authors.lastname: Szmigielski
authors.affiliation: the Department of Oral Immunology and Infectious Diseases, University of Louisville School of Dentistry, Louisville, Kentucky 40202, and.
authors.forename: Barbara
authors.initials: B
authors.lastname: Potempa
authors.affiliation: the Department of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center, Aarhus University, DK-8000 Aarhus, Denmark.
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Enghild
authors.affiliation: the Department of Cell Biology and Physiology, University of New Mexico, Albuquerque, New Mexico 87131.
authors.forename: Eric R
authors.initials: ER
authors.lastname: Prossnitz
authors.affiliation: From the Department of Laboratory Medicine, Medical Protein Chemistry, Lund University, SE-205 02 Malmö, Sweden, anna.blom@med.lu.se.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Blom
authors.affiliation: the Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krakow, Poland.
authors.forename: Jan
authors.initials: J
authors.lastname: Potempa
mesh_terms: D001120:Arginine / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002465:Cell Movement; D002478:Cells, Cultured; D002633:Chemotaxis; D002956:Citrulline / Q000378:metabolism; D015936:Complement C5a / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000235:genetics; D000076342:Protein-Arginine Deiminases; D044087:Receptor, Anaphylatoxin C5a / Q000235:genetics / Q000378:metabolism; D022161:Transport Vesicles / Q000201:enzymology; D020298:U937 Cells
chemical_list: D001426:Bacterial Proteins; D044087:Receptor, Anaphylatoxin C5a; D002956:Citrulline; D015936:Complement C5a; D001120:Arginine; D006867:Hydrolases; D000076342:Protein-Arginine Deiminases; D002118:Calcium
keywords: Bacteria; Chemotaxis; Complement System; Inflammation; Periodontal Disease