Construction and Characterization of Arginine-specific Cysteine Proteinase (Arg-gingipain)-deficient Mutants of Porphyromonas gingivalis

Abstract

Arginine-specific cysteine proteinase (Arg-gingipain; formerly, argingipain) is one of the major extracellular proteinases produced by the oral anaerobic bacterium Porphyromonas gingivalis. To determine whether Arggingipain is important for periodontopathogenicity of the organism, Arg-gingipain-deficient mutants were constructed via gene disruption by use of suicide plasmid systems. First, Southern hybridization analyses suggested that two separate Arg-gingipain-encoding genes designated rgpA and rgpB existed on 12.5-and 7.8-kilobase pair HindIII chromosomal fragments of P. gingivalis ATCC33277, respectively. rgpA and rgpB single mutants were constructed by mobilization of a suicide plasmid. Then, an rgpA rgpB double mutant was isolated by electroporation with a second suicide plasmid. No proteolytic activity for Arg-gingipain was observed in either the cell extract or the culture supernatant of the rgpA rgpB mutant. The chemiluminescence response of polymorphonuclear leukocytes, which is closely related to their bactericidal function, was not inhibited by the culture supernatant of the rgpA rgpB mutant, while the wild type parent showed a significant inhibition of the response. The result suggests that Arggingipain is responsible for disruption of the function of polymorphonuclear leukocytes. In addition, the rgpA rgpB double mutations caused a marked decrease in the hemagglutination of P. gingivalis, indicating that a major part of the hemagglutinin activity of the organism is associated with the two genes. These findings demonstrate that Arg-gingipain makes a significant contribution to the virulence of P. gingivalis.

Activation of Human Prothrombin by Arginine-specific Cysteine Proteinases (Gingipains R) from Porphyromonas gingivalis *

“…From the culture medium of P. gingivalis HG66 we have purified previously two major forms of arginine-specific cysteine proteinases, HRgpA 1 and RgpB, formerly referred to as high molecular mass gingipain R (95-kDa gingipain R1 or HRGP) and 50-kDa gingipain R2 (RGP-2), respectively (24,26). Both of these enzymes are products of two distinct but related genes (27). rgpA encodes a polyprotein which, after post-translational processing/modifications, yields three different forms of the enzyme (28,29); the major one is a non-covalent complex containing separate catalytic and adhesion/hemagglutinin domains (HRgpA).…”

A protein secretion system linked to bacteroidete gliding motility and pathogenesis

“…Bacterial strains and plasmids used in this study are listed in Table S4. P. gingivalis cells were grown anaerobically (10% CO 2 , 10% H 2 , 80% N 2 ) in enriched brain heart infusion medium and on enriched tryptic soy agar (8). For blood agar plates, defibrinated laked sheep blood was added to enriched tryptic soy agar at 5%.…”

“…P. gingivalis secretes extracellular and cell-surface gingipain proteases that are major virulence factors involved in periodontal pathogenesis (6,7). Gingipains consist of Arg-specific cysteine proteinases (Rgp) encoded by rgpA and rgpB, and the Lys-specific cysteine proteinase (Kgp) encoded by kgp (8)(9)(10). Gingipains have signal peptides to allow transit of the cytoplasmic membrane via the Sec machinery, but the mechanism of secretion across the outer membrane is not known.…”

Asp- and Glu-specific Novel Dipeptidyl Peptidase 11 of Porphyromonas gingivalis Ensures Utilization of Proteinaceous Energy Sources

“…An erythromycin-resistant gene fragment from Bacteroides fragilis was amplified by PCR from pUC19-Em with 5ErmF-AM-Cla and 3ErmF-AM-Cla primers (37,38), digested with ClaI, and inserted into a ClaI site of pGEM-T Easy-PGN0607 (designated pGEM-T Easy-PGN0607-Em). Electro-transformation of P. gingivalis ATCC 33277 was carried out with SalI-linearlized pGEM-T Easy-PGN0607-Em, and PGN0607-disrupted strains were selected on ABCM agar supplemented with 1 g/ml menadione and 10 M erythromycin, according to a previously reported method (39).…”