Biochemical characterization of Porphyromonas (Bacteroides) gingivalis collagenase

Abstract

A protease was purified from Porphyromonas gingivalis 1101, a clinical isolate, by sequential sodium dodecyl sulfate-polyacrylamide gel electrophoresis, substrate diffusion gel electrophoresis, and electroelution. The enzyme cleaved radiolabeled human basement membrane type IV collagen and the synthetic collagen peptide substrate for eukaryotic collagenases. It was inactivated by the thiol protease inhibitor N-ethylmaleimide but not by EDTA or EGTA [ethylene glycol-bis(13-aminoethyl ether)-N,N,N',N'-tetraacetic acid] and activated by reducing agents such as 13-mercaptoethanol. The enzyme exists as an active precursor protein of molecular mass 94 kDa and undergoes proteolytic cleavage to 75-, 56-, and 19-kDa forms. Biotin-labeled collagen bound specifically to the 94-kDa form of the protein and to its cleavage products in ligand blots, suggesting a role for this enzyme not only in collagen degradation but also in adhesion to collagenous substrata.Several studies implicate black-pigmented Porphyromonas (Bacteroides)gingivalis as an important etiological agent associated with the pathogenesis of periodontal disease. In the course of many bacterial infections, proteases are putative virulence factors. Examples include zinc metalloproteases from the insect pathogens Serratia spp. (18) and the fish pathogen Vibrio anguillarum (13) and a protease from Legionella pneumophila (1). Such proteases are essential either for the initial invasive stages of the infection or for protection against the host immune response. In human bacterial infections, inactivation of the host immune response by proteases has been demonstrated with the elastase from Pseudomonas aeruginosa (17) and the immunoglobulin protease from Neisseria gonorrhoeae (14).The proteolytic nature of P. gingivalis strains has been well documented in the literature, where they have been shown capable of hydrolyzing gelatin (25), complement (20), and immunoglobulins A and G (7). Several proteases, including an intracellular, trypsinlike protease (23) and a glycylprolyl protease (5), have been isolated and characterized. Since collagen is the major constituent of the gingival connective tissues, perhaps the most important bacterial enzyme associated with the tissue destruction seen in periodontal disease is the collagenase. Although many workers have identified collagenase activity in various P. gingivalis strains, the biochemical nature of the enzyme has not been established. In this work, we report the purification and characterization of the P. gingivalis collagenase.MATERUILS AND METHODS Culture conditions. P. gingivalis was grown on blood agar plates in a gas mix containing 80% N2, 15% C02, and 5% H2 according to Greiner et al. (4).Collagen labeling. Collagen was radiolabeled for the microplate assay (see below) with lodogen (Pierce Chemical Co.) dissolved in chloroform and coated onto Eppendorf tubes (50 mg per tube) (8). The coated tubes were washed briefly in phosphate-buffered saline (PBS) prior to labeling experiments. Subsequently, 100 p,g of collagen in P...

Role of bacterial proteinases in matrix destruction and modulation of host responses

“…Among tested synthetic substrates, it hydrolyzes Pz-peptide but has no activity against chromogenic substrates specific for either dipeptidyl peptidase or trypsin. The enzyme was purified and characterized as a cysteine proteinase (193,332) (17) or prtP [W83] (201). In contrast, two genes, designated kgp (381) and hagD have been cloned and sequenced in strain 381 but apparently they represent, together, the typical Kgp-encoding gene with a catalytic domain interrupted by a termination codon introduced by a single base mutation (129).…”

“…The initial cleavage fragments generated by P. gingivalis collagenase form a unique pattern distinct from the pattern produced by vertebrate or clostridial collagenases (31). A 94-kDa proteinase degrading basement membrane type IV collagen has been purified and characterized but wrongly named collagenase (193) because the term ''collagenase'' is reserved for proteinases able to cleave the helical domain of native collagen type I. In this regard, the enzyme apparently represents a unique cysteine gelatinase.…”

Porphyromonas gingivalis mutant defective in a putative extracytoplasmic function sigma factor shows a mutator phenotype

“…P. gingivalis is implicated as a major pathogen in the development and progression of chronic periodontitis (14). Although this bacterium has numerous potential virulence factors, the proteolytic enzymes are most important because gingipains destroy periodontal tissue directly or indirectly (16). P. gingivalis produces large amounts of Kgp and Rgp proteinase in both cell-free and cell-associated forms.…”

The paradox of painless periodontal disease

“…Prevotella and Treponema spp.) produce proteases, such as collagenases, that were demonstrated to contribute to the proteolytic degradation of periodontal tissues (Steffen and Hentges, 1981;Robertson et al, 1982;Slots and Genco, 1984;Uitto et al, 1988;Grenier et al, 1990;M€ akinen et al, 1990;Lawson and Meyer, 1992;Sojar et al, 1993;Bedi and Williams, 1994). The resulting level of damage would normally be expected to be painful in other tissues, as evidenced by osteoarthritis, both in a collagenase-induced model in rodents (Lee et al, 2009;Adaes et al, 2015) and in human patients (Szebenyi et al, 2006;Torres et al, 2006).…”