Characterization of collagenolytic activity from strains of Bacteroides gingivalis

Abstract

Strains of B. gingivalis were shown to produce collagenolytic activity capable of dissolving reconstituted collagen (type I) fibrils and of cleaving the helical domain of types I. II and III collagens at 22° C. The catalytic activity was dependent on free thiol groups and on metal ions, as indicated by inhibition by thiol blocking reagents and metal chelators. The activity was associated with the bacterial cells and was not secreted to the medium. Under optimal conditions. 100 Units of collagenase per gram cell pellet (wet weight) were released by detergents such as Triton X‐100 and SDS. Zymography of detergent extracts revealed that collagen‐degrading strains, but not an inactive control strain (W), contained a discrete Mr 90 000 gelatin cleaving protease which may be identical to the collagenolytic enzyme. The initial attack on the helical domain of type I collagen occurred near the COOH‐terminus. The a1 and a2 chains were cleaved at the same site, generating a major helical fragment consisting of three shortened (Mr 82 000) a‐chains. Subsequent cleavages of this shortened collagen molecule resulted in generation of multiple fragments from the component a‐chains in the Mr 60 000 to 6000 range. This cleavage pattern was clearly distinct from the characteristic 3/4–1/4 pattern produced by vertebrate collagenases. Type II and III collagens were also cleaved first near the COOH‐terminus, generating fragments of similar size to those produced from type I collagen. In view of its ability to dissolve reconstituted collagen fibrils at 35°C and its ability to attack the helical domain of interstitial collagens in solution at 22°C, we suggest that this enzyme tentatively be classified as a true collagenase.

Specific cell components of Bacteroides gingivalis mediate binding and degradation of human fibrinogen

“…Cell-associated and soluble trypsinlike proteases (6, 9, 23, 26-28, 30, 35) have been identified in a number of B. gingivalis strains. Glycylprolyl dipeptidases have been identified in or purified from B. gingivalis (1,10,13,30), and collagenolytic proteases have also been described (3,27,28,31,33). Trypsinlike proteases ranging in size from Mr 30,000 to 300,000 (6,9,(26)(27)(28) have been reported in B. gingivalis.…”

Collagenase and Collagenase Inhibitors Secreted by Cultured Human Periodontal Ligament Cells.

“…Much of the connective tissue degradation in periodontal diseases is mediated by proteolytic enzymes. Previous studies on such enzymatic tissue degradation have focused on the action of collagenase released by invading polymorphonuclear neutrophils, macrophages and bacteria [4][5][6][7][8][9]. On the other hand, MEIKLE et al [10] have proposed a new hypothesis that metalloproteinases released by interstitial cells of the periodontium not only control the turnover of the matrix, but also are involved in periodontal disease.…”

Salivary Histatin 5 Is an Inhibitor of Both Host and Bacterial Enzymes Implicated in Periodontal Disease

“…This is principally because this microorganism shows many virulence features, such as the release of toxic products of metabolism and outer membrane vesicles containing numerous enzymes involved in invasion and tissue destruction, the elaboration of fimbriae and lipopolysaccharide, the utilization of lectin-type adhesions, and the promotion of hemagglutination and hemolysis (42). Several physiologically important proteins, including collagen (3,18), fibrin and fibrinogen (21), fibronectin (44), plasma protease inhibitors (5), immunoglobulins (41), and complement factors (46), are degraded by proteases from P. gingivalis. Some of these proteolytic activities were previously attributed to trypsin-like proteases, but their isolation and characterization revealed that two distinct cysteine proteinase types occur with strict specificities for cleavage at either arginine (Arggingipains, RgpA and RgpB) or lysine (Lys-gingipain, Kgp) residues (33).…”