Characterization of collagenolytic activity from strains of Bacteroides gingivalis

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.