Contribution of proteases and plasmin‐acquired activity in migration of Peptostreptococcus micros through a reconstituted basement membrane

Abstract

Peptostreptococcus micros is a gram-positive bacterium that has been associated with chronic periodontitis and endodontic infections. The aims of this study were to investigate the production of proteases and the acquisition of plasmin activity by rough and smooth morphotypes of P. micros. The contribution of these properties in the migration of bacteria through a reconstituted basement membrane was also evaluated. Protease activities were determined using chromogenic and fluorogenic substrates as well as by zymography. Plasminogen binding activity was studied using an enzyme-linked immunosorbent assay. The role of proteases and plasmin-acquired activity in tissue penetration was investigated using Matrigel. The rough morphotype strains of P. micros, but not the smooth morphotype strains, were found to possess chymotrypsin-like and gelatinase activities, both of which were inhibited by a serine protease inhibitor. By zymography, three gelatinase bands (165, 129, and 115 kDa) were identified. Both morphotypes of P. micros can bind human plasminogen on their cell surface. Once bound to P. micros, plasminogen activators of bacterial (streptokinase) and human (urokinase) origins were found to activate plasminogen into plasmin. Our results also showed that plasmin activity can be acquired by P. micros following co-incubation with human brain microvascular endothelial cells in culture. When non-coated cells were used, the rough morphotype strain (HG1262), which possesses chymotrypsin-like and gelatinase activities, showed a better capacity to penetrate a reconstituted basement membrane (Matrigel) than the smooth morphotype strain (HG1251). Penetration of the Matrigel by P. micros HG1262 was inhibited by the presence of a serine protease inhibitor. In addition, cells of P. micros with plasmin activity showed a significantly greater tissue penetration capacity. Our study suggests that endogenous proteolytic activities of P. micros as well as plasmin-acquired activity, may facilitate dissemination of bacterial cells to surrounding periodontal tissues and blood vessels.