Character ization of protease from animal periodontal pathogen Porphyromonas gulae

Porphyromonas gulae is an animal-derived oral microorganisms known also to be associated with periodontal disease in humans. We previously reported that P. gulae proteases are possible one of virulence factors related to adhesion and invasion of gingival epithelial cells, and host cell destruction. Here we attempted to characterize bacterial proteases associated with P. gulae. Both alkaline phosphatase and trypsin activity were identified in all examined P. gulae strains and P. gingivalis ATCC33277. Each of the P. gulae strains also showed proteolytic activity in cell extract and/or culture supernatant samples. In addition, there was a significant increase in protease activity level seen in living bacterial cells, dependent on cell number, while there were no significant differences regarding proteolytic activity among the P. gulae strains. The present results indicate that antipain and PMSF are effective inhibitors of P. gulae proteases as well as P. gingivalis. In addition, TLCK and leupeptin significantly inhibited proteolytic activity in a dose-dependent manner. On the other hand, AEBSF, ALLN, aprotinin, bestatin, chymostatin, E64, EDTA, pepstatin, and phosphoramidon showed no inhibitory effects, while those of KYT-1 and KYT-36, P. gingivalis gingipain-specific inhibitors, were negligible. These results suggest that both P. gulae and P. gingivalis produce and secrete trypsin-like serine proteases, while the structure of those proteases differ between the two bacteria.