CYSTEINE PROTEASE OF PORPHYROMONAS-GINGIVALIS-381 ENHANCES BINDING OFFIMBRIAE TO CULTURED HUMAN FIBROBLASTS AND MATRIX PROTEINS

It has been shown that Porphyromonas gingivalis 381, a suspected perio dontopathogen, possesses fimbriae on its cell surface. The organism is also known to produce proteases which can degrade the host cell surfa ce matrix proteins. In this study, we investigated the effect of prote ase on the binding of the purified P. gingivalis fimbriae to cultured fibroblasts or matrix proteins. A protease that can hydrolyze benzoyl- L-arginine p-nitroanilide was obtained from P. gingivalis 381 cells by sonication in phosphate-buffered 0.2% Triton X-100 and was purified b y column chromatography. The molecular size of the protease was estima ted to be 55 kDa by gel filtration or 47 kDa by sodium dodecyl sulfate -polyacrylamide gel electrophoresis analysis. The enzyme activity was markedly inhibited by sulfhydryl reagents, antipain, and leupeptin. Th e protease degraded various host proteins, including collagen and fibr onectin, and cleaved the COOH terminus of the arginine residue in pept ides such as benzoyl-L-arginine p-nitroanilide. However, P. gingivalis fimbriae were not degraded by this protease activity. The enzyme acti vity was enhanced in the presence of reducing agents or CaCl2. When cu ltured fibroblasts were partially treated with the protease, the bindi ng of the purified P. gingivalis fimbriae to the fibroblast monolayer was increased significantly. However, this enhancing effect was suppre ssed upon the addition of antipain and leupeptin. Similarly, binding o f the fimbriae to the collagen or fibronectin immobilized on the micro titer wells was also enhanced. Addition of these host matrix proteins efficiently inhibited the binding of fimbriae to the fibroblast monola yer. The binding assay of fimbriae using dipeptidyl ligand affinity co lumn chromatography demonstrated a clear interaction between fimbriae and the arginine residue. Taken together, these results indicate that the P. gingivalis protease at least partially degrades the host matrix proteins, which, in turn, may lead to an increased exposure of the cr yptic ligands that can result in enhanced fimbria-mediated binding of this organism to periodontal tissues.