Characterization of the proteinase-dependent cytotoxicity of Porphyromonas gingivalis

Cytotoxicity of proteins from Porphyromonns gingivalis towards gingival fibroblasts and epithelial cells was studied by methods measuring vital dye uptake, DNA. synthesis, release of cytoplasmic components, and apoptosis. Microscopic examination of the cells was also performed for detection of morphological changes. Experiments were made with dialyzed culture supernarants of P. gingivalis as well as supernatant fractions obtained by isoelectric focusing. The main cell damage observed was rounding of the cells and detachment from each other and the underlying surface. The cell-damaging activity correlated with the cystein-dependent proteolytic activity of the various supernatant preparations as well as with the occurrence of gelatinolytic protein bands with molecular weights previously reported for the cysteine proteinases gingipains R and K of P. gingivalis. The activity was abolished by heat treatment or by the addition of cysteine proteinase inhibitors. The results indicate that the main cytotoxic effect towards fibroblasts and epithelial cells is degradation of the intercellular matrix. The gingipains released in P, gingivalis culture supernatants are the responsible factors for this degradation.