Protease‐Active Extracellular Protein Preparations From Porphyromonas gingivalis W83 Induce N‐Cadherin Proteolysis, Loss of Cell Adhesion, and Apoptosis in Human Epithelial Cells

BACKGROUND: The protease-induced cytotoxicity of P. gingivalis may partly result from alteration of the extracellular matrix and/or surface receptors that mediate interaction between the host cells and their matrix. While P. gingivalis-induced degradation of E-cadherin has been documented, there is no information on the effects of P. gingivalis proteases on other members of this family of cell adhesion proteins. METHODS: Human epithelial KB cells were exposed to protease-active extracellular protein preparations from isogenic mutants of P. gingivalis. Quantification of apoptosis was performed by visualization of nuclei stained with 4,6'-diamidino-2-phenylindole. Alteration of cell adhesion proteins was examined by immunoblotting of cell lysates using monoclonal antibodies to those proteins. RESULTS: Treated cells exhibited loss of cell adhesion properties with apoptotic cell death subsequently observed. These effects correlated with the different levels of cysteine-dependent proteolytic activities of the isogenic mutants tested. Cleavage of N-cadherin was observed in immunoblots of lysates from detached cells. There was a direct correlation between the kinetics of N-cadherin cleavage and loss of cell adhesion properties. Loss of cell adhesion, as well as N-cadherin cleavage, could be inhibited by preincubation of P. gingivalis protease active extracellular protein preparations with the cysteine protease inhibitor TLCK. In control experiments, the cleavage of N-cadherin was detected after treatment of KB cells with trypsin but not after cell dissociation by a non-enzymatic method. CONCLUSIONS: These results suggest that extracellular proteases from P. gingivalis can induce degradation of N-cadherin, which could have implications for the pathogenicity of this bacterium.