Characterization of Porphyromonas gingivalis-induced degradation of epithelial cell junctional complexes

Porphyromonas gingivalis is considered among the etiological agents of huma n adult periodontitis, Although in vitro studies have shown that P. gingiva lis has the ability to invade epithelial cell lines, its effect on the epit helial barrier junctions is not known. Immunofluorescence analysis of human gingival epithelial cells confirmed the presence of tight-junction (occlud in), adherens junction (E-cadherin), and cell-extracellular matrix junction (pl-integrin) transmembrane proteins. These transmembrane proteins are exp ressed in Madin-Darby canine kidney (MDCK) cells, In addition, MDCK cells p olarize and therefore serve as a useful in vitro model for studies on the e pithelial cell barrier. Using the MDCK cell system, we examined the effect of P. gingivalis on epithelial barrier function, Exposure of the basolatera l surfaces of MDCK cells to P. gingivalis (>10(9) bacteria/ml) resulted in a decrease in transepithelial resistance. Immunofluorescence microscopy dem onstrated decreases in the amounts of immunoreactive occludin, E-cadherin, and pl-integrin at specific times which were related to a disruption of cel l-cell junctions in MDCK cells exposed to basolateral P. gingivalis. Disrup tion of cell-cell junctions was also observed upon apical exposure to bacte ria; however, the effects took longer than those seen upon basolateral expo sure. Cell viability was not affected by either basolateral or apical expos ure to P. gingivalis, Western blot analysis demonstrated hydrolysis of occl udin, E-cadherin, and pl-integrin in lysates derived from MDCK cells expose d to P. gingivalis. Immunoprecipitated occludin and E-cadherin molecules fr om MDCK cell lysates were also degraded by P. gingivalis, suggesting a bact erial protease(s) capable of cleaving these epithelial junction transmembra ne proteins, Collectively, these data suggest that P. gingivalis is able to invade the deeper structures of connective tissues via a paracellular path way by degrading epithelial cell-cell junction complexes, thus allowing the spread of the bacterium, These results also indicate the importance of a c ritical threshold concentration of P. gingivalis to initiate epithelial bar rier destruction.