INDUCTION OF CYTOSKELETAL REARRANGEMENTS AND LOSS OF VOLUME REGULATION IN EPITHELIAL-CELLS BY TREPONEMA-DENTICOLA

The early responses of oral epithelial cells to the adhesion of the oral spirochaete Treponema denticola were studied as a model of microbial perturbation of the plasma membrane. KB cell (ATCC CCL 17) monolayers were incubated with T. denticola (ATCC 35405) in alpha-MEM (minimal essential medium) for periods of 1-4h at 37 degrees C without serum. Control cultures were exposed to bacteria-conditioned alpha-MEM without serum or bacteria or to alpha-MEM alone. At the end of each incubation, detached and attached epithelial cells were harvested and analysed separately. Compared with controls, T. denticola induced in 25% of cells a two-fold, time-dependent increase of detachment by 4h. Detached cells in both T. denticola-exposed and control cultures exhibited 25% reductions in modal diameter, did not exclude propidium iodide, did not readhere, and did not form colonies. In T. denticola-exposed cultures, a larger subset (75%) of cells remained attached to the substratum, demonstrated no significant reduction of colony-forming efficiency and excluded propidium iodide. However, these cells exhibited a 21% reduction in diameter (p < 0.05), a 60% decrease of F-actin (p < 0.001), and a 74% reduction in the proportion expressing desmoplakin II (p < 0.01) after exposure to T. denticola. Flow cytometry showed a small (14%) but significant (p < 0.001) reduction in mean fluorescence intensity due to keratin expression in T. denticola-treated cultures. Exposure of cells to anisosmotic media demonstrated that, in contrast to controls, cultures challenged by bacteria failed to undergo compensatory volume regulation. These data indicate that contact of T. denticola with epithelial cells induces: (1) cell detachment and death in a small subset; (2) loss of cellular volume regulation that is associated with disruption of cytoskeletal proteins in attached cells.