Microtubules Play a Role in the Actinobacillus actinomycetemcomitans Invasion Process.

J.E. LIPPMANN*, D.H. MEYER & P.M. FIVES-TAYLOR. (U. of VT, Burlington, VT).

Many pathogenic organisms are capable of usurping the host cell cytoskeleton in order to carry out their pathogenic life cycle. Previous studies in our lab suggest that Actinobacillus actinomycetemcomitans (Aa) invasion is a dynamic process involving entry, multiplication, transcytosis and spread to adjacent cells via cell-to-cell protrusions. We have recently shown that Aa promotes host cell actin rearrangement at the site of attachment to and entry of KB oral epithelial cells (Adv Dent Res 9: 55-62, 1995). In this study we examined the role of microtubules in the Aa invasion process with both fluorescence microscopy and a viable quantitative assay. Fluorescence microscopy revealed tubulin rearrangement around internalized Aa which suggested a role for microtubules in the invasion process. To confirm these observations, experiments were repeated in the presence of the microtubule inhibitors, taxol and nocodazole. Interestingly, we found increased numbers of Aa which were present in pairs and dividing clusters within the KB cells which suggested that microtubules are involved in exit rather than entry of Aa. Quantitative assays, in which KB cells were either pretreated or treated post infection with the inhibitors, corroborated a role for tubulin in the Aa invasion process. Pretreatment of KB cells with taxol resulted in a 5-fold increase in internalized Aa. Treatment with either taxol or nocodazole after infection also resulted in an increase (up to 8-fold) in intracellular Aa. These results confirmed that the inhibitors had no affect on entry, but instead, they were preventing the exit of Aa from the KB cells. In conclusion, this work provides evidence of a role for tubulin in the Aa invasion process and suggests that Aa transcytosis and spread to adjacent KB cells may be mediated, in part, by tubulin. This work was supported by NIH-NIDR grant RO1DE09760.