Tannerella forsythia invasion in oral epithelial cells requires phosphoinositide 3-kinase activation and clathrin-mediated endocytosis

Tannerella forsythia, a Gram-negative anaerobe implicated in periodontitis, has been detected within human buccal epithelial cells and shown to invade oral epithelial cells in vitro. We have previously shown that this bacterium triggers host tyrosine kinase-dependent phosphorylation and actin-dependent cytoskeleton reorganization for invasion. On the bacterial side, the leucine-rich repeat cell-surface BspA protein is important for entry. The present study was undertaken to identify host signalling molecules during T. forsythia entry into human oral and cervical epithelial cells. Specifically, the roles of phosphatidylinositol 3-kinase (PI3K), Rho-family GTPases, cholesterol-rich membrane microdomains and the endocytic protein clathrin were investigated. For this purpose, cell lines were pretreated with chemical inhibitors or small interfering RNAs (siRNAs) that target PI3Ks, Rho GTPases, clathrin and cholesterol (a critical component of 'lipid rafts), and the resulting effects on T. forsythia uptake were determined. Our studies revealed that T. forsythia entry is dependent on host PI3K signalling, and that purified BspA protein causes activation of this lipid kinase. Bacterial entry also requires the cooperation of host Rac1 GTPase. Finally, our findings indicate an important role for clathrin and cholesterol-rich lipid microdomains in the internalization process