Phosphatidylinositol 3-kinase activation and interaction with focal adhesion kinase in Escherichia coli K1 invasion of human brain microvascular endothelial cells

Invasion of brain microvascular endothelial cells (BMEC) is a prerequisite for successful crossing of the blood-brain barrier by Escherichia coli K1. We have previously demonstrated the requirement of cytoskeletal rearrangeme nts and activation of focal adhesion kinase (FAK) in E. coli K1 invasion of human BMEC (HBMEC), The current study investigated the role of phosphatidy linositol S-kinase (PI3K) activation and PI3K interaction with FAR in E. co li invasion of HBMEC. PI3K inhibitor LY294002 blocked E. coli K1 invasion o f HBMEC in a dose-dependent manner, whereas an inactive analogue LYS03511 h ad no such effect. In HBMEC, E, coli K1 increased phosphorylation of Akt, a downstream effector of PI3K, which was completely blocked by LY294002. In contrast, non-invasive E. coli failed to activate PI3K Overexpression of PI 3K mutants Delta p85 and catalytically inactive p110 in HBMEC significantly inhibited both PI3K/Akt activation and E. coli gl invasion of HBMEC, Stimu lation of HBMEC with E. coli K1 increased PI3K association with FAK. Furthe rmore, PI3K/Akt activation was blocked in HBMEC-overexpressing FAK dominant -negative mutants (FRNK and Phe397FAK). These results demonstrated the invo lvement of PI3K signaling in E. coli K1 invasion of HBMEC and identified a novel role for PI3K interaction with FAR in the pathogenesis off. coli meni ngitis.