PilT is required for PI(3,4,5)P3‐mediated crosstalk between Neisseria gonorrhoeae and epithelial cells

Summary The retractile type IV pilus participates in a number of fundamental bacterial processes, including motil- ity, DNA transformation, fruiting body formation and attachment to host cells. Retraction of the N. gonor- rhoeae type IV pilus requires a functional pilT . Retrac- tion generates substantial force on its substrate ( > 100 pN per retraction event), and it has been spec- ulated that epithelial cells sense and respond to these forces during infection. We provide evidence that piliated, Opa non-expressing Neisseria gonor- rhoeae activates the stress-responsive PI-3 kinase/ Akt (PKB) pathway in human epithelial cells, and activation is enhanced by a functional pilT . PI-3 kinase inhibitors wortmannin and LY294002 reduce cell entry by 81% and 50%, respectively, illustrating the importance of this cascade in bacterial invasion. PI-3 kinase and its direct downstream effectors (PI(3,4,5)P3) and Akt are concentrated in the cell cortex beneath adherent bacteria, particularly at the periphery of the bacterial microcolonies. Further- more, (PI(3,4,5)P3) is translocated to the outer leaflet of the plasma membrane. Finally, we show that (PI(3,4,5)P3) stimulates microcolony formation and upregulates pilT expression in vitro . We conclude that N. gonorrhoeae activation of PI-3 kinase triggers the host cell to produce a lipid second messenger that influences bacterial behaviour.