GC Tfp Retraction and Stress-Responsive Host Signaling

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The N. gonorrhoeas (GC) type IV pilus (Tfp) is a retractile structure that functions in motility, adherence to host cells, and genetic competence. Cycles of retraction, extension, and substrate tethering enable GC to crawl (twitching motility) and influence epithelial cell signaling. Retraction requires PilT, which is hypothesized to function as a Tfp disassembly motor. Retraction occurs often and with substantial force. CG crawl on the epithelial cell surface and aggregate into microcolonies that are themselves motile for long periods during infection. Tfp retraction forces from a microcolony is calculated to fall within the physiologic range known to trigger cellular responses. We therefore tested the hypothesis that epithelial cells sense and respond to Tfp retraction during infection, using a tissue culture model of infection and a genetically-defined pair of wt and pilT strain expressing Tfp but not Opa. Infection activates the stress-responsive PI-3K/Akt pathway, independent of Opa. A functional pilT strongly enhances activation. PI-3K/Akt activation is detected 0.5-2 hours after infection. PIP3, the product of PI-3K, translocates to the outer leaflet of the epithelial membrane and accumulates beneath microcolonies. PIP3 stimulates microcolony formation and increased p/VTmRNA levels. Thus, Tfp retraction triggers the host to produce an effector that, in turn, influences GC motility behavior. Infection also influences epithelial cell gene expression. Over 300 epithelial genes are differentially regulated after 3 hours of infection, a time when GC have formed microcolonies on the cell surface but have not yet entered the cell pilT enhances the expression of a subset of these genes, in a process that involves the MAPK pathway. One consequence of pilT-ehnancement of gene expression is the creation of an environment that allows the cell to withstand apoptosis. These pilT-related responses can be replicated by a magnet pulling on GC membrane-coated magnetic beads seeded on cells. These results suggest that the force of Tfp retraction triggers protective stress-response pathways in the epithelial cell. Based on these findings, we propose a model for the role of Tfp retraction in epithelial cell signaling. We propose to test the model to better understand the role of Tfp retraction in bacterial pathogenesis.