Corneal infection caused by P. aeruginosa (PA) is a sight threatening disease that is difficult to treat. Contact lens wear, the most common predisposing factor for this infection, does not normally cause full thickness epithelial injury to the cornea. Yet little is known about mechanisms that PA use to traverse corneal epithelial cells and reach the underlying stroma. Since traversal represents an early step in pathogenesis, insight into the mechanisms involved could lead to development of preventative measures that completely avoid the devastating effects of PA infection. Twitching motility (tm), a property of bacterial pili, has been found to play a role in corneal infection caused by PA; preliminary data show it is required for traversal across corneal epithelial cells in culture. This project will be focused on the mechanisms by which tm contributes to traversal. The hypothesis to be tested is that tm plays a role in the exit of PA from cells it has invaded and that it is also involved in travel between cells. Exit from corneal epithelial cells will be studied using standard plaque assays, modified gentamicin survival assay and quantitative deconvolution fluorescence microscopy (dm). Extracellular travel will be studied using time-lapse video microscopy and dm.