Summary: P. aeruginosa is a leading cause of blinding eye disease. Contributing to disease pathogenesis is invasion of corneal epithelial cells, as we have demonstrated in vitro and in vivo. Here, we report that P. aeruginosa can establish multiple niches inside corneal epithelial cells, some colonizing the cytoplasm and other vacuoles – often in the same cell. Reporter expression results show different phenotypic states in these alternate locations, the pattern suggesting “acute” infection mode for cytoplasmic bacteria and “chronic” (biofilm forming) mode for those in vacuoles. While genetic regulation of the switch between these two states has been extensively studied in vitro, how this might work inside a cell is unknown. Our published and preliminary data show that “acute” and “chronic” modes cooperate to determine corneal epithelial cell responses, to allow P. aeruginosa to penetrate the multilayered corneal epithelium, to colonize the cornea, and to cause pathology. Here, we will test the hypothesis that their unique intracellular niches contribute to their cooperative efforts during corneal infection. The three aims will explore significance of intracellular diversification to the bacteria, to the host corneal epithelial cell, and to disease pathogenesis. Approaches will utilize multiple novel tools developed using funding from this grant, including bacterial constructs, imaging technologies, and infection models. The two consultants are leading experts in bacterial gene regulation, respectively covering both phenotypes. Importantly, our data suggest that vacuolar P. aeruginosa transition into biofilms, which can be difficult to kill or even to culture. A vacuole would enclose a biofilm inside two host membranes - vacuolar and plasma. This intracellular phenotype alone could explain why P. aeruginosa infections are notoriously difficult to manage and why disease can continue despite apparent sterilization of the ulcer.