Project Summary/Abstract
The healthy corneal epithelium is an effective barrier to pathogenic and environmental bacteria. Because of
this, corneal infection or keratitis models often rely on bypassing the epithelium altogether by introducing
microbes directly into the corneal stroma, where they initiate immune cell responses that can damage tissue
and cause vision-threatening scarring. A limitation of this experimental approach is that epithelial defense
mechanisms are unable to be identified or investigated. The bacterial pathogen Pseudomonas aeruginosa is
the most common infection associated with soft contact lens wear, suggesting it is uniquely capable of causing
infection in the stroma even when the epithelium remains intact. One way in which P. aeruginosa colonizes the
epithelium is by invading and replicating inside epithelial cells, which has been investigated in cultured cells,
and observed in mouse corneal infection models. Preliminary data show that the toxin ExoS produced by P.
aeruginosa suppresses caspase-4 mediated pyroptosis of invaded corneal epithelial cells, buying time for
bacteria to replicate in a protected niche. Additional data indicate that a different inflammasome pathway is
also active in corneal epithelial cells, and leads to production of the cytokine IL-1ß, which ExoS also
suppresses. Therefore, a novel role for ExoS in corneal infection could be to prolong a niche for bacteria to
replicate within corneal epithelial cells, while limiting secretion of cytokines that recruit immune cells. Using
imaging methods to selectively monitor invaded cells, and CRISPR-Cas9 to manipulate corneal epithelial cells
genetically, we will answer three outstanding questions regarding both corneal epithelial defense, and bacterial
subversion of it: 1. How does ExoS block caspase-4-mediated pyroptosis? 2. How do corneal epithelial cells
detect and respond to P. aeruginosa? 3. What is the significance of epithelial cell inflammasome activation and
pyroptosis in protecting the eye from developing keratitis in the stroma? Successful completion of these aims
will identify a new mechanism of host defense at the ocular surface, further our understanding of
inflammasomes in epithelial cells, and elucidate how a uniquely devastating corneal pathogen is able to
overcome host defenses.