Cystic Fibrosis Transmembrane Conductance Regulator-Mediated Corneal Epithelial Cell Ingestion of Pseudomonas aeruginosa Is a Key Component in the Pathogenesis of Experimental Murine Keratitis

In several recent studies, our laboratory has provided evidence that the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) can serve as an epithelial cell receptor for internalization of both Pseudomonas aeruginosa (24, 26) and Salmonella typhi (23). The complete outer-core polysaccharide portion of the lipopolysaccharide (LPS) of P. aeruginosa was identified as a ligand for CFTR in these studies (24, 26). We have proposed that CFTR-mediated lung epithelial cell internalization of P. aeruginosa is part of the host defense mechanism for clearing P. aeruginosa from this tissue. Since many severe cases of CF are associated with CFTR mutations that greatly reduce or eliminate expression of CFTR protein, this deficiency may underlie the inability of many CF patients to clear P. aeruginosa from the lung. P. aeruginosa is also the bacterial pathogen most commonly involved in keratitis associated with eye trauma and with the use of contact lenses (3, 27, 32, 34). Numerous P. aeruginosa and host factors have been reported to contribute to pathogenesis, including binding of bacterial pili and LPS to asialo-GM1 receptors (12), elaboration of bacterial and host proteases (6, 15, 29, 36), and intracellular uptake of P. aeruginosa by corneal epithelial cells (9, 11). However, where corneal epithelial cell ingestion of P. aeruginosa falls in the spectrum of critical to irrelevant to the pathologic process is not known. Although we have shown that the LPS outer-core polysaccharide is the bacterial ligand for corneal epithelial cell uptake of P. aeruginosa (39), it has not been established whether CFTR is expressed in corneal epithelium and can serve as a receptor for bacteria during eye infection. We have also shown that addition of complete core LPS inhibits the ingestion of P. aeruginosa by corneal epithelial cells in vitro, but addition of this polysaccharide to infectious inocula of P. aeruginosa applied to the injured eyes of mice resulted in only modest effects on corneal infection and eye pathology (40). These modest effects were felt to be due to the difficulty that a monovalent, exogenous LPS inhibitor added to the infectious inoculum of P. aeruginosa would have in competing with a highly repetitive endogenous LPS ligand on the bacterial surface for binding and internalization into corneal cells. This situation would render the bacterial cells fairly insensitive to competition by the soluble inhibitor. Following the identification of the first extracellular domain of CFTR as a lung epithelial cell receptor for P. aeruginosa, we investigated whether CFTR could also serve as a receptor for uptake of P. aeruginosa during eye infections and the role that this uptake played in pathogenesis. Use of CFTR-specific peptides and the availability of transgenic mouse strains with Cftr mutations provided better experimental systems for further study of P. aeruginosa corneal cell internalization in the pathogenic process than the competition experimental protocols used previously (40). If CFTR peptides could effectively block internalization of P. aeruginosa by corneal cells, then delivery of the peptides along with the infectious inoculum followed by multiple, subsequent applications of CFTR peptides could help determine the role of corneal cell internalization in the pathologic process. Transgenic mice either heterozygous or homozygous for the ΔF508 Cftr allele provide a clear system for testing the hypothesis that CFTR-mediated uptake of P. aeruginosa is critical for the development of infection and disease. The ΔF508 Cftr allele encodes a temperature-sensitive variant of CFTR that fails to be exported to the plasma membrane of cells (4, 5). Mutant Cftr animals also provide a system for bacteriologic and histologic analysis of the progression of infection and development of eye pathology. In this work, we evaluated whether CFTR is expressed in corneal cells, whether CFTR serves as a receptor for uptake of P. aeruginosa during experimental eye infection, and the role of epithelial cell internalization of P. aeruginosa during infection on pathogenesis of experimental keratitis.