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

Abstract

Previous findings indicate that the cystic fibrosis transmembrane conductance regulator (CFTR) is a ligand for Pseudomonas aeruginosa ingestion into respiratory epithelial cells. In experimental murine keratitis, P. aeruginosa enters corneal epithelial cells. We determined the importance of CFTR-mediated uptake of P. aeruginosa by corneal cells in experimental eye infections. Entry of noncytotoxic (exoU) P. aeruginosa into human and rabbit corneal cell cultures was inhibited with monoclonal antibodies and peptides specific to CFTR amino acids 108 to 117. Immunofluorescence microscopy and flow cytometry demonstrated CFTR in the intact murine corneal epithelium, and electron microscopy showed that CFTR binds to P. aeruginosa following corneal cell ingestion. In experimental murine eye infections, multiple additions of 5 nM CFTR peptide 103-117 to inocula of either cytotoxic (exoU +) or noncytotoxic P. aeruginosa resulted in large reductions in bacteria in the eye and markedly lessened eye pathology. Compared with wild-type C57BL/6 mice, heterozygous ΔF508 Cftr mice infected with P. aeruginosa had an approximately 10-fold reduction in bacterial levels in the eye and consequent reductions in eye pathology. Homozygous ΔF508 Cftr mice were nearly completely resistant to P. aeruginosa corneal infection. CFTR-mediated internalization of P. aeruginosa by buried corneal epithelial cells is critical to the pathogenesis of experimental eye infection, while in the lung, P. aeruginosa uptake by surface epithelial cells enhances P. aeruginosa clearance from this tissue.

The Role of Pseudomonas aeruginosa Lipopolysaccharide in Bacterial Pathogenesis and Physiology

“…Experiments using epithelial cell lines carrying either the wildtype or ∆F508 variants of CFTR indicated that the internalization of P. aeruginosa was reduced when mutant but not wildtype CFTR was expressed, suggesting that CFTR mutations may promote P. aeruginosa infection [44]. In contrast, CFTR-dependent internalization of P. aeruginosa in corneal epithelial cells is necessary for this bacterium to cause keratitis [48][49][50]. Similarly, in Salmonella enterica serovar Typhi, entry into intestinal epithelial cells is also mediated by CFTR, and it has been documented that CF patients possessing mutant forms of this protein might be protected from contracting typhoid fever [51].…”

Staphylococcus aureusCorneal Infections: Effect of the Panton-Valentine Leukocidin (PVL) and Antibody to PVL on Virulence and Pathology

“…Primary cultures of human epithelial corneal cells were established from corneal rims prepared and cultured in flasks as described by Zaidi et al 31 Cells were seeded at 2 3 10 5 cells per well in 24-well plates and after 18 hours exposed to a 1:20 or 1:10 dilution of the cell-free, sterile supernatant from S. aureus strains LAC, LACDPVL, MW2, or MW2DPVL grown overnight at 378C in YCP broth. Aliquots from the wells were collected every 5 minutes and lactate dehydrogenase (LDH) release determined by a commercially available assay (Tox-7 in vitro cytotoxicity assay; Sigma, St. Louis, MO).…”

Disruption of CFTR-Dependent Lipid Rafts Reduces Bacterial Levels and Corneal Disease in a Murine Model ofPseudomonas aeruginosaKeratitis

“…The epithelial cells were grown in 96-well tissue culture plates as described 24 and incubated with 0, 0.5, 2, or 5 mM CD for 30 minutes, then infected for 3 hours with various P. aeruginosa strains at a multiplicity of infection of ϳ10:1. In brief, approximately 10 6 CFU of P. aeruginosa was added to monolayers of 10 5 cultured corneal cells.…”

“…To determine the internalized colony-forming units, gentamicin-exclusion assays were used as described elsewhere. 21,24,[35][36][37] Three-hundred micrograms gentamicin per milliliter was added to the infected epithelial cell cultures to kill extracellular bacteria. After 1 hour of exposure to the antibiotic, the cells were washed, and intracellular bacteria were released from them by lysis with 0.5% Triton X-100, diluted, and plated for bacterial enumeration.…”

“…[21][22][23] The bacterial ligand has been identified as the outer-core oligosaccharide of the bacterial lipopolysaccharide (LPS), 22 and the main epithelial cell receptor implicated in binding of this bacterium is the cystic fibrosis transmembrane conductance regulator (CFTR). [23][24][25] Disruption of the bacteria-CFTR interaction reduces disease severity in a murine model of scratch-injury keratitis, 22,24 and transgenic CF mice are markedly resistant to P. aeruginosa keratitis. 24 One recent report has disputed that CFTR is the P. aeruginosa receptor for corneal epithelial and conjunctival cells 26 ; however, the lack of CFTR-negative animals and cells as comparators raises questions as to whether the conclusions dismissing CFTR as the corneal epithelial cell receptor for P. aeruginosa have been adequately justified by the experimental data.…”