Disruption of CFTR-dependent lipid rafts reduces bacterial levels and corneal disease in a murine model of Pseudomonas aeruginosa keratitis.

It is now well appreciated that cholesterol-rich membrane microdomains or lipid rafts are locations on the plasma membranes of eukaryotic cells often used by bacterial,1 viral,2 and protozoal parasites3 to gain access to the cell’s interior. This process is important in the pathogenesis of diverse organisms such as Neisseria gonorrheae, Escherichia coli, and Pseudomonas aeruginosa,1 human immunodeficiency and herpes simplex viruses,2 and Plasmodium spp.3 The broad use of such host cell features among diverse pathogens suggests that therapies aimed at disrupting the ability of parasites to use lipid rafts could be effective in preventing or treating infections. Although no general approach has yet been devised, it is of note that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, broadly known as statin drugs, which reduce cholesterol levels and thus the ability of cells to form cholesterol-rich membrane microdomains, have been suggested by observational studies to improve outcomes in sepsis and other types of infection.4,5 However, since these drugs have multiple biological effects,6 any benefit attributable to statins in the context of sepsis has not been associated with any specific mechanism of action of these drugs. Nonetheless, disruption of cholesterol-rich membrane microdomains and reduction of microbial uptake into host cells when this interaction promotes pathogenesis is a plausible, if unproven, effect of statins in modulating infection. One important infectious process shown to involve lipid-raft uptake of a pathogen and subsequent disease is microbial keratitis,7,8 a sight-threatening infection often associated with both the extended wear of contact lenses9–12 and the use of soft daily-wear, frequent-replacement lenses.13 Keratitis also occurs in the setting of eye trauma, ocular disease, and even systemic disease.14,15 A prominent pathogen in these settings is Pseudomonas aeruginosa,11,16 which presents a particularly difficult therapeutic challenge owing to the rapid progression of infection, the need to deliver antibiotics locally via instillation or injection into the eye, and the antibiotic resistance that commonly occurs in strains of this pathogen. Therefore, new adjunctive therapies, derived from an understanding of the basic aspects of pathogenesis of the microbe–host interaction, could markedly augment the clinical treatment of this serious eye disease. Several recent studies have shown that P. aeruginosa enters epithelial cells of the lung17–20 and cornea7,8 through cholesterol-rich membrane microdomains. Uptake of P. aeruginosa into corneal cells promotes pathogenesis and bacterial survival.21–23 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).23–25 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 cells26; 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. Disruption of lipid rafts by extraction of cholesterol from the plasma membrane of epithelial cells by agents such as β-cyclodextrin (CD) usually reduces the amount of bacteria that can enter the epithelial cells.17,27,28 However, neither the potential prophylactic use of such an agent to prevent eye infections in a setting where there is increased risk of infection (i.e., trauma, surgery, or otokeratology) nor therapeutic use of this modality in the setting of established P. aeruginosa infection has been tried. To extend further the understanding of the interaction of P. aeruginosa and corneal epithelial cells and evaluate whether an intervention that disrupts this interaction has therapeutic potential, we used confocal laser microscopy to visualize the binding of P. aeruginosa to CFTR in lipid rafts of corneal epithelial cells expressing wild-type (WT)-CFTR, as well as the organism’s interaction with isogenic cells carrying only mutant ΔF508 CFTR alleles. We further evaluated the ability of CD to inhibit epithelial cell association with and uptake of invasive strains of P. aeruginosa and its effect on the cytotoxicity of exotoxin U-positive (ExoU+) P. aeruginosa, by using these CFTR-isogenic cells. We also determined whether prophylactic or therapeutic topical administration of CD to the scratch-injured eye of mice infected with P. aeruginosa ameliorated the pathologic effects of infection and modulated bacterial levels within the cornea.