betadex and Pseudomonas-Infections

Pseudomonas aeruginosa enters corneal epithelial cells in vitro via membrane microdomains or lipid rafts. Bacterial entry, mediated by the cystic fibrosis transmembrane conductance regulator (CFTR), promotes infection and disease. This study was conducted to determine whether P. aeruginosa and CFTR are colocalized to rafts in isogenic corneal cells expressing wild-type (WT) or mutant DeltaF508-CFTR and whether disruption of the rafts both in vitro and in vivo affects the bacterial levels and the course of the disease.. Transformed human corneal epithelial cells from a patient homozygous for DeltaF508-CFTR, and the same cells corrected with WT-CFTR, were exposed to six isolates of P. aeruginosa-three invasive and three cytotoxic strains-in the presence of beta-cyclodextrin (CD), which disrupts rafts. Association and cellular uptake of the invasive strains were measured, as was lactate dehydrogenase release induced by the cytotoxic strains. Scratch-injured mouse eyes were infected with the six P. aeruginosa strains, and the effect of prophylactic or therapeutic administration of CD on bacterial levels and disease was evaluated.. P. aeruginosa and CFTR were colocalized with lipid rafts in cells with WT-CFTR, and CD treatment of these cells disrupted bacterial association, internalization, and cytotoxic effects. Cells expressing DeltaF508-CFTR were marginally affected by CD. Prophylactic and therapeutic topical application of CD ameliorated corneal disease and reduced the bacterial count in the eye.. P. aeruginosa enters human corneal epithelial cells via lipid rafts containing CFTR, and disruption of raft-mediated uptake of this organism by CD protects against disease and reduces bacterial levels in the mouse model of keratitis.

Topics: Animals; Bacterial Adhesion; beta-Cyclodextrins; Blotting, Western; Cell Line, Transformed; Colony Count, Microbial; Corneal Ulcer; Cystic Fibrosis Transmembrane Conductance Regulator; Disease Models, Animal; Epithelium, Corneal; Eye Infections, Bacterial; Fluorescent Antibody Technique; Humans; Membrane Microdomains; Mice; Microscopy, Confocal; Pseudomonas aeruginosa; Pseudomonas Infections; RNA, Small Interfering

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein is an epithelial cell receptor for the outer core oligosaccharide of the Pseudomonas aeruginosa LPS. Bacterial binding leads to CFTR-dependent bacterial internalization, initiation of NF-kappaB nuclear translocation, cellular desquamation, and eventual apoptosis of the infected cells, all of which are critical for innate immune resistance to infection with this pathogen. Lack of this reaction in CF patients underlies their hypersusceptibility to chronic P. aeruginosa infection. In this study we tested whether these epithelial cell responses are dependent upon the localization of CFTR to lipid rafts. Confocal microscopy showed that green fluorescent protein-tagged CFTR (GFP-CFTR) and the lipid raft marker ganglioside GM1 colocalized at sites of P. aeruginosa contact and internalization. GFP-CFTR localized to low density Triton X-100-insoluble fractions in lysates of Madin-Darby canine kidney GFP-CFTR cells, and P. aeruginosa infection increased the levels of GFP-CFTR in these fractions as determined by Western blot. Cells expressing GFP-DeltaF508-CFTR did not have rafts with detectable CFTR protein. Extraction of cell surface cholesterol via cyclodextrin treatment of the cells inhibited CFTR entry into rafts. In addition, cyclodextrin treatment of both human and canine epithelial cells inhibited cellular ingestion of P. aeruginosa, NF-kappaB nuclear translocation, and apoptosis. These results indicate that lipid raft localization of CFTR is required for signaling in response to P. aeruginosa infection. Such signaling is needed for the coordination of innate immunity to P. aeruginosa lung infection, a process that is defective in CF.

Topics: Active Transport, Cell Nucleus; Animals; Apoptosis; beta-Cyclodextrins; Cell Line; Cell Line, Transformed; Cyclodextrins; Cystic Fibrosis Transmembrane Conductance Regulator; Dogs; Down-Regulation; Epithelial Cells; G(M1) Ganglioside; Humans; Membrane Microdomains; Mutation; NF-kappa B; Octoxynol; Pseudomonas aeruginosa; Pseudomonas Infections; Signal Transduction; Solubility; Up-Regulation

Pseudomonas aeruginosa infection is a serious complication in patients with cystic fibrosis and in immunocompromised individuals. Here we show that P. aeruginosa infection triggers activation of the acid sphingomyelinase and the release of ceramide in sphingolipid-rich rafts. Ceramide reorganizes these rafts into larger signaling platforms that are required to internalize P. aeruginosa, induce apoptosis and regulate the cytokine response in infected cells. Failure to generate ceramide-enriched membrane platforms in infected cells results in an unabated inflammatory response, massive release of interleukin (IL)-1 and septic death of mice. Our findings show that ceramide-enriched membrane platforms are central to the host defense against this potentially lethal pathogen.

Topics: Animals; Apoptosis; beta-Cyclodextrins; Bone Marrow Transplantation; Cells, Cultured; Ceramides; Cyclodextrins; Cystic Fibrosis Transmembrane Conductance Regulator; Enzyme Activation; Epithelial Cells; fas Receptor; Fibroblasts; Filipin; Fluorescent Dyes; Humans; Ionophores; Membrane Microdomains; Mice; Nystatin; Pseudomonas aeruginosa; Pseudomonas Infections; Signal Transduction; Sphingomyelin Phosphodiesterase