title: Regulation of Pseudomonas aeruginosa internalization after contact lens wear in vivo and in serum-free culture by ocular surface cells.
abstract: OBJECTIVE
  To determine the effects of contact lenses (CLs) and Pseudomonas aeruginosa (PA) infection on localization of cystic fibrosis transmembrane conductance regulator (CFTR) on corneal surface epithelial cells and the association between lipid raft formation and CFTR in mediating PA binding and internalization in ocular surface epithelium.
  
  
  METHODS
  CFTR immunolocalization was evaluated in vivo in rabbit corneal-conjunctival epithelium (with/without CL wear) before and after PA exposure and in serum-free human corneal epithelial cell culture (hTCEpi). Lipid raft formation was visualized with Alexa555-conjugated cholera toxin beta-subunit. Lipid raft involvement in PA internalization was assayed in vivo by gentamicin survival assays after topical filipin pretreatment. Involvement of CFTR in PA binding and internalization was evaluated by blockade with CFTR peptides or LPS.
  
  
  RESULTS
  CL wear in vivo enhanced anti-CFTR staining, but CFTR localization did not correlate with the PA binding by ocular surface cells. Conjunctival epithelial cells stained for CFTR but did not bind or internalize PA. Corneal epithelial cells in vivo did not stain for CFTR unless challenged by contact lens-induced hypoxia. PA internalization by hTCEpi was significantly inhibited by LPS (P < 0.01), but not by CFTR peptides. Remarkably, normal conjunctival epithelial cells showed lipid raft formation and CFTR staining but did not bind PA. Inhibition of raft formation by filipin blocked PA internalization in vivo after CL wear.
  
  
  CONCLUSIONS
  CFTR is not the predominant receptor for ocular surface PA infection, and after hypoxic CL challenge, neither lipid rafts nor CFTR localization alone predicts PA binding; however, lipid rafts are critical to CL-mediated PA internalization.
authors.affiliation: Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, 75390, USA.
authors.forename: Naoka
authors.initials: N
authors.lastname: Yamamoto
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Yamamoto
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Petroll
authors.forename: James V
authors.initials: JV
authors.lastname: Jester
authors.forename: H Dwight
authors.initials: HD
authors.lastname: Cavanagh
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D015169:Colony Count, Microbial; D003228:Conjunctiva / Q000166:cytology; D003261:Contact Lenses; D016895:Culture Media, Serum-Free; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004847:Epithelial Cells / Q000378:metabolism*; D019573:Epithelium, Corneal / Q000378:metabolism*; D015818:Eye Infections, Bacterial / Q000378:metabolism* / Q000382:microbiology; D005372:Filipin / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D021962:Membrane Microdomains / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D011552:Pseudomonas Infections / Q000378:metabolism* / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D011817:Rabbits
chemical_list: D016895:Culture Media, Serum-Free; D008070:Lipopolysaccharides; D008565:Membrane Proteins; C106354:flotillins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005372:Filipin