title: Internalization of Pseudomonas aeruginosa is mediated by lipid rafts in contact lens-wearing rabbit and cultured human corneal epithelial cells.
abstract: OBJECTIVE
  The internalization of Pseudomonas aeruginosa (PA) in nasal and tracheal epithelium has recently been shown to involve the formation of cholesterol- and sphingolipid-rich plasma membrane domains (lipid rafts). The purpose of this study was to investigate the role of lipid rafts in PA internalization by corneal epithelium in vivo, in vitro, and after contact lens wear.
  
  
  METHODS
  Lipid raft formation was evaluated in rabbit corneas with and without contact lens wear and a human corneal epithelial (hTCEpi) cell line before and after PA infection with cornea-pathogenic strains by staining with FITC-conjugated cholera toxin beta-subunit, known to bind the lipid raft component GM1. Bacterial internalization was assessed by gentamicin survival assay. The role of lipid rafts in PA internalization was evaluated by pretreatment of hTCEpi cells with cholesterol metabolism inhibitors. The interaction of PA with lipid rafts was confirmed by flow cytometry.
  
  
  RESULTS
  Contact lens wear in rabbits induced lipid raft formation in occasional surface corneal epithelial cells. Subsequent PA exposure showed preferential binding to lipid raft-forming cells, leading to lipid raft aggregation and PA internalization. A similar sequence of lipid raft formation and PA internalization was also observed in hTCEpi for all PA strains. Internalization of all PA strains was blocked by three cholesterol metabolism inhibitors (P < 0.01). Flow cytometry showed an association of PA with rafts.
  
  
  CONCLUSIONS
  These findings demonstrate that contact-lens-mediated PA internalization involves lipid raft formation. Also, hTCEpi cells may be used as an experimental model for studying further the molecular mechanism(s) of PA infection in the corneal epithelium.
authors.affiliation: Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, 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: H Dwight
authors.initials: HD
authors.lastname: Cavanagh
authors.forename: James V
authors.initials: JV
authors.lastname: Jester
mesh_terms: D000818:Animals; D000924:Anticholesteremic Agents / Q000494:pharmacology; D001422:Bacterial Adhesion / Q000502:physiology; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D003261:Contact Lenses; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D019573:Epithelium, Corneal / Q000382:microbiology* / Q000473:pathology; D015818:Eye Infections, Bacterial / Q000382:microbiology* / Q000473:pathology; D005434:Flow Cytometry; D006801:Humans; D007634:Keratitis / Q000382:microbiology / Q000473:pathology; D008563:Membrane Lipids / Q000502:physiology; D021962:Membrane Microdomains / Q000502:physiology*; D011552:Pseudomonas Infections / Q000382:microbiology* / Q000473:pathology; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D011817:Rabbits
chemical_list: D000924:Anticholesteremic Agents; D008563:Membrane Lipids; D002772:Cholera Toxin; D002784:Cholesterol