Regulation of Pseudomonas aeruginosa internalization after contact lens wear in vivo and in serum-free culture by ocular surface cells

Naoka Yamamoto, Nobutaka Yamamoto, Matthew W. Petroll, James V. Jester, H. Dwight Cavanagh

Research output: Contribution to journalArticle

17 Scopus citations

Abstract

PURPOSE. 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 L-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.

Original languageEnglish (US)
Pages (from-to)3430-3440
Number of pages11
JournalInvestigative Ophthalmology and Visual Science
Volume47
Issue number8
DOIs
StatePublished - Aug 1 2006

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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