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N. Yamamoto, N. Yamamoto, J.V. Jester, H.D. Cavanagh; Internalization of Pseudomonas Aeruginosa in Corneal Epithelium is Mediated by Lipid Rafts . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2122.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We previously showed that the internalization of P. aeruginosa (PA) in corneal epithelial cells (in vivo, in vitro) appears to involve the formation of sphingolipid–rich plasma membrane domains forming lipid raft platforms (ARVO, 2004). The purpose of this study was to investigate whether the internalization of PA through lipid raft (LR) formation is a common mechanism among corneally invasive PA strains. Methods: Two corneally invasive isolates, 6294 and 6487 (gifts of Dr. Suzanne M. Fleiszig), and one non–corneal isolate, known to be infectious to the cornea (ATCC27853) of PA were used in this study. LR formation was visualized in an immortalized human corneal epithelial cell line (hTCEpi) before and after PA infection by staining with the FITC–conjugated ß–subunit of cholera toxin (ß–CT) known to bind to LR component, ganglioside GM1, followed by confocal microscopy. Bacterial internalization was quantified by gentamicin survival assay. The role of LR in PA internalization was evaluated by pretreatment of hTCEpi cells with cholesterol metabolism inhibitors, methyl–ß–cyclodextrin, filipin and nystatin prior to PA infection. Following exposure of PA to hTCEpi cell lysate and complexing with FITC–conjugated ß–CT, the specific interaction of PA with LR was tested by FACS. Results: Infection with all PA strains produced LR reorganization, aggregation and PA internalization at the same LR membrane sites in hTCEpi cells. Quantification of PA internalization showed that all cholesterol metabolism inhibitors significantly decreased PA internalization of 3 strains in a dose–dependent manner (p<0.01). FACS analysis showed that exposure of PA to cell lysate significantly increased bacterial fluorescence (p<0.05), confirming specific binding of LR to PA. Conclusions: These findings with 3 infectious strains of PA, one non–corneal but infectious and two corneal isolates, suggest that LR formation is required for infection of invasive PA strains in corneal epithelium.
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