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J. Zhang, A. Kumar, F.–S.X. Yu; Integrity of Lipid Rafts Is Required for Pseudomonas aeruginosa Internalization and TLR2 Signaling in Human Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5084.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Lipid microdomains or lipid rafts of the plasma membrane that are characterized by a high cholesterol and sphingolipid content have been implicated in host–pathogen interaction and in signal transduction. In this study, we sought to determine whether lipid rafts are required for Pseudomonas (P.) aeruginosa internalization and Toll–like receptor–mediated signaling in human corneal epithelial cells (HCECs). Methods: HUCL cells, a telomerase–immortalized human corneal epithelial cell line, were challenged with P. aeruginosa strain PAO1 or TLR2 agonist Pam3Cys–Ser–(Lys)4 (Pam3). Lipid rafts were isolated from Triton X–100 insoluble material of HUCL cells using equilibrium density gradient centrifugation. Methyl–ß–cyclodextrin (MßCD, a cholesterol sequestering drug) and water soluble cholesterol were used for cholesterol depletion and repletion from the plasma membrane, respectively. Lipid raft marker flotinin, IΚB–α phosphorylation and degradation were detected by Western blotting. The internalization of PAO1 expressing green fluorescent protein was observed under a fluorescent microscope and measured with bacterial colony counting. Results: Cholesterol depletion with MßCD significantly reduced the internalization of PAO1 into HCECs. The effect of MßCD on PAO1 intenalization was reversed by cholesterol replenishment, suggesting that the effects of MßCD are limited to plasma membrane cholesterol removal. MßCD treatment also blocked bacterium–induced NF–ΚB activation assessed by IΚB–α phosphorylation and degradation. In HCECs, Pam3 induced IΚB–α phosphorylation and degradation in a TLR2–dependent manner; MßCD treatment blocked Pam3–induced NF–ΚB activation and cholesterol repletion restored Pam3–induced NF–ΚB activation. Conclusions: The integrity of lipid rafts is important for P. aeruginosa internalization and infection as well as for TLR–mediated NF–ΚB signaling in HCECs.
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