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J. Ramirez, J. LeDue, C. Tam, R. Borazjani, D. Evans, S. Fleiszig; Traversal of Corneal Epithelium by Cytotoxic Strains of Pseudomonas aeruginosa. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3895.
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For in vitro studies, SV40 human corneal epithelial (HCE) cells were grown airlifted on Transwell filters (3 µm pores) to form multilayers. Epithelial traversal assays were used to compare traversal capacity of the cytotoxic P. aeruginosa strain PA14 to that of an isogenic non cytotoxic exoU mutant (PA14 exoU). Transepithelial Resistance (TER) was measured with an EVOM meter. For in vivo studies, eyes of C57BL/6 mice were tissue paper-blotted (enables fluorescein staining), inoculated, and bacterial interactions with the epithelium assessed 16 h later by deconvolution fluorescence microscopy.
Epithelial traversal by this cytotoxic strain P. aeruginosa in vitro involves ExoU. The loss of TER associated with traversal by wild-type bacteria suggests that epithelial cell death and/or loss of tight junction integrity is involved in traversal by cytotoxic P. aeruginosa. Since the exoU mutant was also able to traverse (albeit at lower levels) and this occurred in the absence of TER loss, additional mechanisms are involved in in vitro traversal. In vivo, ExoU promotes epithelial attachment for this cytotoxic strain, but (under the conditions examined) it does not enable penetration. Whether the phospholipase activity of ExoU is involved in these capacities is to be determined.
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