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D. J. Evans, D. K. Augustin, S. M. J. Fleiszig; Differential Gene Expression in P. aeruginosa Following Traversal of Multilayered Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3903.
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© ARVO (1962-2015); The Authors (2016-present)
Traversal of the corneal epithelium is likely to be an important step in the pathogenesis of contact lens-associated microbial keratitis. We have previously shown that P. aeruginosa which had traversed corneal epithelial cells in vitro acquired an enhanced capacity for traversal when compared to naïve bacteria. Here, we tested the hypothesis that enhanced traversal involves differential bacterial gene expression.
Multilayered telomerase-immortalized human corneal epithelial cells were grown on 3 µm pore-size Transwell filters and P. aeruginosa (PA01, ~106 CFU) were added to the apical surface. After 8 h, bacteria that had traversed cells were collected from the basal compartment. Naïve bacteria were grown in tissue culture media without cell exposure. Affymetrix Genechip® P. aeruginosa Genome Arrays were used to compare gene expression in translocated versus naïve bacteria. Genes that were upregulated > 2.7-fold (based on phoP/Q baseline) were selected for functional analysis using transposon mutants purchased through the University of Washington P. aeruginosa Mutant Collection.
Microarray data revealed that 456 genes (of 1800 analyzed) were upregulated after traversal. These fell into the following categories: hypothetical proteins (193), conserved hypothetical proteins (96), motility (44), transcriptional regulators (27), secreted proteins (22), outer-membrane components (19), adhesion (18), two-component systems (16), efflux pumps (8), protease (6), chemotaxis (4), and secretion systems (3). Mutants in 65 genes of these genes were examined for traversal capacity. Of these, 11 mutants showed loss of function, including 4 known involved in virulence factor regulation. Also of interest, 6 mutants had enhanced capacity to traverse, including 1 known to negatively regulate exotoxin A.
Numerous genes are up and down-regulated after P. aeruginosa traverses corneal epithelial cells. Determining which of these genes actually modulate traversal capacity of the bacteria is likely to identify virulence factors that play critical (and early) roles in corneal infection when it occurs in the absence of overt stromal exposure (e.g. contact lens wear). These would be excellent targets for strategies aimed at preventing infection.
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