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I. Alarcon, S.M. J. Fleiszig; Does Twitching Motility Participate in P. aeruginosa Escape From Corneal Epithelial Cells After Invasion? . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5089.
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Purpose: We have shown that twitching motility is necessary for P. aeruginosa virulence in the cornea, and that twitching motility mutants are less able to traverse corneal epithelial cells in culture, despite a normal capacity for epithelial cell invasion. The hypothesis we are testing is that twitching motility contributes to exit from epithelial cells after bacterial invasion. Methods: Rabbit corneal epithelial cell layers were grown to confluence on 24–well plates or semi permeable filters in vitro. Wild–type P. aeruginosa invasive strain PAK was compared to a pilU (twitching motility) mutant of PAK in three different assays, each involving incubation times varying between 1and 6 h using inocula of 106 CFU bacteria. a) Gentamicin survival assays were used to measure bacterial invasion of corneal epithelial cells. b) The number of viable intracellular bacteria persisting within cells following a 3 h invasion period was determined by extended gentamicin survival assays. c) The number of bacteria crossing (traversing) multilayered corneal epithelium over time was quantified by viable counts of the basolateral chamber. Results: The twitching mutant invaded more than wild–type bacteria when longer incubation times were used, (1–2 h p > 0.14 and 3–6 h p < 0.02; a 3–fold difference after 6 h). Both wild–type and mutant bacteria survived and replicated within cells after invasion, but the number of twitching mutants recovered over time increased at a greater rate compared to twitching competent bacteria (increasing to a 4–fold difference after 6 h, p < 0.01). Conclusions: Twitching motility mutants accumulate within corneal epithelial cells. This might involve an increased capacity for survival/replication within cells. However, it is more likely that accumulation is due to a reduced capacity for cellular exit after invasion, which could explain why fewer twitching mutants traverse corneal epithelial cells.
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