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Victoria Hritonenko, Amber Jolly, Courtney Maloney, Allison Farfel, David Evans, Suzanne Fleiszig; ExoS, a type III secreted toxin, is produced intracellularly by P. aeruginosa after corneal epithelial cell invasion, and is triggered by exposure to cell lysates. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5215.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously shown that the type 3 secretion system (T3SS) of Pseudomonas aeruginosa contributes to the pathogenesis of P. aeruginosa keratitis. We have also shown that ExoS, one of the known T3SS effectors, is required for intracellular survival by P. aeruginosa. Known triggers for T3SS expression include low calcium conditions and contact with live mammalian cells. Here, we tested the hypothesis that the environment inside epithelial cells can trigger effector expression.
To localize ExoS using immunofluorescence microscopy, corneal epithelial cells were grown on glass coverslips, inoculated with 107 CFU/ml PAO1ΔΔΔexoSTY + pUCPexoS-HA or a translocon mutant (PAO1ΔpopBΔΔΔexoSTY + pUCPexoS-HA) for 3 h, followed by gentamicin solution (4 h) to kill extracellular bacteria. ExoS was localized in samples using antibody to HA (tagged to ExoS). In other experiments, bacteria were exposed to epithelial cell lysates and ExoS expression measured by Western blot. Lysates were prepared using freeze-thaw cycles, and after removal of cell debris, were inoculated with ~103 CFU/mL PAO1 for 12 h. Bacteria were then removed by centrifugation, and supernatants examined and compared to uninoculated control lysate. The impact of challenge with bacterial supernatant (containing bacterial antigens) prior to lysate preparation was also explored.
ExoS was found in the host cytosol of P. aeruginosa infected cells, even with a translocon mutant that cannot inject effectors across host cell membranes. ExoS expression was induced by exposure to cell lysates prepared in 300 μl PBS (20 ± 9.7%) compared to low calcium inducing conditions, but not when the lysate was diluted 3-fold. ExoS was found associated with lysate supernatants, not bacterial pellets. Intriguingly, the size of the band recognized by the ExoS antibody increased (by ~ 4 kDa) when lysates were prepared from cells pre-exposed to bacterial antigens.
The data show that P. aeruginosa expresses ExoS intracellularly after it invades corneal epithelial cells. The induction of ExoS expression by epithelial cell lysates suggests biochemical triggers for expression, which can now be identified. The significance of the increased size of ExoS when triggered by lysates from cells pre-exposed to bacterial antigens is to be determined.
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