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Aaron B. Sullivan, Connie K. Tam, David J. Evans, Suzanne M. Fleiszig; Mechanisms Of P. aeruginosa Traversal Of The Corneal Epithelium In Situ. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3651.
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© ARVO (1962-2015); The Authors (2016-present)
We recently developed methods to enable corneal epithelial penetration by bacteria to be visualized and quantified in eyeballs of mice without tissue fixation or labeling. Here, the methods were used to determine the role of the type III secretion (T3S) system for P. aeruginosa.
Two different methods were used to enable corneal epithelial susceptibility to P. aeruginosa penetration using freshly excised eyeballs from mice; 1) After rinsing, corneas were blotted with a Kimwipe™, and 100 mM EGTA for 1 h at 37°C; or 2) MyD88 knockout mouse eyes were used. Corneas were inoculated with GFP-labeled 200 µl ~109CFU/mL P. aeruginosa strain PAO1 or an isogenic T3S (ΔexsA) mutant for 6 h, 37°C. After rinsing, corneas were imaged using 2-photon microscopy (Mai Tai laser 720 nm, NADPH autofluorescence), confocal reflection microscopy (HeNe laser 633 nm) and confocal GFP labeling (Argon Laser 488nm). Epithelial thickness and depth of bacterial penetration were compared. Efficiency of T3S was examined using QPCR and Coomassie staining of secreted proteins. Methods were in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Declaration of Helsinki.
The parent wildtype PAO1 penetrated the epithelium of blotted/EGTA treated corneas to the underlying basal lamina 6 h post challenge, accompanied by severe epithelial damage. T3S mutants adhered, without penetrating, and visible cell damage was not observed (p < 0.05 for differences in traversal and also corneal thickness). Surprisingly, both parent and T3S mutant traversed MyD88 knockout mouse corneal epithelium. Comparison of three sources of wild-type PAO1 in the blot/EGTA model showed varying capacity to penetrate and damage the epithelium, correlating with differences in T3S efficiency.
The T3S is required for epithelial traversal for corneas made susceptible by blotting+EGTA treatment, but not in MyD88 deficiency. Thus, there are multiple pathways to susceptibility, and the role specific virulence factors play varies accordingly. Specifically, the data show that P. aeruginosa can use T3S independent means to traverse corneal epithelium if MyD88 dependent defenses are compromised, and illustrate that virulence factor expression can vary among "identical" strains from different sources.
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