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Abby Kroken, David J Evans, Suzanne M J Fleiszig; Corneal epithelial cells suppress intracellular replication by clearing cytoplasmic Pseudomonas aeruginosa. Invest. Ophthalmol. Vis. Sci. 2016;57(12):321.
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© ARVO (1962-2015); The Authors (2016-present)
The corneal epithelium is extremely resistant to Pseudomonas aeruginosa, whereas the same microbe kills cultured epithelial cells within hours. Here, we investigated whether corneal cells have unique defenses by comparing cultured telomerase-immortalized corneal epithelial cells to transformed cell lines often used to study P. aeruginosa pathogenesis.
HeLa cells or human telomerase-immortalized corneal epithelial cells were infected with invasive P. aeruginosa strain PAO1 or type three secretion mutants, all expressing GFP under control of the ExoS promoter to discriminate intracellular from extracellular bacteria. Extracellular bacteria were eliminated with amikacin at 3 hours. Infections were viewed using time-lapse fluorescence microscopy and analyzed with ImageJ. Invasion was scored visually and defined by presence of intracellular P. aeruginosa that replicated at least once.
Compared to HeLa cells, corneal epithelial cells were more resistant to bacterial invasion by a P. aeruginosa mutant lacking ExoS, a toxin that blocks phagocytosis by impairing actin polymerization and vesicle trafficking (37 ± 8% of HeLa cells versus 18 ± 4% of corneal cells invaded). The presence of ExoS reduced HeLa cell invasion (20 ± 4% of cells invaded), but had little impact on the percent of corneal cells invaded (18 ± 10%). HeLa cells supported high levels of cytoplasmic bacterial replication before loss of plasma membrane integrity and cell death. Mixed phenotypes occurred in corneal epithelial cells: some cells formed membrane blebs to which bacteria trafficked and replicated (as we have previously described), whereas other cells appeared to clear intracellular bacteria, as fluorescent bacterial bodies in the cytosol disappeared from detection.
The anti-phagocytic toxin ExoS does not impact invasion of P. aeruginosa into corneal cells as it does in HeLa cells, suggesting that corneal epithelial cells resist ExoS toxicity at the level of individual cells. When invasion does occur, replication of bacteria may be restricted within corneal cells by clearance of bacteria from the cytoplasm. Our findings suggest that factors within corneal epithelial cells, independent of their barrier function in situ, participate in corneal resistance to microbial pathogenesis. Further studies will identify mechanisms of intracellular microbe control by the corneal epithelium.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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