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Abby Kroken, David J Evans, Suzanne M J Fleiszig; Corneal epithelial cells suppress vacuolar escape of P. aeruginosa. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4050.
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© ARVO (1962-2015); The Authors (2016-present)
Previously we reported that invasive strains of P. aeruginosa invade corneal epithelial cells wherein they replicate and induce formation of plasma membrane blebs to which they traffic. These events depend on the bacterial Type Three Secretion System (T3SS), specifically the T3SS toxin ExoS. Other investigators have studied ExoS using HeLa cells (not normally targeted by P. aeruginosa) and PA103 (does not natively express ExoS), and have not noted these phenomena. Here, we tested the hypothesis that the impact of ExoS depends on bacterial strain and cell type.
Epithelial cell lines (HeLa or corneal) were infected with P. aeruginosa strain PAO1 (natively expresses ExoS) or PA103 (engineered to express ExoS). Intracellular bacteria were quantified using a gentamicin protection assay. A T3SS-driven GFP reporter was used to monitor T3SS expression in individual bacteria. Images were captured using time lapse wide-field or confocal microscopy.
In both cell types, PAO1 and PA103 expressing ExoS induced membrane blebbing, but only PAO1 invaded, replicated intracellularly, or trafficked to blebs. In contrast to corneal cells, HeLa cells even supported intracellular replication of PAO1 mutants lacking all known T3SS toxins (including ExoS). That replication remained dependent on the T3SS, which was expressed intracellularly, and occurred in the cytoplasm, without blebs, and in cells possessing acidified vacuoles - all differentiating it from ExoS-dependent replication in corneal cells. T3SS machinery mutants remained in vacuoles in HeLa cells, showing that vacuolar escape is mediated by a T3SS component or unknown effector, but not ExoS. This contrasts with ExoS-dependent vacuolar escape in corneal cells.
ExoS has been mostly studied using PA103 and HeLa cells. Our data show that neither accurately model how natively encoded ExoS influences P. aeruginosa interactions with corneal epithelial cells. This explains why its role in intracellular survival was overlooked by investigators using those tools. Our data show that; 1) corneal epithelial cells have innate defenses against internalized bacteria that are lacking in HeLa cells and which ExoS can overcome, and 2) unknown T3SS factors allow intracellular replication in cells lacking those innate defenses. Identifying mechanisms involved could lead to novel strategies to combat infection.
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