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Robert M Q Shanks, Kimberly M Brothers; Identification of bacterial factors that induce autophagy in corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2020;61(7):456.
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© ARVO (1962-2015); The Authors (2016-present)
Autophagy is a cellular recycling process that contributes to defending mammalian cells against microbial pathogens. A previous study demonstrated that ocular pathogens including Serratia marcescens activate autophagy in a corneal epithelial cells. In this study we used a genetic approach to identify bacterial factors produced by S. marcescens that are responsible for activating autophagy to determine how bacteria influence host responses.
An ocular surface LC3-GFP autophagy reporter cell line was challenged with normalized secretomes of mutant bacterial strains. S. marcescens mutant strains with defects in biosynthesis of surface or secreted factors (biosurfactant, fimbriae, flagellin, lipopolysaccharide, prodigiosin, serralysin protease, serratamolide, S-layer, and type I and VI secretions systems) and genes whose products regulate transcription of these products (crp, eepR, gumB, and hexS) were tested for reduced ability to induce autophagy. LC3-GFP phenotypes were analyzed using ImageJ software.
Of the tested transcription factor mutants, two genes, eepR and gumB, were required for S. marcescens to fully induce autophagy. Subsequent analyses support the model that the eepR and gumB autophagy induction defects are due to a lack of prodigiosin biosynthesis. Mutants in the prodigiosin biosynthetic operon (pigA and pigB) were defective in autophagy induction, and induced expression of the pig operon restored autophagy induction to the eepR mutant. Furthermore, purified prodigiosin was sufficient to induce autophagy. The gumB mutant was more defective in autophagy production than the eepR mutant due to the inability to generate both prodigiosin and the cytolysin ShlA.
Results presented here demonstrate an independent contribution of prodigiosin and the ShlA cytolysin to autophagy induction by an ocular surface cell line in response to bacteria. Together these data provide insight into mechanisms human cells use to respond to bacterial pathogens.
This is a 2020 ARVO Annual Meeting abstract.
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