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Kimberly Brothers, Shenghuo Tian, Paul R Kinchington, Regis P Kowalski, Robert M Q Shanks; The Impact of Ocular Pathogens on Autophagy. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5760. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Autophagy is a cellular process for the recycling of cellular proteins, damaged organelles, and the digestion of microbes. The goal of this study was to develop a corneal autophagy reporter cell line and to use it to determine how ocular pathogens influence host response through the autophagy pathway.
Human corneal limbal epithelial (HCLE) cells were modified using lentivirus to express LC3-GFP from the CMV Promoter as a reporter for autophagy or GFP alone as a control. Growth medium and normalized sterile bacterial culture filtrates (secretomes) were added to LC3-GFP HCLE cells incubated for 3 hours and imaged by confocal microscopy. To induce autophagy 18.3 μM rapamycin was used. To inhibit autophagy, 5000 μM 3-methyladenine was used. To test the role of S. marcescens prodigiosin in autophagy the prodigiosin biosynthetic operon was placed under control of a tightly tunable promoter in S. marcescens using plasmid pMQ262. Various S. marcescens mutants were tested. Clinical keratitis isolates from Acinetobacter baumannii, Escherichia coli, Moraxella sp., Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, and Stenotrophomonas maltophilia were tested.
Control experiments using known autophagy modulators support that the reporter cell line was functional. HCLE cells treated with PIC3611 and K904 S. marcescens secretomes resulted in increased LC3 “puncta” while mutants with reduced pigment, pigA, eepR, and gumB, had fewer LC3 puncta. Secretomes from strains with a prodigiosin inducible operon demonstrated an increased level of LC3 puncta compared to uninduced samples suggesting S. marcescens prodigiosin induces autophagy. Other bacterial keratitis isolates had variable ability to induce autophagy in HCLE cells.
S. marcescens and other bacteria that cause keratitis induced autophagy in corneal cells suggesting that this is a broad response to ocular pathogens. Prodigiosin is absent in most clinical isolates suggesting these S. marcescens isolates may have lost pigmentation to evade recognition by the host. This study implicates a role for autophagy in bacterial interactions with the ocular surface and suggests a new role for the biologically active pigment, prodigiosin.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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