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OMIG Abstract 8
Bacterial Isolates Induce Autophagy in a Human Ocular Surface Cell Line
Kimberly M. Brothers, Regis P. Kowalski, Shenghe Tian, Paul R. Kinchington, and Robert M.Q. Shanks
The Charles T. Campbell Laboratory, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
Purpose: Autophagy protects cells against invading intracellular pathogenic bacteria; however, it has not been determined if ocular clinical isolates of bacteria induce this pathway. The goal of this study was to develop a human autophagy reporter corneal cell line to test the hypothesis that ocular pathogens induce autophagy.
Methods: An LC3-GFP autophagy reporter was introduced into human corneal limbal epithelial (HCLE) cells using lentivirus transduction. The cell line was validated using the autophagy activator rapamycin and inhibitor 3-methyladenine (3’MA). Normalized secretomes from bacterial ocular isolates were used to challenge LC3-GFP cells. Secretome-induced cytotoxicity to HCLE cells was assessed in the presence and absence of 3’MA using Presto Blue® reagent.
Results: Rapamycin and 3’MA experiments show that the reporter cell line was functional. A significant increase in LC3-GFP was observed with Escherichia coli, Serratia marcescens, Staphylococcus aureus MRSA, and MSSA secretomes. HCLE cells were more susceptible to cytotoxic factors produced by S. marcescens and MRSA keratitis isolates when autophagy was inhibited with 3’MA.
Conclusions: Secretomes of E. coli, S. marcescens, and S. aureus MRSA and MSSA keratitis isolates, but not other tested species, induced autophagy in LC3-GFP HCLE cells indicating autophagy induction in response to a subset of Gram-negative and Gram-positive ocular isolates. Results provide evidence of a difference between MRSA and MSSA in the activation of autophagy. 3’MA inhibition of autophagy in human corneal cells rendered them more susceptible to bacterial cytotoxic factors, suggesting a protective role for autophagy against bacterial ocular pathogens.
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