The Charles T. Campbell Ophthalmic Microbiology Laboratory, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
The Charles T. Campbell Ophthalmic Microbiology Laboratory, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Exp Eye Res. 2018 Mar;168:12-18. doi: 10.1016/j.exer.2017.12.010. Epub 2017 Dec 27.
Autophagy protects cells from intracellular pathogens, but can be exploited by some infectious agents to their benefit. Currently it is not known if bacteria induce autohpagy in cells of the cornea. The goal of this study was to develop an ocular surface autophagy reporter cell line and determine whether ocular bacterial pathogens influence host responses through autophagy induction. The cell line was made using lentivirus transduction of an LC3-GFP fusion protein in human corneal limbal epithelial (HCLE) cells. LC3-GFP puncta in HCLEs were induced by rapamycin and ammonium chloride treatments, and prevented by the autophagy inhibitors 3-methyladenine (3'MA) and bafilomycin. Importantly, secretomes from Escherichia coli, Serratia marcescens, Staphylococcus aureus, methicillin sensitive (MSSA) and resistant (MRSA), were found to induce autophagy, whereas other bacteria, including Acinetobacter baumannii, Achromobacter xylosoxidans, Enterococcus faecalis, Klebsiella pneumoniae, Moraxella sp., and Stenotrophomonas maltophilia, did not. Our data indicates differences between tested ocular isolates of MRSA and MSSA in the activation of autophagy. HCLEs treated with 3'MA were slightly more susceptible to cytotoxic factors produced by S. marcescens and MRSA keratitis isolates, by contrast, bafilomycin A1 treatment caused no difference. This work demonstrates the successful development and validation of an autophagy reporter corneal cell line and indicates differences between ocular bacterial isolates in the activation of autophagy.
自噬可以保护细胞免受细胞内病原体的侵害，但某些传染性病原体可以利用自噬来获益。目前尚不清楚细菌是否会在角膜细胞中诱导自噬。本研究的目的是开发一种眼部表面自噬报告细胞系，并确定眼部细菌病原体是否通过诱导自噬来影响宿主反应。该细胞系通过慢病毒转导在人角膜缘上皮（HCLE）细胞中引入 LC3-GFP 融合蛋白而制成。雷帕霉素和氯化铵处理可诱导 HCLE 中的 LC3-GFP 斑点形成，而自噬抑制剂 3-甲基腺嘌呤（3-MA）和巴弗洛霉素则可阻止 LC3-GFP 斑点形成。重要的是，我们发现大肠杆菌、粘质沙雷氏菌、金黄色葡萄球菌（包括耐甲氧西林金黄色葡萄球菌（MRSA）和甲氧西林敏感金黄色葡萄球菌（MSSA））的分泌液可诱导自噬，而其他细菌，包括鲍曼不动杆菌、木糖氧化无色杆菌、粪肠球菌、肺炎克雷伯菌、莫拉菌属和嗜麦芽窄食单胞菌则不能。我们的数据表明，测试的 MRSA 和 MSSA 眼部分离株在自噬激活方面存在差异。用 3-MA 处理的 HCLE 对粘质沙雷氏菌和 MRSA 角膜炎分离株产生的细胞毒性因子的敏感性略有增加，相比之下，巴弗洛霉素 A1 处理则没有差异。这项工作证明了自噬报告角膜细胞系的成功开发和验证，并表明眼部细菌分离株在自噬激活方面存在差异。
