Abstract
BackgroundThe cellular process of autophagy is essential for maintaining the health of ocular tissue. Dysregulation of autophagy is associated with several ocular diseases including keratoconus and macular degeneration. It is known that autophagy can be used to respond to microbial infections and that certain microbes can exploit the autophagic process to their benefit. In this study, a genetic approach was used to identify surface-associated and secreted products generated by the opportunistic pathogen Serratia marcescens involved in activation of autophagy.MethodsA recombinant human corneal limbal epithelial cell line expressing a LC3-GFP fusion protein was challenged with normalized secretomes from wild-type and mutant S. marcescens derivatives. LC3-GFP fluorescence patterns were used to assess the ability of wild-type and mutant bacteria to influence autophagy. Purified prodigiosin was obtained from stationary phase bacteria and used to challenge ocular cells.ResultsMutations in the global regulators eepR and gumB genes highly reduced the ability of the bacteria to activate autophagy in corneal cells. This effect was further narrowed down to the secreted cytolysin ShlA and the biologically active pigment prodigiosin. Purified prodigiosin and ShlA from Escherichia coli further supported the role of these factors in activating autophagy in human corneal cells. Additional genetic data indicate a role for flagellin and type I pili, but not the nuclease, S-layer protein, or serratamolide biosurfactant in activation of autophagy.ConclusionsThis work identifies specific bacterial components that activate autophagy and give insight into potential host-pathogen interactions or compounds that can be used to therapeutically manipulate autophagy.
Highlights
The cellular process of autophagy is essential for maintaining the health of ocular tissue
Analysis of autophagy induced by keratitis isolates Bacterial stocks (Table 1) were stored at − 80 °C and single colonies were obtained on lysogeny broth (LB) agar
S. marcescens secretome induction of autophagy is inhibited by 3-methyladenine Our previous study showed that a subset of ocular bacterial pathogens induced autophagy in a corneal cell line, and that among the strongest induction was observed with S. marcescens [18]
Summary
The cellular process of autophagy is essential for maintaining the health of ocular tissue. Dysregulation of autophagy is associated with several ocular diseases including keratoconus and macular degeneration. It is known that autophagy can be used to respond to microbial infections and that certain microbes can exploit the autophagic process to their benefit. A genetic approach was used to identify surfaceassociated and secreted products generated by the opportunistic pathogen Serratia marcescens involved in activation of autophagy. A recent study measured activation of autophagy in mouse corneas following infection with the fungus Aspergillus fumigatus and positively correlated autophagy with the severity of infectious pathology [12]. Data from a study using the bacterium Pseudomonas aeruginosa, suggest that it benefits from activating autophagy as a means of escaping extracellular killing in macrophages [13]. In general, activation of autophagy is thought to protect cells from microbial infection [14, 15]. It is known that a few bacterial proteins such as TlpE from P. aeruginosa, bacterial macrolide, rapamycin, TLRligands, and proinflammatory cytokines can activate autophagy [15,16,17], but knowledge of the scope of infectious components that activate autophagy is limited [15]
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