Biologically active pigment and ShlA cytolysin of Serratia marcescens induce autophagy in a human ocular surface cell line

Abstract

Background: 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. In this study, a genetic approach was used to identify surfaceassociated and secreted products generated by the opportunistic pathogen Serratia marcescens involved in activation of autophagy. Methods: A 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. Results: Mutations 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. Conclusions: This 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.

The roles of autophagy and mitophagy in corneal pathology: current knowledge and future perspectives

“…In addition, host response varied with respect to the bacterial strain, with S. marcescens exhibiting the strongest response (146). Another study by the same group showed that S. marcescens mutants that lack function of eepR and gumB genes, both of which regulate multiple heat-stable secondary metabolites, were unable to promote autophagy in HCLE cells (147). Since targets of eepR and gumB include genes involved in the synthesis of prodigiosin (pigD), the authors tested a strain of S. marcescens with a mutation in pigD.…”

Bacterial pore-forming toxins

“…In addition to causing cellular lysis [154], ShlA can promote blebbing and autophagy of ocular epithelial cells [155,156]. In non-phagocytic epithelial cells, ShlA mediates extracellular induction of autophagy, likely to promote bacterial intracellular survival [157].…”

IgaA protein, GumB, has a global impact on the transcriptome and surface proteome of Serratia marcescens

“…Like IgaA in S. enterica, the Escherichia coli protein, YrfF, is an essential gene (8,9). In Serratia sp ATCC39006 the IgaA protein regulates CRISPR-based immunity to plasmid and phage DNA (10), and in S. marcescens the IgaA family protein GumB regulates multiple phenotypes including microbial pathogenesis, biosynthesis of antimicrobial secondary metabolites, capsular polysaccharide, and the shlA cytolysin (11)(12)(13)(14).…”