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Robert M Q Shanks, Kimberly Brothers, Nicholas A Stella, Kira L Lathrop, Eric G Romanowski, Regis P Kowalski; Inside and out: the roles of a stress response-cytolysin in corneal epithelial cell damage and intrastromal inflammation. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6412.
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Keratitis is a vision threatening condition, frequently caused by bacterial infections. Bacteria must penetrate the epithelial layer to initiate infection; therefore, microbial mechanisms to breach this barrier are key to establish corneal infection. We used a genetic approach to investigate the mechanisms by which keratitis isolates of Proteus mirabilis and Serratia marcescens damage the epithelium and alter corneal epithelial wound closure. The importance of this system in pathogenesis was evaluated using a rabbit keratitis model.
Mutations were identified in the genome of S. marcescens keratitis isolates that rendered the bacteria unable to induce cellular blebbing and death of human corneal limbal epithelial cells (HCLE). Video microscopy and confocal microscopy of bright field and calcein AM-stained cells were performed. Cytotoxicity was additionally measured using Presto Blue. The corneal stromas of New Zealand White rabbits were inoculated with 3000 CFU of S. marcescens and isogenic mutants, and corneas were evaluated at 48 hours for clinical inflammatory signs, bacterial proliferation, and pro-inflammatory cytokine production.
The major epithelium damaging factor produced by S. marcescens and P. mirabilis were ShlA-family cytolysins. Cellular death was consistent with necroptosis and could be prevented using antioxidants. We determined that expression of the shlA gene was controlled by a stress response transcriptional regulation system. This stress response system includes the protein GumB. The gumB mutant was unable to kill epithelial cells in vitro. The gumB mutant could proliferate within the stroma of rabbits, but the mutant was severely defective in instigating a strong inflammatory response.
Our study identifies ShlA-family cytolysins as key factors for the Enterobacteriaceae family of bacteria to damage the corneal epithelium, and that the cytolysins are regulated by a stress response system necessary for the bacterium to induce corneal inflammation. Results from this study suggest approaches to reduce pathogenesis in bacterial keratitis.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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