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Robert M Q Shanks, Nicholas A Stella, Kimberly Brothers, Eric G Romanowski; Cytotoxicity to Corneal Cells in vitro by Serratia marcescens is Mediated by Transcription Factors FlhC and FlhD and Requires the ShlA Hemolysin.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6269.
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The bacterium Serratia marcescens is a common contaminant of contact lenses and can cause vision threatening ocular infections. A mutation in a non-cytotoxic S. marcescens isolate was previously reported to increase expression of the flhDC operon by ~10-fold. We observed that this mutant strain, designated scrp-31r, was highly cytotoxic to human corneal limbal epithelial cells (HCLE). The FlhC and FlhD proteins form a heterodimeric transcription factor that regulates flagella, phospholipase A (PhlA), and a hemolysin (ShlA). The purpose of this study was to determine the mechanism of scrp-31r associated cytotoxicity to ocular cells.
S. marcescens strain PIC3611 (wild type) and scrp-31r were used in this study. The flhD, phlA, and shlA genes were mutated by plasmid integration. The flhDC operon was cloned under control of an arabinose-inducible promoter. Strain PIC3611, scrp-31r and mutant derivatives were applied to HCLE cells at a multiplicity of infection (MOI) of 20 and 200, incubated for 2-4 h, the bacteria were removed, and HCLE viability was assessed with fluorescent viability dye resazurin (Presto Blue).
To verify that the elevated toxicity of scrp-31r was due to flhDC expression, two approaches were taken: 1) the flhD gene was mutated in scrp-31r, and 2) the flhDC operon was expressed from a multicopy plasmid in the wild type. Mutation of flhD attenuated scrp-31r cytotoxicity, and multicopy expression of flhDC increased cytotoxicity. These results support that flhDC mediates cytotoxicity to ocular cells. To determine the mechanism of elevated cytotoxicity, candidate ocular virulence factors phlA and shlA were mutated in scrp-31r. The shlA gene, but not the phlA gene was necessary for the scrp-31r cytotoxicity phenotype.
This genetic study provides novel evidence that the S. marcescens hemolysin, ShlA, is an ocular virulence factor.
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