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Robert M. Shanks, Nicholas A. Stella, Roni M. Lahr, Shaoru Wang, Tara Veverka, Regis P. Kowalski, Xinyu Liu; Serratamolide as a novel hemolytic factor produced by Serratia marcescens. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6127.
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Serratia marcescens is a bacterium that commonly causes vision-threatening keratitis and hospital acquired infections and is a common contaminant of contact lens cases. Bacterial hemolysins are important virulence factors. The goal of this study was to identify a hemolytic agent produced by keratitis isolates and laboratory strains of S. marcescens.
Hemolysis was measured using TSA plates with 5% v/v sheep erythrocytes or fresh blood from C57BL/6 mice. Random mutagenesis of S. marcescens was performed using a mariner-based transposon. Mutants defective in extracellular hemolysin production were isolated on blood agar plates. Transposon insertion sites were determined by marker rescue and sequencing. Complementation analysis and over expression was performed with arabinose-inducible genetic constructs. Serratamolide was extracted with ethyl acetate and purified by preparative HPLC and verified as pure with high-resolution mass spectroscopy (HR-MS) and 1H NMR analysis. Cytotoxicity to human airway epithelial and human corneal limbal epthithelial (HCLE) cells was performed using Alamar Blue viability dye. The presence of the swrW gene in human keratitis and contact lens isolates was determined using PCR.
Random mutations in the swrW gene conferred a hemolysis defective phenotype in S. marcescens. The swrW mutant defect was complemented by the wild-type swrW gene on a plasmid. The SwrW protein is involved in production of the cyclic lipopeptide serratamolide. Purified serratamolide was hemolytic to rabbit and mouse erythrocytes, and cytotoxic to human airway and ocular cells in vitro. The swrW gene was found in the majority of contact-lens associated keratitis isolates.
Serratamolide is a novel hemolysin produced by S. marcescens ocular isolates and may contribute to the ability of contact lens associated bacteria to cause infections.
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