Abstract
Serratia marcescens is a common contaminant of contact lens cases and lenses. Hemolytic factors of S. marcescens contribute to the virulence of this opportunistic bacterial pathogen. We took advantage of an observed hyper-hemolytic phenotype of crp mutants to investigate mechanisms of hemolysis. A genetic screen revealed that swrW is necessary for the hyper-hemolysis phenotype of crp mutants. The swrW gene is required for biosynthesis of the biosurfactant serratamolide, previously shown to be a broad-spectrum antibiotic and to contribute to swarming motility. Multicopy expression of swrW or mutation of the hexS transcription factor gene, a known inhibitor of swrW expression, led to an increase in hemolysis. Surfactant zones and expression from an swrW-transcriptional reporter were elevated in a crp mutant compared to the wild type. Purified serratamolide was hemolytic to sheep and murine red blood cells and cytotoxic to human airway and corneal limbal epithelial cells in vitro. The swrW gene was found in the majority of contact lens isolates tested. Genetic and biochemical analysis implicate the biosurfactant serratamolide as a hemolysin. This novel hemolysin may contribute to irritation and infections associated with contact lens use.
Highlights
Serratia marcescens is a nosocomial pathogen [1,2,3], a common contaminant of contact lens cases and is associated with a number of ocular conditions including keratitis, conjunctivitis, and contact lens acute red eye (CLARE) [4]
The cyaA and crp mutant strains exhibited dramatically increased zones of hemolysis on blood agar plates compared to the parental, wild-type (WT) strain CMS376 [18], that produces small zones of hemolysis after several days of incubation at 30uC (Fig 1A)
We focused on crp mutants, for simplicity
Summary
Serratia marcescens is a nosocomial pathogen [1,2,3], a common contaminant of contact lens cases and is associated with a number of ocular conditions including keratitis, conjunctivitis, and contact lens acute red eye (CLARE) [4]. ShlA is a key virulence factor and a pore-forming hemolysin [10,11], whereas PhlA is phospholipase, one of whose cleavage products is lysophospholipid, a surfactact that can lyse red blood cells [12]. Regulators of the shlA hemolysin gene include the FlhDC flagellar biosynthesis regulator and RssAB, a two component transcriptional regulator [11]. RssAB is a negative regulator of flhDC expression, whereas FlhDC is a positive regulator of the shlA hemolysin operon, shlBA [11]. It would be predicted that crp mutants should have reduced hemolytic activity through a reduction of both shlA and phlA expression. We observed that crp mutants exhibited increased levels of hemolytic activity, suggesting another mechanism of hemolysis. We used a genetic approach to gain insight into the mechanism of hemolysis exhibited by crp mutants. Genetic and biochemical analysis in this study support the model that the biosurfactant serratamolide is a hemolysin
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