Serratamolide is a Hemolytic Factor Produced by Serratia marcescens

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.

Serratia marcescens Cyclic AMP Receptor Protein Controls Transcription of EepR, a Novel Regulator of Antimicrobial Secondary Metabolites

“…Previous studies demonstrated cAMP-CRP regulation of flagellum and secondary metabolite production by S. marcescens strain PIC3611 (18,21,23). Direct binding of cAMP-CRP to the promoter of the flagellar master regulator operon, flhDC, was observed, but a positive interaction between cAMP-CRP and promoters of the genes required for biosynthesis of the secondary metabolites prodigiosin (pigA-N) or serratamolide (swrW) was not (18).…”

“…When this construct integrates, the promoter region is duplicated such that the lacZ gene becomes a reporter for eepR expression, and the native eepR gene comes under the control of the regulatory elements in the 263 bp of DNA upstream of the ORF, maintaining EepR, which may be necessary for eepR expression. For pigA and swrW promoter analysis, transcriptional lacZ fusions to internal fragments of the pigB and swrW genes (plasmids pMQ268 and pMQ223) were targeted to the chromosome by homologous recombination, verified by PCR, and used as previously described (18,21,23).…”

“…Zones of biosurfactant around colonies on swarming agar plates were measured 18 to 20 h after inoculation of the bacteria onto the surface of the agar as previously reported (23,25). Quantitative analysis of serratamolide from culture supernatants by high-performance liquid chromatography-mass spectrometry (HPLC-MS) was carried out as previously described (25).…”

“…Mutation of crp in CMS376 results in higher levels of hemolysis than that of the isogenic parental strain due to increased production of the biosurfactant serratamolide (23), also known as serrawettin W1 (41). Since EepR/S appears to function with CRP in a regulatory pathway that controls prodigiosin production and EepR and EepS are required for the hemolysis phenotype of crp mutants (Fig.…”

“…Whereas recombinant S. marcescens CRP bound directly to the promoter of flhDC to regulate flagellum production (18), interactions were not detected between recombinant CRP and promoters of the prodigiosin biosynthetic operon (pigA-N) or the swrW gene, required for serratamolide production (18,21,23). Based on these observations, we hypothesized an intermediate regulatory protein, regulated by cAMP-CRP, that in turn regulates expression of pigA-N and swrW.…”