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.

Figure 1. Hyper-hemolysis phenotype of crp mutants and genetic analysis. A.

S. marcescens with mutations in cyaA or crp exhibit a hyper-hemolysis phenotype compared to the isogenic WT strain on TSA agar with 5% sheep red blood cells at 48 hours. B. Complementation of cyaA and crp hyper-hemolysis phenotypes with wild-type copies of the respective genes on multicopy plasmids (pcyaA = pMQ157, pcrp = pMQ166). Asterisks indicate significantly larger zones (72h) than the WT (p<0.05, ANOVA with Tukey’s post-test). C. Double mutant hemolysis phenotypes show that expected hemolysin genes, phlA and shlA are not required for the crp mutant phenotype. The swrW gene is required, and a control for insertion mutagenesis (fimC) is included.

Figure 2. Isolation of swrW and its role in surfactant production and hemolysis. A.

Sample genetic screen plate shows crp mutants with random transposon insertions. The white arrow indicates a colony deficient in secreted hemolysis production with a transposon insertion that mapped to the swrW gene. This image is illuminated from the back, so that the gold surface coloration is not apparent. B. Surface coloration of crp swrW double mutants is metallic gold compared to the red-orange color of the crp mutant. C. Surfactant zones (mm) measured from the colony to the maximum extent of the surfactant zone (n≥4 per genotype). Asterisk represents a statistically significant increase in surfactant zone compared to the WT (p<0.05) by ANOVA with Tukey’s post-test. D. Mutation of swrW reduced or eliminated the ability of laboratory strain Nima and three of five clinical keratitis isolates to make zones of hemolysis on blood agar plates. Representative images from reproducible experiments are shown.

Figure 3. Genetic evidence that serratamolide mediates hemolysis. A.

Hemolysis and swarming by a mutant known to have elevated serratamolide production (hexS) is increased, and these phenotypes require SwrW. B. Elevated expression of a swrW promoter reporter in the crp mutant. Top, expression measured using a plasmid based-tdtomato reporter construct at t = 20 hrs. Asterisk indicates statistical significance (p<0.05) by the Student’s T-test. A representative experiment is shown (n = 4). Error bars indicate one standard deviation. Bottom, semi-quantitative RT-PCR analysis of RNA from WT and Δcrp mutant strains measured relative expression of swrW and internal standard 16S RNA from stationary phase cultures (OD₆₀₀ = ∼3.5). C. Arabinose-inducible expression of the swrW gene in an swrW transposon mutant strain restores hemolysis. D. Swarming motility defect of the swrW mutant is restored by induced expression of the swrW gene.

Figure 4. Serratamolide purification and verification of biological activity. A.

Structure of serratamolide. B. HPLC trace of spent supernatants from a swrW mutant with either an empty vector (swrW+vector) or a swrW expression plasmid (swrW+pswrW). The expected peak for serratamolide is indicated by an arrow. C. Swarming motility of an swrW mutant treated with DMSO or purified serratamolide. This shows that the purified compound restores swarming motility as expected.

Figure 5. Serratamolide is hemolytic to erythrocytes and cytotoxic to epithelial cells in vitro. A.

Hemolysis of DMSO and serratamolide (1 mg/ml) to sheep red blood cells. Wells were cut into a TSA+sheep blood agar plate, and DMSO or serratamolide was added to the well and incubated for 24 hours. White arrow indicates zone of hemolysis around serratamolide treated well. B. Hemolysis of murine red blood cells in solution by serratamolide (µg/ml) incubated for 10 seconds. Distilled water was used as a complete lysis control (Lysis). A representative experiment is shown. Error bars indicate one standard deviation. C. Cytotoxicity to immortalized human airway (A549) and corneal (HCLE) epithelial cells was measured using alamar blue fluorescence that provided a positive output for viability of cells. The average of eight independent replicates is shown for each cell line from two separate experiments. Error bars indicate one standard deviation.