The unusual mode of action of the polyketide glycoside antibiotic cervimycin C [II]

Cervimycins A-D are bis-glycosylated polyketide antibiotics produced by Streptomyces tendae HKI 0179 with bactericidal activity against Gram-positive bacteria. In this study, cervimycin C (CmC) treatment caused a spaghetti-like phenotype in Bacillus subtilis 168, with elongated curved cells, which stayed joined after cell division, and exhibited a chromosome segregation defect, resulting in ghost cells without DNA. Electron microscopy of cervimycin treated S. aureus (3 x MIC) revealed swollen cells, misshapen septa, cell wall thickening, and a rough cell wall surface. Incorporation tests in B. subtilis indicated an effect on DNA biosynthesis at high cervimycin concentrations. Indeed, artificial down-regulation of the DNA gyrase subunit B gene (gyrB) increased the activity of cervimycin in agar diffusion tests, and, in high concentrations (starting at 62.5 x MIC), the antibiotic inhibited S. aureus DNA gyrase supercoiling activity in vitro. To obtain a more global view on the mode of action of CmC, transcriptomics and proteomics of cervimycin treated versus untreated S. aureus cells were performed. Interestingly, 3 x MIC of cervimycin did not induce the SOS response, which would indicate disturbance of the DNA gyrase activity in vivo. Instead, cervimycin induced the expression of the CtsR/HrcA heat shock operon and the expression of autolysins, exhibiting similarity to the ribosome-targeting gentamicin. In summary, we identified the DNA gyrase as a target, but at low concentrations electron microscopy and omics data revealed a more complex mode of action of cervimycin, that comprised induction of the heat shock response, indicating protein stress in the cell. A synergistic effect was seen when sodium chloride (NaCl) was added during cervimycin MIC testing of S. aureus SG511. Thus, transcriptomics of S. aureus cells under osmotic stress (1 M NaCl) were performed. The stress response was compared to the cervimycin stress response to identify factors which might contribute to the synergistic antibacterial effect of the two molecules. Overall design: Comparative gene expression profiling analysis of RNA-seq data for S. aureus SG511 cells grown in TSB supplemented with 1 M NaCl versus cells grown in TSB without NaCl.