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. 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.