JAC Advance Access originally published online on September 10, 2009
Journal of Antimicrobial Chemotherapy 2009 64(5):949-959; doi:10.1093/jac/dkp325
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Original research |
Response of Staphylococcus aureus to subinhibitory concentrations of a sequence-selective, DNA minor groove cross-linking pyrrolobenzodiazepine dimer
1 School of Pharmacy, University of London, London WC1N 1AX, UK 2 School of Biological Sciences, University of Southampton, Southampton SO16 7PX, UK 3 Division of Cellular and Molecular Medicine, St George's, University of London, London SW17 0RE, UK
Received 29 April 2009; returned 22 June 2009; revised 6 August 2009; accepted 10 August 2009
* Corresponding author. Tel/Fax: +44-20-7753-5867; E-mail: peter.taylor{at}pharmacy.ac.uk
Objectives: ELB-21 is a pyrrolo[2,1-c][1,4]benzodiazepine dimer with potent antistaphylococcal activity; it binds covalently to guanine residues on opposing strands of duplex DNA, interfering with regulatory proteins and transcription elongation in a sequence-selective manner. Transcriptional and proteomic alterations induced by exposure of Staphylococcus aureus clinical isolate EMRSA-16 to ELB-21 were determined in order to define more precisely the bactericidal mechanism of the drug.
Methods: DNase I footprinting was used to identify high-affinity DNA binding sites. Microarrays and gel electrophoresis were used to assess the ELB-21-induced phenotype.
Results: High-affinity interstrand binding sites in which guanine residues were separated by 4 bp, and also some intrastrand cross-linking sites of variable length were identified. Exposure of EMRSA-16 to 0.015 mg/L ELB-21 elicited a 2-fold or greater up-regulation of 168 genes in logarithmic phase and 181 genes in stationary phase; the majority of genes affected were associated with resident prophages 
Sa2 and Sa3, pathogenicity island SaPI4 and DNA damage repair. ELB-21 induced a marked increase in the number of viable phage particles in culture supernatants. The expression of only a limited number of genes showed a >50% reduction. Sixteen extracellular and four intracellular proteins were differentially expressed during logarithmic and stationary phases, including RecA, proteins associated with staphylococcal pathogenesis (IsaA, CspA), cell division and wall synthesis.
Conclusions: ELB-21 kills S. aureus by forming multiple interstrand and intrastrand DNA cross-links, resulting in induction of the DNA damage response, derepression of resident prophages and modulation of a limited number of genes involved with cell wall synthesis.
Keywords: S. aureus , DNA cross-linking , DNA damage response , sequence-selective DNA binding