Response of Staphylococcus aureus to subinhibitory concentrations of a sequence-selective, DNA minor groove cross-linking pyrrolobenzodiazepine dimer.
OBJECTIVE
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.
Doyle
Marie
M
School of Pharmacy, University of London, London, UK.
Feuerbaum
Eva-Anne
EA
Fox
Keith R
KR
Hinds
Jason
J
Thurston
David E
DE
Taylor
Peter W
PW
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000096:biosynthesis*; D001483:Base Sequence; D001569:Benzodiazepines / Q000494:pharmacology*; D001665:Binding Sites; D002473:Cell Wall / Q000378:metabolism; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D006801:Humans; D050296:Microbial Viability / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D020411:Oligonucleotide Array Sequence Analysis; D039002:Prophages / Q000187:drug effects; D020543:Proteome / Q000032:analysis; D011758:Pyrroles / Q000494:pharmacology*; D013211:Staphylococcus aureus / Q000187:drug effects*; D014775:Virus Activation / Q000187:drug effects
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D020543:Proteome; D011758:Pyrroles; C438462:pyrrolo(2,1-c)(1,4)benzodiazepine; D001569:Benzodiazepines