title: Role of the extended alpha4 domain of Staphylococcus aureus gyrase A protein in determining low sensitivity to quinolones.
abstract: Fluoroquinolones target two bacterial type II topoisomerases, DNA gyrase and topoisomerase IV. Acquired resistance to quinolones occurs stepwise, with the first mutation occurring in the more sensitive target enzyme. To limit the emergence of resistance, quinolones should ideally possess dual activities against the two enzymes. For reasons that are as yet unclear, Staphylococcus aureus gyrase is less sensitive to quinolones than topoisomerase IV, counter to its greater sensitivity in Escherichia coli, thereby limiting the use of quinolones for the treatment of staphylococcal infections. Mutations in the alpha4-helix domain of the GyrA subunit of gyrase are important in determining quinolone resistance. We replaced an extended region encompassing the alpha4 domain in the E. coli GyrA protein with its homolog in S. aureus and tested for its ability to complement a thermosensitive gyrase and its catalytic and noncatalytic properties. Purified gyrase reconstituted with chimeric GyrA was more resistant to ciprofloxacin than wild-type gyrase at both inhibition of catalytic activity and stimulation of cleavage complexes, and this difference was more apparent in the presence of K+-glutamate. The chimeric GyrA subunit was able to complement thermosensitive gyrase, similar to wild-type GyrA. Without supplemental K+-glutamate the MICs of ciprofloxacin for thermosensitive E. coli complemented with chimeric DNA gyrase were equal to those for E. coli complemented with wild-type gyrase but were twofold higher in the presence of K+-glutamate. Our findings suggest that the extended alpha4 domain of S. aureus GyrA is responsible, at least in part, for the increased resistance of S. aureus gyrase to quinolones and that this effect is modulated by K+-glutamate.
authors.affiliation: Division of Infectious Diseases, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114-2696, USA.
authors.forename: Jacob
authors.initials: J
authors.lastname: Strahilevitz
authors.forename: Ari
authors.initials: A
authors.lastname: Robicsek
authors.forename: David C
authors.initials: DC
authors.lastname: Hooper
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology*; D001483:Base Sequence; D002939:Ciprofloxacin / Q000494:pharmacology; D027081:DNA Gyrase / Q000737:chemistry* / Q000302:isolation & purification; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology; D024841:Fluoroquinolones / Q000494:pharmacology*; D018698:Glutamic Acid / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000201:enzymology; D059005:Topoisomerase II Inhibitors
chemical_list: D000890:Anti-Infective Agents; D024841:Fluoroquinolones; D059005:Topoisomerase II Inhibitors; D018698:Glutamic Acid; D002939:Ciprofloxacin; D027081:DNA Gyrase