Inhibitory activities of quinolones against DNA gyrase and topoisomerase IV purified from Staphylococcus aureus

Abstract

In order to clarify the mechanism of action of quinolones against Staphylococcus aureus, GrlA and GrlB proteins of topoisomerase IV encoded by genes with or without mutations were purified separately as fusion proteins with maltose-binding protein in Escherichia coli. The reconstituted enzymes showed ATP-dependent decatenation and relaxing activities but had no supercoiling activity. The inhibitory effects of quinolones on the decatenation activity of topoisomerase IV were determined by quantitative electrophoresis with kinetoplast DNA as a substrate. The 50% inhibitory concentrations (IC50s) of levofloxacin, DR-3354, DU-6859a, DV-7751a, ciprofloxacin, sparfloxacin, and tosufloxacin against topoisomerase IV of S. aureus FDA 209-P were 2.3, 97, 0.45, 1.5, 2.5, 7.4, and 1.8 microg/ml, respectively, and were correlated well with their MICs. The IC50s of these drugs were from 2 to 20 times lower than those for the DNA gyrase. These results support genetic evidence that the primary target of new quinolones is topoisomerase IV in quinolone-susceptible strains of S. aureus. Three altered proteins of topoisomerase IV containing Ser-->Phe changes at codon 80 or Glu-->Lys changes at codon 84 of grlA, or both, were also purified. The inhibitory activities of quinolones against the topoisomerase IV which contained a single amino acid change were from 8 to 95 times weaker than those against the nonaltered enzyme. These results suggest that the mutations in the corresponding genes confer quinolone resistance.

Curing bacteria of antibiotic resistance: reverse antibiotics, a novel class of antibiotics in nature

“…Recombinant plasmids pMALc-2-SAGA and pMALc-2-SAGB carrying wild-type gyrA and gyrB genes, respectively, were constructed by cloning the PCR-amplified type II topoisomerase genes of FDA 209P into the plasmid pMAL-c2 expression vector (New England Biolabs, Beverly, MA) [15]. Plasmid pMALc-2-SAGAmut84 carrying gyrA(Ser84Leu) was constructed from pMALc-2-SAGA using a site-directed mutagenesis system Mutan-K (TaKaRa Bio Inc., Otsu, Japan).…”

Mechanism of quinolone resistance in Staphylococcus aureus

“…15,17 The activity of each enzyme was measured electrophoretically. The reaction mixture for topoisomerase IV, containing 78 mM Tris-HCl (pH7.5), 120 mM KCl, 10 mM MgCl 2 , 100 µg/ml bovine serum albumin, 2 mM dithiothreitol (DTT), 1 mM ATP, 0.5 µg of Factor Xa, and appropriate amounts of GrlA and GrlB proteins (to give 1 U of decatenated activity), was incubated at 37°C for 1 h, and then 0.4 µg of kinetoplast DNA (kDNA; Nippon Gene, Toyama, Japan) was added.…”

“…We found a mutation in the grlB gene (GAT(Asp)-432 AE AAT(Asn)) in a clinical isolate highly resistant to quinolones that also had GrlA80 and GyrA84 alterations. 15 A novel mutation (Asp-432 AE Asn) in grlB is considered to correspond to the Asp-437 AE Asn of gyrB. Furthermore, Fournier and Hooper 12 reported a GrlB mutation at codon 470 (Asn AE Asp), and the strain containing the altered GrlB became slightly resistant to quinolones (two to eight-fold) and susceptible to novobiocin compared with the parent strain.…”

“…15,17 Also, we discuss the relationship between the antibacterial activity and the enzyme-inhibitory activity of quinolones.…”

Bacterial targets and antibiotics: genome-based drug discovery

“…A further consideration when examining the response of the cell to an antibiotic is the specificity of the antibiotic. For example, quinolones, inhibitors of DNA gyrase, also inhibit topoisomerase IV [28] and at higher concentrations, translation [29]. The scarcity of information concerning the pyrimidine pathway is indicative of the limitations in the sensitivity of the methodology.…”