INHIBITORY ACTIVITIES OF QUINOLONES AGAINST DNA GYRASE AND TOPOISOMERASE-IV PURIFIED FROM STAPHYLOCOCCUS-AUREUS

In order to clarify the mechanism of action of quinolones against Stap hylococcus 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 reconst ituted enzymes showed ATP-dependent decatenation and relaxing activiti es but had no supercoiling activity. The inhibitory effects of quinolo nes on the decatenation activity of topoisomerase IV were determined b y quantitative electrophoresis with kinetoplast DNA as a substrate. Th e 50% inhibitory concentrations (IC(50)s) of levofloxacin, DR-3354, DU -6859a, DV-7751a, ciprofloxacin, sparfloxacin, and tosufloxacin agains t topoisomerase IV of S. aureus FDA 209-P were 2.3, 97, 0.45, 1.5, 2.5 , 7.4, and 1.8 mu g/ml, respectively, and were correlated well with th eir MICs. The IC(50)s of these drugs were from 2 to 20 times lower tha n those for the DNA gyrase. These results support genetic evidence tha t the primary target of new quinolones is topoisomerase IV in quinolon e-susceptible strains of S. aureus. Three altered proteins of topoisom erase IV containing Ser-->Phe changes at codon 80 or Glu-->Lys changes at codon 84 of grlA, or both, were also purified. The inhibitory acti vities of quinolones against the topoisomerase IV which contained a si ngle amino acid change were from 8 to 95 times weaker than those again st the nonaltered enzyme. These results suggest that the mutations in the corresponding genes confer quinolone resistance.