Cleavable-complex formation by wild-type and quinolone-resistant Streptococcus pneumoniae type II topoisomerases mediated by gemifloxacin and other fluoroquinolones

Abstract

Gemifloxacin is a recently developed fluoroquinolone with potent activity against Streptococcus pneumoniae. We show that the drug is more active than moxifloxacin, gatifloxacin, levofloxacin, and ciprofloxacin against S. pneumoniae strain 7785 (MICs, 0.03 to 0.06 microg/ml versus 0.25, 0.25, 1, and 1 to 2 microg/ml, respectively) and against isogenic quinolone-resistant gyrA-parC mutants (MICs, 0.5 to 1 microg/ml versus 2 to 4, 2 to 4, 16 to 32, and 64 microg/ml, respectively). Gemifloxacin was also the most potent agent against purified S. pneumoniae DNA gyrase and topoisomerase IV in both catalytic inhibition and DNA cleavage assays. The drug concentrations that inhibited DNA supercoiling or DNA decatenation by 50% (IC(50)s) were 5 to 10 and 2.5 to 5.0 microM, respectively. Ciprofloxacin and levofloxacin were some four- to eightfold less active against either enzyme; moxifloxacin and gatifloxacin showed intermediate activities. In assays of drug-mediated DNA cleavage by gyrase and topoisomerase IV, the same order of potency was seen: gemifloxacin > moxifloxacin > gatifloxacin > levofloxacin approximately ciprofloxacin. For gemifloxacin, the drug concentrations that caused 25% linearization of the input DNA by gyrase and topoisomerase IV were 2.5 and 0.1 to 0.3 microM, respectively; these values were 4-fold and 8- to 25-fold lower than those for moxifloxacin, respectively. Each drug induced DNA cleavage by gyrase at the same spectrum of sites but with different patterns of intensity. Finally, for enzymes reconstituted with quinolone-resistant GyrA S81F or ParC S79F subunits, although cleavable-complex formation was reduced by at least 8- to 16-fold for all the quinolones tested, gemifloxacin was the most effective; e.g., it was 4- to 16-fold more active than the other drugs against toposiomerase IV with the ParC S79F mutation. It appears that the greater potency of gemifloxacin against both wild-type and quinolone-resistant S. pneumoniae strains arises from enhanced stabilization of gyrase and topoisomerase IV complexes on DNA.

FIG. 1.

Drug-promoted DNA breakage by wild-type S. pneumoniae DNA gyrase (A) and the mutant enzyme reconstituted with GyrA S81F (B). Supercoiled plasmid pBR322 DNA (0.4 μg) was incubated with recombinant S. pneumoniae GyrB (1.7 μg) and either wild-type GyrA (A) or GyrA S81F (B) (in each case, 0.45 μg) in the absence or presence of gemifloxacin (GEMI), moxifloxacin (MOXI), gatifloxacin (GATI), or levofloxacin (LEVO) at the indicated concentrations. Following treatment with SDS and proteinase K, the DNA products were examined in 1% agarose gels. Lanes a and b, supercoiled and linear pBR322, respectively; S, L, and N, supercoiled, linear, and nicked plasmid pBR322 bands, respectively.

FIG. 2.

Quinolone-mediated DNA cleavage by S. pneumoniae topoisomerase IV (A) and by its mutant ParC S79F complex (B). Plasmid pBR322 (0.4 μg) was incubated with ParE (1.7 μg) and either wild-type ParC (A) or ParC S79F (B) (0.45 μg). Samples were processed and analyzed as described in the Fig. 1 legend. The abbreviations and lane contents are as described in the Fig. 1 legend.

FIG. 3.

Site specificity of quinolone-promoted DNA cleavage by S. pneumoniae DNA gyrase. EcoRI-cut pBR322 DNA (0.4 μg) was incubated with GyrA (0.45 μg) and GyrB (1.7 μg) in the absence or presence of quinolones at the indicated concentrations (in micromolar). After treatment with SDS and proteinase K, DNA samples were examined by electrophoresis in 1% agarose. Lanes M, b, and a; DNA size markers, linear pBR322, and supercoiled pBR322, respectively. GEMI, gemifloxacin; GATI, gatifloxacin; MOXI, moxifloxacin; CIP, ciprofloxacin.