Activities of fluoroquinolones against Streptococcus pneumoniae type II topoisomerases purified as recombinant proteins

Abstract

Streptococcus pneumoniae topoisomerase IV and DNA gyrase have been purified from a fluoroquinolone-susceptible Streptococcus pneumoniae strain, from first-step mutants showing low-level resistance to ciprofloxacin, sparfloxacin, levofloxacin, and ofloxacin, and from two clinical isolates showing intermediate- and high-level fluoroquinolone resistance by a gene cloning method that produces recombinant proteins from Escherichia coli. The concentrations of ciprofloxacin, sparfloxacin, levofloxacin, or ofloxacin required to inhibit wild-type topoisomerase IV were 8 to 16 times lower than those required to inhibit wild-type DNA gyrase. Furthermore, low-level resistance to these fluoroquinolones was entirely due to the reduced inhibitory activity of fluoroquinolones against topoisomerase IV. For all the laboratory strains, the 50% inhibitory concentration for topoisomerase IV directly correlated with the MIC. We therefore propose that with S. pneumoniae, ciprofloxacin, sparfloxacin, levofloxacin, and ofloxacin target topoisomerase IV in preference to DNA gyrase. Sitafloxacin, on the other hand, was found to be equipotent against either enzyme. This characteristic is unique for a fluoroquinolone. A reduction in the sensitivities of both topoisomerase IV and DNA gyrase are required, however, to achieve intermediate- or high-level fluoroquinolone resistance in S. pneumoniae.

FIG. 1

pMAL-c2 vector.

FIG. 2

SDS-PAGE analysis of ParC and ParE subunits from S. pneumoniae C3LN4. Proteins at various steps were electrophoresed in an SDS–10% polyacrylamide gel and silver stained. Lane 1, molecular marker (116-, 97-, 66-, and 45-kDa proteins); lane 2, affinity-purified MBP-ParC fusion protein; lane 3, affinity-purified MBP-ParE fusion protein; lane 4, factor Xa digest of MBP-ParC; lane 5, factor Xa digest of MBP-ParE; lane 6, affinity-purified ParC; lane 7, affinity-purified ParE.

FIG. 3

Inhibitory activity of ciprofloxacin on the decatenation reaction of topoisomerase IV from S. pneumoniae C3LN4. Lane 1, drug-free control; lanes 2 to 8, ciprofloxacin at 2, 4, 6, 8, 10, 12, and 14 μg per ml, respectively; kDNA, catenated kinetoplast DNA; monomer, decatenated monomer DNA.

FIG. 4

Inhibitory activity of ciprofloxacin on the supercoiling reaction of DNA gyrase from S. pneumoniae C3LN4. Lane 1, drug-free control; lanes 2 to 10, ciprofloxacin at 8, 16, 24, 32, 40, 48, 56, 64, and 72 μg per ml, respectively; Sc, supercoiled pBR322 DNA; Rel, relaxed pBR322 DNA.

FIG. 5

Correlation between topoisomerase IC₅₀ and MIC determined by using S. pneumoniae C3LN4 and its fluoroquinolone-resistant mutants. ●, topoisomerase IV (correlation = 0.97); ○, DNA gyrase (correlation = 0.57).