Drug interactions with Bacillus anthracis topoisomerase IV: biochemical basis for quinolone action and resistance

Abstract

Bacillus anthracis, the causative agent of anthrax, is considered a serious threat as a bioweapon. The drugs most commonly used to treat anthrax are quinolones, which act by increasing the levels of DNA cleavage mediated by topoisomerase IV and gyrase. Quinolone resistance most often is associated with specific serine mutations in these enzymes. Therefore, to determine the basis for quinolone action and resistance, we characterized wild-type B. anthracis topoisomerase IV, the GrlA(S81F) and GrlA(S81Y) quinolone-resistant mutants, and the effects of quinolones and a related quinazolinedione on these enzymes. Ser81 is believed to anchor a water-Mg(2+) bridge that coordinates quinolones to the enzyme through the C3/C4 keto acid. Consistent with this hypothesized bridge, ciprofloxacin required increased Mg(2+) concentrations to support DNA cleavage by GrlA(S81F) topoisomerase IV. The three enzymes displayed similar catalytic activities in the absence of drugs. However, the resistance mutations decreased the affinity of topoisomerase IV for ciprofloxacin and other quinolones, diminished quinolone-induced inhibition of DNA religation, and reduced the stability of the enzyme-quinolone-DNA ternary complex. Wild-type DNA cleavage levels were generated by mutant enzymes at high quinolone concentrations, suggesting that increased drug potency could overcome resistance. 8-Methyl-quinazoline-2,4-dione, which lacks the quinolone keto acid (and presumably does not require the water-Mg(2+) bridge to mediate protein interactions), was more potent than quinolones against wild-type topoisomerase IV and was equally efficacious. Moreover, it maintained high potency and efficacy against the mutant enzymes, effectively inhibited DNA religation, and formed stable ternary complexes. Our findings provide an underlying biochemical basis for the ability of quinazolinediones to overcome clinically relevant quinolone resistance mutations in bacterial type II topoisomerases.

Figure 1

DNA relaxation activities of wild-type, GrlA^S81F, and GrlA^S81Y B. anthracis topoisomerase IV. The ability of wild-type (WT, black squares), GrlA^S81F (S81F, red circles), and GrlA^S81Y (S81Y, blue triangles) topoisomerase IV to relax negatively supercoiled pBR322 plasmid DNA is shown. Error bars represent the standard deviation of three or more independent experiments. The inset shows an agarose gel of a typical 30 min relaxation time course catalyzed by wild-type topoisomerase IV. The positions of negatively supercoiled (replicative form I, FI) and nicked (replicative form II, FII) plasmids are indicated.

Figure 2

DNA decatenation activities of wild-type, GrlA^S81F, and GrlA^S81Y topoisomerase IV. The ability of wild-type (WT, black squares), GrlA^S81F (S81F, red circles), and GrlA^S81Y (S81Y, blue triangles) topoisomerase IV to decatenate kinetoplast DNA is shown. Error bars represent the standard deviation of three or more independent experiments. The inset shows an agarose gel of a typical 30 min decatenation time course catalyzed by wild-type topoisomerase IV. The positions of kDNA (K) and monomeric free circles (FC) resulting from decatenation of kDNA are indicated.

Figure 3

DNA cleavage activities of wild-type, GrlA^S81F, and GrlA^S81Y topoisomerase IV. The ability of wild-type (WT, black squares), GrlA^S81F (S81F, red circles), and GrlA^S81Y (S81Y, blue triangles) topoisomerase IV to cleave negatively supercoiled pBR322 plasmid DNA is shown. Assays were carried out in the presence of 1 mM CaCl₂. Error bars represent the standard deviation of three or more independent experiments. The inset shows an agarose gel of a typical DNA cleavage assay mediated by wild-type topoisomerase IV in the absence of divalent metal ion (None) or in the presence of Mg²⁺, Ca²⁺, or Mn²⁺. The positions of negatively supercoiled (replicative form I, FI), nicked (replicative form II, FII), and linear (replicative form III, FIII) plasmids are indicated.

Figure 4

Structures of the quinolones and quinazolinedione utilized in this study. Ciprofloxacin, levofloxacin, moxifloxacin, norfloxacin, and sparfloxacin are clinically-relevant fluoroquinolones (10). CP-115,953 is an experimental fluoroquinolone that displays high activity against both prokaryotic and eukaryotic type II enzymes (42, 56, 57). 8-Methyl-quinazoline-2,4-dione is a quinolone-related compound that previously has been shown to overcome quinolone-resistance mutations, including the GyrA^S83W mutation, in E. coli gyrase (60).

Figure 5

Effects of quinolones and 8-methyl-quinazoline-2,4-dione on the DNA cleavage activities of wild-type, GrlA^S81F, and GrlA^S81Y topoisomerase IV. DNA cleavage mediated by wild-type (WT), GrlA^S81F (S81F), and GrlA^S81Y (S81Y) topoisomerase IV in the presence of drugs are shown in the left, center, and right panels, respectively. Results with ciprofloxacin (Cipro, blue circles), levofloxacin (Levo, yellow circles), CP-115,953 (953, open green squares), norfloxacin (Nor, open diamonds), moxifloxacin (Moxi, black diamonds), sparfloxacin (Spar, purple triangles), and 8-methyl-quinazoline-2,4-dione (Dione, red squares) are shown for wild-type topoisomerase IV. Data with ciprofloxacin, levofloxacin, CP-115,953, and 8-methyl-quinazoline-2,4-dione are shown for the mutant enzymes. Error bars represent the standard deviation of three or more independent experiments.

Figure 6

DNA cleavage induced by GrlA^S81F and GrlA^S81Y topoisomerase IV at high quinolone concentrations. A titration is shown for ciprofloxacin and GrlA^S81F (S81F, red circles) or GrlA^S81Y (S81Y, blue triangles) topoisomerase IV. Results with low concentrations of ciprofloxacin (up to 30 μM, the concentration that generated maximal DNA cleavage) and the wild-type enzyme (WT, black squares) are shown for reference. The inset shows results for levofloxacin (Levo, solid bars) and CP-115,953 (953, stippled bars) with wild-type, GrlA^S81F, and GrlA^S81Y topoisomerase IV (black, red, and blue, respectively). Quinolone concentrations that generated maximal levels of DNA cleavage (20 μM and 300 μM for the wild-type and mutant enzymes, respectively) were used. Error bars represent the standard deviation of three or more independent experiments.

Figure 7

Effects of Mg²⁺ on DNA cleavage mediated by wild-type and GrlA^S81F topoisomerase IV in the presence of ciprofloxacin and 8-methyl-quinazoline-2,4-dione. Results are shown for 50 μM ciprofloxacin (Cipro, left panel) and 10 μM 8-methyl-quinazoline-2,4-dione (Dione, right panel) with the wild-type (WT, black squares) and GrlA^S81F (S81F, red circles) enzymes. DNA cleavage for each drug-enzyme pair was normalized to 100% at 10 mM Mg²⁺ to facilitate direct comparisons. Error bars represent the standard deviation of three or more independent experiments.

Figure 8

Effects of quinolones and 8-methyl-quinazoline-2,4-dione on sites of DNA cleavage mediated by wild-type, GrlA^S81F, and GrlA^S81Y topoisomerase IV. An autoradiogram of a polyacrylamide gel identifying DNA sites cleaved by the wild-type (WT), GrlA^S81F (S81F), and GrlA^S81Y (S81Y) enzymes is shown. Reactions contained no enzyme (No topo), or topoisomerase IV in the presence of the indicated concentrations of Mg²⁺, Ca²⁺, or ciprofloxacin (Cipro), levofloxacin (Levo), CP-115,953 (953), or 8-methyl-quinazoline-2,4-dione (Dione). Mg²⁺ (10 mM) was used in all drug-containing reactions. The autoradiogram is representative of three or more independent experiments.

Figure 9

Competition between ciprofloxacin and 8-methyl-quinazoline-2,4-dione. The ability of 0-150 μM ciprofloxacin to compete with 20 μM 8-methyl-quinazoline-2,4-dione for GrlA^S81F topoisomerase IV was determined using DNA cleavage assays. Both drugs were added to reaction mixtures simultaneously. The relative contribution of the quinazolinedione (Dione, left axis) to the total level of DNA cleavage was calculated as follows: (% DNA cleavage with both drugs – % DNA cleavage with ciprofloxacin only) ÷ (% DNA cleavage with quinazolinedione only – % DNA cleavage with ciprofloxacin only). The relative contribution of ciprofloxacin (Cipro) to the total level of DNA cleavage (1 minus the above equation) can be read from the right axis. Error bars represent the standard deviation of three or more independent experiments.

Figure 10

Effects of quinolones and 8-methyl-quinazoline-2,4-dione on the DNA religation activities of wild-type, GrlA^S81F, and GrlA^S81Y topoisomerase IV. Results for assays carried out in the absence of drugs (No Drug, open triangle) or in the presence of ciprofloxacin (Cipro, blue circles), levofloxacin (Levo, yellow circles), CP-115,953 (953, open green squares), or 8-methyl-quinazoline-2,4-dione (Dione, red squares) are shown. DNA religation mediated by wild-type (WT), GrlA^S81F (S81F), and GrlA^S81Y (S81Y) topoisomerase IV are shown in the left, center, and right panels, respectively. Religation was assessed by monitoring the loss of double-stranded DNA breaks (linear product) over time. Cleavage at time zero was set to 100%. Quinolone concentrations were 20 μM in assays that examined wild-type topoisomerase IV and were increased to 200 μM in assays that examined the GrlA^S81F and GrlA^S81Y enzymes. The concentration of 8-methyl-quinazoline-2,4-dione was 20 μM in all assays. Reactions carried out in the absence of drugs replaced Mg²⁺ with Ca²⁺ in order to achieve readily quantifiable levels of DNA cleavage. Error bars represent the standard deviation of three or more independent experiments.

Figure 11

Effects of ciprofloxacin and 8-methyl-quinazoline-2,4-dione on the persistence of ternary enzyme-drug-DNA cleavage complexes formed with wild-type, GrlA^S81F, and GrlA^S81Y topoisomerase IV. Results are shown for ciprofloxacin (Cipro, left panel) and 8-methyl-quinazoline-2,4-dione (Dione, right panel) with the wild-type (WT, black squares), GrlA^S81F (S81F, red circles), and GrlA^S81Y (S81Y, blue triangles) enzymes. Initial DNA cleavage-religation reactions were allowed to come to equilibrium and were then diluted 20-fold with DNA cleavage buffer. The persistence of cleavage complexes was assessed by monitoring the loss of double-stranded DNA breaks (linear product) over time. Cleavage at time zero was set to 100%. The concentration of ciprofloxacin was 20 μM in assays that examined wild-type topoisomerase IV and was increased to 200 μM in assays that examined the GrlA^S81F and GrlA^S81Y enzymes. The concentration of 8-methyl-quinazoline-2,4-dione was 20 μM in all assays. Error bars represent the standard deviation of three or more independent experiments.