L. Mark Fisher

Mycobacterium tuberculosis DNA Gyrase: Interaction with Quinolones and Correlation with Antimycobacterial Drug Activity

ABSTRACT Genome studies suggest that DNA gyrase is the sole type II topoisomerase and likely the unique target of quinolones in Mycobacterium tuberculosis. Despite the emerging importance of quinolones in the treatment of mycobacterial disease, the slow growth and high pathogenicity of M. tuberculosis have precluded direct purification of its gyrase and detailed analysis of quinolone action. To address these issues, we separately overexpressed the M. tuberculosis DNA gyrase GyrA and GyrB subunits as His-tagged proteins in Escherichia coli from pET plasmids carrying gyrA and gyrB genes. The soluble 97-kDa GyrA and 72-kDa GyrB subunits were purified by nickel chelate chromatography and shown to reconstitute an ATP-dependent DNA supercoiling activity. The drug concentration that inhibited DNA supercoiling by 50% (IC50) was measured for 22 different quinolones, and values ranged from 2 to 3 μg/ml (sparfloxacin, sitafloxacin, clinafloxacin, and gatifloxacin) to >1,000 μg/ml (pipemidic acid and nalidixic acid). By comparison, MICs measured against M. tuberculosis ranged from 0.12 μg/ml (for gatifloxacin) to 128 μg/ml (both pipemidic acid and nalidixic acid) and correlated well with the gyrase IC50s (R2 = 0.9). Quinolones promoted gyrase-mediated cleavage of plasmid pBR322 DNA due to stabilization of the cleavage complex, which is thought to be the lethal lesion. Surprisingly, the measured concentrations of drug inducing 50% plasmid linearization correlated less well with the MICs (R2 = 0.7). These findings suggest that the DNA supercoiling inhibition assay may be a useful screening test in identifying quinolones with promising activity against M. tuberculosis. The quinolone structure-activity relationship demonstrated here shows that C-8, the C-7 ring, the C-6 fluorine, and the N-1 cyclopropyl substituents are desirable structural features in targeting M. tuberculosis gyrase.

Novel Gyrase Mutations in Quinolone-Resistant and -Hypersusceptible Clinical Isolates of Mycobacterium tuberculosis: Functional Analysis of Mutant Enzymes

ABSTRACT Mutations in the DNA gyrase GyrA2GyrB2 complex are associated with resistance to quinolones in Mycobacterium tuberculosis. As fluoroquinolones are being used increasingly in the treatment of tuberculosis, we characterized several multidrug-resistant clinical isolates of M. tuberculosis carrying mutations in the genes encoding the GyrA or GyrB subunits associated with quinolone resistance or hypersusceptibility. In addition to the reported putative quinolone resistance mutations in GyrA, i.e., A90V, D94G, and D94H, we found that the GyrB N510D mutation was also associated with ofloxacin resistance. Surprisingly, several isolates bearing a novel combination of gyrA T80A and A90G changes were hypersusceptible to ofloxacin. M. tuberculosis GyrA and GyrB subunits (wild type [WT] and mutants) were overexpressed in Escherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. Mutant proteins were produced similarly from engineered gyrA and gyrB alleles by mutagenesis. MICs, enzyme inhibition, and drug-induced DNA cleavage were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. Mutant gyrase complexes bearing GyrA A90V, D94G, and D94H and GyrB N510D were resistant to quinolone inhibition (MICs and 50% inhibitory concentrations [IC50s] at least 3.5-fold higher than the concentrations for the WT), and all, except the GyrB mutant, were less efficiently trapped as a quinolone cleavage complex. In marked contrast, gyrase complexes bearing GyrA T80A or A90G were hypersusceptible to the action of many quinolones, an effect that was reinforced for complexes bearing both mutations (MICs and IC50s up to 14-fold lower than the values for the WT). This is the first detailed enzymatic analysis of hypersusceptibility and resistance in M. tuberculosis.

Structural Basis of Gate-DNA Breakage and Resealing by Type II Topoisomerases

Type II DNA topoisomerases are ubiquitous enzymes with essential functions in DNA replication, recombination and transcription. They change DNA topology by forming a transient covalent cleavage complex with a gate-DNA duplex that allows transport of a second duplex though the gate. Despite its biological importance and targeting by anticancer and antibacterial drugs, cleavage complex formation and reversal is not understood for any type II enzyme. To address the mechanism, we have used X-ray crystallography to study sequential states in the formation and reversal of a DNA cleavage complex by topoisomerase IV from Streptococcus pneumoniae, the bacterial type II enzyme involved in chromosome segregation. A high resolution structure of the complex captured by a novel antibacterial dione reveals two drug molecules intercalated at a cleaved B-form DNA gate and anchored by drug-specific protein contacts. Dione release generated drug-free cleaved and resealed DNA complexes in which the DNA gate instead adopts an unusual A/B-form helical conformation with a Mg2+ ion repositioned to coordinate each scissile phosphodiester group and promote reversible cleavage by active-site tyrosines. These structures, the first for putative reaction intermediates of a type II topoisomerase, suggest how a type II enzyme reseals DNA during its normal reaction cycle and illuminate aspects of drug arrest important for the development of new topoisomerase-targeting therapeutics.

Potent Antipneumococcal Activity of Gemifloxacin Is Associated with Dual Targeting of Gyrase and Topoisomerase IV, an In Vivo Target Preference for Gyrase, and Enhanced Stabilization of Cleavable Complexes In Vitro

ABSTRACT We investigated the roles of DNA gyrase and topoisomerase IV in determining the susceptibility of Streptococcus pneumoniaeto gemifloxacin, a novel fluoroquinolone which is under development as an antipneumococcal drug. Gemifloxacin displayed potent activity against S. pneumoniae 7785 (MIC, 0.06 μg/ml) compared with ciprofloxacin (MIC, 1 to 2 μg/ml). Complementary genetic and biochemical approaches revealed the following. (i) The gemifloxacin MICs for isogenic 7785 mutants bearing either parC orgyrA quinolone resistance mutations were marginally higher than wild type at 0.12 to 0.25 μg/ml, whereas the presence of both mutations increased the MIC to 0.5 to 1 μg/ml. These data suggest that both gyrase and topoisomerase IV contribute significantly as gemifloxacin targets in vivo. (ii) Gemifloxacin selected first-stepgyrA mutants of S. pneumoniae 7785 (gemifloxacin MICs, 0.25 μg/ml) encoding Ser-81 to Phe or Tyr, or Glu-85 to Lys mutations. These mutants were cross resistant to sparfloxacin (which targets gyrase) but not to ciprofloxacin (which targets topoisomerase IV). Second-step mutants (gemifloxacin MICs, 1 μg/ml) exhibited an alteration in parC resulting in changes of ParC hot spot Ser-79 to Phe or Tyr. Thus, gyrase appears to be the preferential in vivo target. (iii) Gemifloxacin was at least 10- to 20-fold more effective than ciprofloxacin in stabilizing a cleavable complex (the cytotoxic lesion) with either S. pneumoniaegyrase or topoisomerase IV enzyme in vitro. These data suggest that gemifloxacin is an enhanced affinity fluoroquinolone that acts against gyrase and topoisomerase IV in S. pneumoniae, with gyrase the preferred in vivo target. The marked potency of gemifloxacin against wild type and quinolone-resistant mutants may accrue from greater stabilization of cleavable complexes with the target enzymes.

Ciprofloxacin and the fluoroquinolones: New concepts on the mechanism of action and resistance

Ciprofloxacin, a new fluoroquinolone, is a potent, broad-spectrum antibacterial agent. It rapidly blocks bacterial deoxyribonucleic acid (DNA) replication by inhibiting DNA gyrase, an essential prokaryotic enzyme that catalyzes chromosomal DNA supercoiling. Molecular genetic approaches have been used to study the interaction of 4-quinolones with DNA gyrase from quinolone-sensitive strains and from uropathogenic quinolone-resistant clinical isolates of Escherichia coli. An important mutational locus in the gyrase A gene that confers resistance to ciprofloxacin and other quinolones has been identified, and a new, rapid method to examine clinical isolates for the presence of mutations at this position has been devised. A quinolone resistant gyrA gene has been previously cloned and sequenced from an E. coli clinical isolate. Genetic analysis indicated that resistance resulted from a Ser-83----Trp change in the 875 residue gyrase A protein: two other changes observed in the protein, Asp-678----Glu and Ala-828----Ser, were neutral. GyrA genes carrying these mutations have now been expressed, corresponding mutant gyrase A proteins purified, and their quinolone resistance properties tested by complementing with gyrase B protein and studying the resulting gyrase activity in an adenosine triphosphate-dependent DNA supercoiling assay. The in vitro DNA supercoiling activity of the A (Ser-83----Trp) mutant subunit complemented with wild-type gyrase B subunit was highly resistant to ciprofloxacin and other 4-quinolones. In contrast, A subunit carrying codon 678 and 828 changes reconstituted a quinolone-sensitive gyrase activity. Thus, quinolone-resistant gyrase A proteins may be readily obtained for study by using high-copy gyrA plasmids. In addition, other quinolone-resistant strains of E. coli have been examined for the presence of mutations at gyrase A codons 82 and 83 using a new analytical method based on a restriction fragment length polymorphism (RFLP). This analysis revealed that seven of eight resistant clinical isolates of E. coli examined carried gyrA mutations at codon 82 or 83, whereas five sensitive strains appeared to possess wild-type sequence. Thus, mutations at codon 83 (and possibly 82) in the gyrA gene frequently confer resistance to 4-quinolones, including ciprofloxacin. The RFLP method described should prove useful in examining strains for such mutations. These results are discussed with regard to the mode of interaction of the 4-quinolones with gyrase.

Probing the Differential Interactions of Quinazolinedione PD 0305970 and Quinolones with Gyrase and Topoisomerase IV

ABSTRACT Quinazoline-2,4-diones, such as PD 0305970, are new DNA gyrase and topoisomerase IV (topo IV) inhibitors with potent activity against gram-positive pathogens, including quinolone-resistant isolates. The mechanistic basis of dione activity vis-à-vis quinolones is not understood. We present evidence for Streptococcus pneumoniae gyrase and topo IV that PD 0305970 and quinolones interact differently with the enzyme breakage-reunion and Toprim domains, DNA, and Mg2+-four components that are juxtaposed in the topoisomerase cleavage complex to effect DNA scission. First, PD 0305970 targets primarily gyrase in Streptococcus pneumoniae. However, unlike quinolones, which select predominantly for gyrA (or topo IV parC) mutations in the breakage-reunion domain, unusually the dione selected for novel mutants with alterations that map to a region of the Toprim domain of GyrB (R456H and E474A or E474D) or ParE (D435H and E475A). This “dione resistance-determining region” overlaps the GyrB quinolone resistance-determining region and the region that binds essential Mg2+ ions, each function involving conserved EGDSA and PLRGK motifs. Second, dione-resistant gyrase and topo IV were inhibited by ciprofloxacin, whereas quinolone-resistant enzymes (GyrA S81F and ParC S79F) remained susceptible to PD 0305970. Third, dione-promoted DNA cleavage by gyrase occurred at a distinct repertoire of sites, implying that structural differences with quinolones are sensed at the DNA level. Fourth, unlike the situation with quinolones, the Mg2+ chelator EDTA did not reverse dione-induced gyrase cleavage nor did the dione promote Mg2+-dependent DNA unwinding. It appears that PD 0305970 interacts uniquely to stabilize the cleavage complex of gyrase/topo IV perhaps via an altered orientation directed by the bidentate 3-amino-2,4-dione moiety.

Quinolone Resistance Mutations in Streptococcus pneumoniae GyrA and ParC Proteins: Mechanistic Insights into Quinolone Action from Enzymatic Analysis, Intracellular Levels, and Phenotypes of Wild-Type and Mutant Proteins

ABSTRACT Mutations in DNA gyrase and/or topoisomerase IV genes are frequently encountered in quinolone-resistant mutants ofStreptococcus pneumoniae. To investigate the mechanism of their effects at the molecular and cellular levels, we have used anEscherichia coli system to overexpress S.pneumoniae gyrase gyrA and topoisomerase IV parC genes encoding respective Ser81Phe and Ser79Phe mutations, two changes widely associated with quinolone resistance. Nickel chelate chromatography yielded highly purified mutant His-tagged proteins that, in the presence of the corresponding GyrB and ParE subunits, reconstituted gyrase and topoisomerase IV complexes with wild-type specific activities. In enzyme inhibition or DNA cleavage assays, these mutant enzyme complexes were at least 8- to 16-fold less responsive to both sparfloxacin and ciprofloxacin. The ciprofloxacin-resistant (Cipr) phenotype was silent in a sparfloxacin-resistant (Spxr) S.pneumoniae gyrA (Ser81Phe) strain expressing a demonstrably wild-type topoisomerase IV, whereas Spxr was silent in a CiprparC (Ser79Phe) strain. These epistatic effects provide strong support for a model in which quinolones kill S. pneumoniae by acting not as enzyme inhibitors but as cellular poisons, with sparfloxacin killing preferentially through gyrase and ciprofloxacin through topoisomerase IV. By immunoblotting using subunit-specific antisera, intracellular GyrA/GyrB levels were a modest threefold higher than those of ParC/ParE, most likely insufficient to allow selective drug action by counterbalancing the 20- to 40-fold preference for cleavable-complex formation through topoisomerase IV observed in vitro. To reconcile these results, we suggest that drug-dependent differences in the efficiency by which ternary complexes are formed, processed, or repaired in S. pneumoniae may be key factors determining the killing pathway.

Target specificity of the new fluoroquinolone besifloxacin in Streptococcus pneumoniae, Staphylococcus aureus and Escherichia coli

OBJECTIVES Besifloxacin is a new fluoroquinolone in development for ocular use. We investigated its mode of action and resistance in two major ocular pathogens, Streptococcus pneumoniae and Staphylococcus aureus, and in the reference species Escherichia coli. METHODS Primary and secondary targets of besifloxacin were evaluated by: (i) mutant selection experiments; (ii) MIC testing of defined topoisomerase mutants; and (iii) inhibition and cleavable complex assays with purified S. pneumoniae and E. coli DNA gyrase and topoisomerase IV enzymes. RESULTS Enzyme assays showed similar besifloxacin activity against S. pneumoniae gyrase and topoisomerase IV, with IC(50) and CC(25) of 2.5 and 1 microM, respectively. In contrast to ciprofloxacin and moxifloxacin, besifloxacin was equally potent against both S. pneumoniae and E. coli gyrases. DNA gyrase was the primary target in all three species, with substitutions observed at positions 81, 83 and 87 in GyrA and 426 and 466 in GyrB (E. coli numbering). Topoisomerase IV was the secondary target. Notably, resistant mutants were not recovered at 4-fold besifloxacin MICs for S. aureus and S. pneumoniae, and S. aureus topoisomerase mutants were only obtained after serial passage in liquid medium. Besifloxacin MICs were similarly affected by parC or gyrA mutations in S. aureus and S. pneumoniae and remained below 1 mg/L in gyrA-parC double mutants. CONCLUSIONS Although mutant selection experiments indicated that gyrase is a primary target, further biochemical and genetic studies showed that besifloxacin has potent, relatively balanced activity against both essential DNA gyrase and topoisomerase IV targets in S. aureus and S. pneumoniae.

Complementation of temperature-sensitive topoisomerase II mutations in Saccharomyces cerevisiae by a human TOP2β construct allows the study of topoisomerase IIβ inhibitors in yeast

Abstract We show herein that human DNA topoisomerase IIβ is functional in yeast. It can complement a yeast temperature-sensitive mutation in topoisomerase II. The effect on human topoisomerase IIβ of a number of topoisomerase II inhibitors was analysed in a yeast in vivo system and compared with that of human topoisomerase IIα and wild-type yeast topoisomerase II. A drug permeable yeast strain (JN394 top2 – 4) was used to analyse the in vivo effects of known anti-topoisomerase II agents on human topoisomerase IIβ transformants. A parallel analysis on human topoisomerase IIα transformants provides the first in vivo analysis of the responses of yeast bearing the individual isoforms to these drugs. The strain was analysed at 35°C, a non-permissive temperature at which only plasmid-borne topoisomerase II is active. A shuttle vector with either human topoisomerase IIβ, human topoisomerase IIα or yeast topoisomerase II under the control of a GAL1 promoter was used. The key findings were that amsacrine produced comparable levels of cell killing with both α and β, whilst etoposide, doxorubicin and mitoxantrone produced higher degrees of cell killing with α than with β or yeast topoisomerase II. Merbarone had the greatest effect on the yeast strain bearing plasmid-borne yeast topoisomerase II. Suramin, quercetin and genistein showed little cell killing in this system. This yeast in vivo system provides a powerful way to analyse the effects of anti-topoisomerase II agents on transformants bearing the individual human isoforms. This system also provides a means of analysing putative drug-resistance mutations in human topoisomerase IIβ or to select for drug-resistance mutations in human topoisomerase IIβ.

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 μg/ml versus 0.25, 0.25, 1, and 1 to 2 μg/ml, respectively) and against isogenic quinolone-resistant gyrA-parC mutants (MICs, 0.5 to 1 μg/ml versus 2 to 4, 2 to 4, 16 to 32, and 64 μg/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% (IC50s) were 5 to 10 and 2.5 to 5.0 μM, 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 ≈ 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 μM, 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.