Bacillus anthracis GrlA^V96A Topoisomerase IV, a Quinolone Resistance Mutation That Does Not Affect the Water-Metal Ion Bridge

Abstract

The rise in quinolone resistance is threatening the clinical use of this important class of broad-spectrum antibacterials. Quinolones kill bacteria by increasing the level of DNA strand breaks generated by the type II topoisomerases gyrase and topoisomerase IV. Most commonly, resistance is caused by mutations in the serine and acidic amino acid residues that anchor a water-metal ion bridge that facilitates quinolone-enzyme interactions. Although other mutations in gyrase and topoisomerase IV have been reported in quinolone-resistant strains, little is known regarding their contributions to cellular quinolone resistance. To address this issue, we characterized the effects of the V96A mutation in the A subunit of Bacillus anthracis topoisomerase IV on quinolone activity. The results indicate that this mutation causes an ∼3-fold decrease in quinolone potency and reduces the stability of covalent topoisomerase IV-cleaved DNA complexes. However, based on metal ion usage, the V96A mutation does not disrupt the function of the water-metal ion bridge. A similar level of resistance to quinazolinediones (which do not use the bridge) was seen. V96A is the first topoisomerase IV mutation distal to the water-metal ion bridge demonstrated to decrease quinolone activity. It also represents the first A subunit mutation reported to cause resistance to quinazolinediones. This cross-resistance suggests that the V96A change has a global effect on the structure of the drug-binding pocket of topoisomerase IV.

INTRODUCTION

Quinolone resistance has been increasing steadily since the 1990s and is threatening the clinical efficacy of this class of broad-spectrum antibacterials (1–3). The most common form of quinolone resistance is target mediated and results from specific point mutations in gyrase and topoisomerase IV (1, 3–6).

Gyrase and topoisomerase IV are type II topoisomerases, and both are encoded by nearly all bacterial species (3, 5, 7–14). These enzymes alter DNA topology by passing an intact double helix through a transient break that they generate in a separate segment of DNA (3, 7, 8, 10–13, 15). Both are heterotetramers, consisting of two A subunits (GyrA in gyrase and GrlA in topoisomerase IV) and two B subunits (GyrB in gyrase and GrlB in topoisomerase IV). The A subunits contain the active site tyrosine residues involved in DNA cleavage and ligation, and the B subunits bind ATP, which is required for overall catalytic activity (3, 5, 7, 8, 10, 11, 13, 15). Quinolones, including ciprofloxacin, take advantage of DNA cleavage mediated by type II topoisomerases and kill bacteria by increasing the levels of enzyme-generated strand breaks (1, 3–6, 16). Gyrase and topoisomerase IV are essential for cell survival (3, 7, 8, 10–13), and both appear to be physiological targets for quinolone antibacterials (3, 4, 17–21).

Most often, target-mediated quinolone resistance is caused by mutations in a highly conserved serine (originally described as Ser83 in Escherichia coli GyrA [22, 23]) or acidic residue (located four positions downstream) in the A subunit (1, 3–6, 24–32). Recent structural and functional studies with topoisomerase IV (33–35) suggest that a water-metal ion bridge facilitates the most important interaction between clinically relevant quinolones and the enzyme. This water-metal ion bridge is formed when the C-3/C-4 keto acid of the drug chelates a divalent metal ion, which interacts with the protein through water molecules that are coordinated by the conserved serine and acidic amino acid residues. In Bacillus anthracis topoisomerase IV, mutations in the bridge-anchoring residues (Ser81 and Glu85) cause drug resistance that is specific to quinolones (34–36). Resistance is caused by a partial or complete loss of bridge function that is accompanied by a marked decrease in quinolone affinity (34, 35).

In addition to mutations in the bridge-anchoring amino acids, substitutions in other topoisomerase IV residues (in the A subunit and the TOPRIM domain of the B subunit) have been reported in quinolone-resistant strains (18, 26–29, 32, 37). Typically, these substitutions are seen in combination with mutations in the serine or acidic residue, and none of them have been characterized in a purified system. Consequently, the contributions of these mutations to cellular quinolone resistance are not clear.

In order to determine the effects of a mutation that is not in a bridge-anchoring residue on drug activity, we characterized B. anthracis GrlA^V96A topoisomerase IV. This V96A mutation was found in a laboratory strain selected for quinolone resistance (27). Results indicate that the V96A mutation causes moderate (∼3-fold) resistance to quinolones and decreases the stability of covalent topoisomerase IV-cleaved DNA complexes, but it does not disrupt bridge function. In contrast to mutations in the bridge-anchoring residues, GrlA^V96A topoisomerase IV displays cross-resistance to quinazolinediones (quinolone-related drugs that do not utilize the water-metal ion bridge). Thus, drug-enzyme interactions can be modulated by alterations in GrlA that do not involve the water-metal ion bridge.

MATERIALS AND METHODS

Enzymes, DNA, and chemicals.

Wild-type B. anthracis GrlA and GrlB and drug-resistant GrlA^V96A were expressed and purified as described previously (38). In all assays, topoisomerase IV was used as a 1:1 mixture of GrlA to GrlB.

Negatively supercoiled pBR322 plasmid DNA was prepared from E. coli using a Plasmid Mega kit (Qiagen), as described by the manufacturer.

Ciprofloxacin was obtained from LKT Laboratories, stored at −20°C as a 40 mM stock solution in 0.1 N NaOH, and diluted 5-fold with 10 mM Tris-HCl (pH 7.9) immediately prior to use. 3-Amino-7-[(3S)-3-(aminomethyl)-1-pyrrolidinyl]-1-cyclopropyl-6-fluoro-8-methyl-2,4(1H,3H)-quinazolinedione [8-methyl-3′-(AM)P-dione] (UIJR-1-048) was synthesized using established methods, as reported previously (39). The quinazolinedione was stored at 4°C as a 20 mM stock solution in 100% dimethyl sulfoxide (DMSO). All other chemicals were analytical reagent grade.

DNA relaxation.

DNA relaxation assays were based on the protocol of Fortune and Osheroff (40) as modified by Aldred et al. (34). Reaction mixtures (20 μl) contained 18.75 nM wild-type or GrlA^V96A B. anthracis topoisomerase IV and 5 nM negatively supercoiled pBR322 in 40 mM HEPES (pH 7.6), 100 mM potassium glutamate, 10 mM magnesium acetate [Mg(OAc)₂], 50 mM NaCl, and 1 mM ATP and were incubated at 37°C. Relaxation was stopped at times ranging from 0 to 30 min by adding 3 μl of 0.77% SDS and 77.5 mM EDTA. Samples were mixed with 2 μl of agarose gel loading buffer (60% sucrose, 10 mM Tris-HCl [pH 7.9], 0.5% bromophenol blue, and 0.5% xylene cyanol FF), heated at 45°C for 5 min, and subjected to electrophoresis in 1% agarose gels in 100 mM Tris-borate (pH 8.3) and 2 mM EDTA. Gels were stained with 0.75 μg/ml ethidium bromide for 30 min. DNA bands were visualized with medium-range UV light and quantified using an Alpha Innotech digital imaging system. The percent relaxed DNA was determined by the loss of supercoiled DNA substrate.

DNA cleavage.

DNA cleavage reactions were carried out using the procedure of Fortune and Osheroff (40) as modified by Aldred et al. (34). The reaction mixtures contained 75 nM wild-type or GrlA^V96A topoisomerase IV and 10 nM negatively supercoiled pBR322 in a total of 20 μl of cleavage buffer (40 mM Tris-HCl [pH 7.9], 10 mM MgCl₂, 50 mM NaCl, and 2.5% [vol/vol] glycerol). In some reactions, the concentration dependence of MgCl₂ was examined or the divalent metal ion was replaced with CaCl₂, MnCl₂, or NiCl₂. The reaction mixtures were incubated at 37°C for 10 min, and enzyme-DNA cleavage complexes were trapped by adding 2 μl of 5% SDS followed by 2 μl of 250 mM EDTA (pH 8.0). Proteinase K (2 μl of a 0.8 mg/ml solution) was added, and samples were incubated at 45°C for 45 min to digest the enzyme. The samples were mixed with 2 μl of agarose gel loading buffer, heated at 45°C for 5 min, and subjected to electrophoresis in 1% agarose gels in 40 mM Tris-acetate (pH 8.3) and 2 mM EDTA containing 0.5 μg/ml ethidium bromide. DNA bands were visualized and quantified as described above. DNA cleavage was monitored by the conversion of supercoiled plasmid to linear molecules.

Assays that monitored the DNA cleavage activities of wild-type and GrlA^V96A B. anthracis topoisomerase IV in the absence of drugs substituted 10 mM CaCl₂ for 10 mM MgCl₂ in the cleavage buffer in order to increase the level of DNA scission (34, 35).

Persistence of topoisomerase IV-DNA cleavage complexes.

The persistence of topoisomerase IV-DNA cleavage complexes established in the presence of drugs was determined using the procedure of Gentry et al. (41) as modified by Aldred et al. (34). Initial reaction mixtures contained 375 nM wild-type or GrlA^V96A B. anthracis topoisomerase IV, 50 nM DNA, and the indicated concentrations of ciprofloxacin or 8-methyl-3′-(AM)P-dione in a total of 20 μl of DNA cleavage buffer. Reaction mixtures were incubated at 37°C for 10 min and then diluted 20-fold with DNA cleavage buffer warmed to 37°C. Samples (20 μl) were removed at times ranging from 0 to 300 min, and DNA cleavage was stopped with 2 μl of 5% SDS followed by 2 μl of 250 mM EDTA (pH 8.0). Samples were digested with proteinase K and processed as described above for the DNA cleavage assays. The levels of DNA cleavage were set to 100% at time zero, and the persistence of cleavage complexes was determined by the loss of the linear reaction product over time.

RESULTS

Characterization of B. anthracis GrlA^V96A topoisomerase IV.

The overwhelming majority of mutations that cause quinolone resistance in topoisomerase IV are in the conserved serine and acidic amino acid residues that anchor the water-metal ion bridge (1, 3–6, 24–32). Mutations in other residues also have been reported (18, 26–29, 32, 37, 42–44), but very little is understood about their impact on drug activity. Therefore, to examine the potential mechanisms of drug resistance that do not involve the water-metal ion bridge, we characterized B. anthracis GrlA^V96A topoisomerase IV. The V96A alteration was found in a laboratory culture of B. anthracis that was selected for quinolone resistance (27). Because the strain also carries an uncharacterized mutation in gyrase (GyrA^E89R), it is impossible to determine what contribution (if any) the GrlA^V96A mutation in topoisomerase IV makes to the observed cellular drug resistance. Based on crystallographic studies (33, 45), V96 is not in close enough proximity to either the quinolone or the water-metal ion bridge to participate directly in drug-topoisomerase IV interactions (Fig. 1).

FIG 1.

Crystal structure of a moxifloxacin-Acinetobacter baumannii topoisomerase IV cleavage complex (33). Moxifloxacin is shown in black. The noncatalytic Mg²⁺ ion that is chelated by the C-3/C-4 keto acid of the quinolone and participates in the bridge interaction is shown in green. The four water molecules that fill out the coordination sphere of the Mg²⁺ ion are shown in blue. Ribbons representing the amino acid backbone of the enzyme are shown in yellow. Residues reported to contribute to quinolone resistance in B. anthracis (26–28) are superimposed on the A. baumannii structure and are shown in red. The serine and acidic amino acids (S81 and E85) in B. anthracis topoisomerase IV) that anchor the water-metal ion bridge are indicated, and the residue that is equivalent to B. anthracis V96 is circled. Protein Data Bank accession no. 2XKK was visualized using the Discovery Studio 3.5 visualizer (Accelrys Software, Inc.).

Cellular sensitivity to quinolones can be decreased if a mutation diminishes the activity of topoisomerase IV. Therefore, we assessed the ability of GrlA^V96A topoisomerase IV to relax and cleave plasmid DNA in the absence of drug. The mutant enzyme relaxed DNA at the same rate as the wild-type enzyme (Fig. 2, left). Furthermore, GrlA^V96A topoisomerase IV retained wild-type levels of DNA cleavage activity (Fig. 2, right). Therefore, if the V96A mutation contributes to quinolone resistance, it does not do so by decreasing the native activities of the type II enzyme.

FIG 2.

DNA relaxation and cleavage activities of wild-type and GrlA^V96A B. anthracis topoisomerase IV in the absence of drugs. The abilities of wild-type (WT) (●) and GrlA^V96A (V96A) (○) topoisomerase IV to relax (left panel) and to cleave (right panel) negatively supercoiled pBR322 plasmid DNA are shown. Error bars represent the standard deviations from three or more independent experiments.

Effects of drugs on B. anthracis GrlA^V96A topoisomerase IV.

To determine whether the V96A mutation alters the sensitivity of B. anthracis topoisomerase IV to quinolones, the ability of ciprofloxacin to enhance DNA cleavage mediated by the mutant enzyme was examined (Fig. 3, left). GrlA^V96A topoisomerase IV displayed resistance to the quinolone, albeit at a lower level than that reported for B. anthracis enzymes containing mutations at Ser81 or Glu85 (34, 35). The drug concentration required to induce 50% maximal DNA cleavage was ∼3-fold higher for the V96A mutant enzyme compared to that of the wild-type topoisomerase IV (∼6 versus ∼2 μM). Equivalent levels of cleavage were seen with the two enzymes at 100 μM ciprofloxacin (Fig. 3, left, inset). These results indicate that the V96A mutation causes quinolone resistance by decreasing drug potency (i.e., affinity for the enzyme) rather than efficacy (i.e., maximal drug-induced DNA cleavage). A similar ∼3-fold decrease in drug potency with GrlA^V96A topoisomerase IV was seen with a ciprofloxacin derivative that substituted a 3′-(aminomethyl)pyrrolidine [3′-(AM)P] group for the piperazine ring at C-7 (50% maximal cleavage was achieved at 0.6 μM with the wild type [WT] versus 1.7 μM with V96A) (data not shown). This C-7 ring substitution can overcome resistance caused by mutations in the bridge-anchoring serine and acidic residues by establishing novel contacts within the enzyme-DNA cleavage complex (34–36, 39, 46). The inability of the 3′-(AM)P-substituted ciprofloxacin to overcome resistance suggests that the V96A mutation alters drug-enzyme interactions through a mechanism that does not involve the water-metal ion bridge.

FIG 3.

Effects of ciprofloxacin and 8-methyl-3′-(AM)P-dione on the DNA cleavage activities of wild-type and GrlA^V96A topoisomerase IV from B. anthracis. DNA cleavage mediated by wild-type (WT) (●) and GrlA^V96A (V96A) (○) topoisomerase IV in the presence of a quinolone (left panel) or a quinazolinedione (right panel) is shown. The insets show the level of cleavage induced by the enzymes in the presence of 100 μM drug. Error bars represent the standard deviations from three or more independent experiments. The molecular structures of the two compounds are shown above their respective panels.

To further explore this possibility, the effects of 8-methyl-3′-(AM)P-dione on B. anthracis GrlA^V96A topoisomerase IV were examined. Quinazolinediones are related to quinolones but lack the C-3/C-4 keto acid that chelates divalent metal ions. Consequently, these drugs cannot utilize the water-metal ion bridge. Quinazolinediones that retain high activities against bacterial type II enzymes with mutations in the bridge-anchoring serine and acidic residues have been reported (34–36, 46, 47). In all cases, these drugs contain a 3′-(AM)P (or related) group at the C-7 position (see Fig. 3) (34–36, 39, 46, 47). The effects of 8-methyl-3′-(AM)P-dione on DNA cleavage mediated by GrlA^V96A topoisomerase IV are shown in Fig. 3 (right). Similar to the level of resistance seen with ciprofloxacin, the concentration of the quinazolinedione required to induce 50% maximal DNA cleavage was ∼3-fold higher for the V96A mutant enzyme compared to that for wild-type B. anthracis topoisomerase IV (∼3 versus ∼1 μM). Furthermore, at 100 μM drug, 8-methyl-3′-(AM)P-dione induced ∼20% more cleavage complexes with GrlA^V96A than with the wild-type enzyme (Fig. 3, right, inset). Thus, the V96A mutation, which is distal to the bridge-anchoring residues, had similar effects on the activities of both the quinolone and the quinazolinedione.

Finally, the effects of the V96A mutation on the persistence (i.e., stability) of drug-induced cleavage complexes were determined. Persistence was assessed by establishing DNA cleavage-religation equilibria with topoisomerase IV in the presence of ciprofloxacin or 8-methyl-3′-(AM)P-dione, diluting reaction mixtures 20-fold, and monitoring the loss of cleavage complexes over time (Fig. 4, left and right, respectively). With the V96A mutant enzyme, the half-life (t_1/2) for ciprofloxacin-induced DNA cleavage complexes decreased ∼4-fold, and for the quinazolinedione, it decreased ∼2-fold. Thus, the resistance that accompanies the V96A mutation in B. anthracis topoisomerase IV appears to result from the formation of less stable drug-induced cleavage complexes.

FIG 4.

Effects of ciprofloxacin and 8-methyl-3′-(AM)P-dione on the persistence of cleavage complexes formed by wild-type and GrlA^V96A B. anthracis topoisomerase IV. The persistence of ternary enzyme-drug-DNA cleavage complexes formed with the wild-type (WT) (●) and mutant (V96A) (○) enzymes in the presence of the quinolone (20 μM for WT, 50 μM for V96A) or the quinazolinedione (20 μM) is shown (left and right, respectively). The data table (right, inset) lists the t_1/2 of the DNA cleavage complexes formed with each drug-enzyme combination. The initial DNA cleavage-religation reactions were allowed to come to equilibrium, and reaction mixtures were then diluted 20-fold with DNA cleavage buffer. The levels of DNA cleavage at time zero were set to 100%, and results were quantified by monitoring the loss of double-stranded DNA breaks over time. Error bars represent the standard deviations from three or more independent experiments.

Whereas mutations in the serine and acidic amino acid residues that anchor the water-metal ion bridge cause resistance that is specific to quinolones (34–36, 46, 47), the GrlA^V96A mutation affects both quinolones and quinazolinediones. Furthermore, the level of resistance caused by this mutation is lower than that seen in the presence of Ser81 or Glu85 mutations in B. anthracis topoisomerase IV (34–36). Finally, the only quinazolinedione resistance mutations reported to date occur in the TOPRIM domain of the B subunit of gyrase and topoisomerase IV (46, 48). B. anthracis GrlA^V96A represents the first mutation in the A subunit of the bacterial type II enzyme that has been demonstrated to confer quinazolinedione resistance.

Effects of the GrlA^V96A mutation on the water-metal ion bridge interaction.

The location of Val96 in the crystal structure (see Fig. 1) (33), together with the results from the above-mentioned studies, suggests that the GrlA^V96A mutation alters drug sensitivity through a mechanism that does not involve disruption of the water-metal ion bridge that coordinates the drug with topoisomerase IV. Previous studies found that mutations that impaired the function of the water-metal ion bridge decreased the affinity of the Mg²⁺ ion used in the bridge, raising the concentration of the divalent cation required to fully support quinolone-induced DNA cleavage (34, 35). These mutations also restricted the variety of metal ions that could support bridge and quinolone functions (35). Because quinazolinediones do not utilize the water-metal ion bridge, the metal ion requirements for these drugs were not affected by mutations in the bridge-anchoring residues (34, 35).

To determine whether the V96A mutation affects the ability of the water-metal ion bridge to function, the requirement for metal ions was determined. In contrast to mutations in Ser81 or Glu85 in B. anthracis topoisomerase IV, the concentration of Mg²⁺ required to support quinolone-induced DNA cleavage was not altered by the presence of the V96A mutation (Fig. 5, left). This finding strongly suggests that the mutation does not affect the ability of ciprofloxacin to utilize the bridge. As a control, a parallel experiment was carried out with the metal-ion-independent quinazolinedione (Fig. 5, right). Once again, the V96A mutation had no effect on the Mg²⁺ concentration required to support drug-induced DNA cleavage.

FIG 5.

Effects of Mg²⁺ concentration on DNA cleavage mediated by wild-type and GrlA^V96A topoisomerase IV from B. anthracis in the presence of ciprofloxacin and 8-methyl-3′-(AM)P-dione. Results are shown for 50 μM ciprofloxacin (Cipro) (left panel) and 10 μM 8-methyl-3′-(AM)P-dione (Dione) (right panel) with the wild-type (WT) (○) and GrlA^V96A (V96A) (●) enzymes. DNA cleavage was monitored by the appearance of linear DNA and for each drug-enzyme pair was normalized to 100% at 10 mM Mg²⁺ to facilitate direct comparisons. Error bars represent the standard deviations from three or more independent experiments.

To further examine the effects of the V96A mutation on metal ion usage by quinolones, the abilities of Mn²⁺ and Ni²⁺ to support drug activity against B. anthracis GrlA^V96A topoisomerase IV were assessed (Fig. 6, left and right, respectively). A previous study demonstrated that Mn²⁺ and Ni²⁺ can support quinolone activity against wild-type B. anthracis topoisomerase IV but not against enzymes carrying mutations in the bridge-anchoring serine and glutamic acid residues (35). In contrast, the V96A mutation had no effect on the abilities of Mn²⁺ and Ni²⁺ to support ciprofloxacin-induced DNA cleavage. As above, the mutation also did not affect the metal ion requirements for quinazolinedione activity (Fig. 6). Taken together, the above results indicate that the V96A mutation alters the activities of quinolones without impairing the function of the water-metal ion bridge.

FIG 6.

Effects of Mn²⁺ and Ni²⁺ on drug-induced DNA cleavage mediated by GrlA^V96A B. anthracis topoisomerase IV. Results are shown for cleavage mediated by the mutant enzyme in the presence of ciprofloxacin (circles) or 8-methyl-3′-(AM)P-dione (squares) and Mn²⁺ (left) or Ni²⁺ (right). Metal ions were utilized at the concentration that yielded maximal enzyme activity (5 mM for Mn²⁺, 10 mM for Ni²⁺). Error bars represent the standard deviations from three or more independent experiments.

DISCUSSION

Although the most common mutations in gyrase and topoisomerase IV associated with quinolone resistance are in the serine and acidic residues that anchor the water-metal ion bridge, other mutations also have been observed in quinolone-resistant bacterial strains (18, 26–29, 32, 37, 42–44). However, little is known about how (or if) these mutations contribute to drug resistance. To address this issue, we analyzed the effects of drugs on B. anthracis GrlA^V96A topoisomerase IV. The results indicate that the mutant enzyme displays moderate resistance to ciprofloxacin without altering the function of the water-metal ion bridge. Thus, even though the water-metal ion bridge facilitates the primary contact between clinically relevant quinolones and B. anthracis topoisomerase IV, drug interactions can be modulated by subtle changes in protein structure.

Quinazolinediones are related to quinolones, enhance gyrase- and topoisomerase IV-mediated DNA cleavage, and share an interaction domain with quinolones in these enzymes (34, 35, 45). However, they do not utilize the water-metal ion bridge to promote interactions in the cleavage complex (34–36, 46, 47). Consequently, the activities of quinazolinediones are not affected by mutations that disrupt the water-metal ion bridge. Previously, the only mutations demonstrated to cause quinazolinedione resistance were found in the B subunit of gyrase or topoisomerase IV (46, 48). At least one of these mutations was specific for quinazolinediones and did not decrease the sensitivities of the enzymes to clinically relevant quinolones. As described above, GrlA^V96A topoisomerase IV displayed similar levels of resistance to 8-methyl-3′-(AM)P-dione and to ciprofloxacin. Therefore, V96A represents the first quinazolinedione resistance mutation found in the A subunit of a bacterial type II enzyme. Furthermore, this finding suggests that the mutation at V96 has a global effect on the drug interaction site.

Although V96 appears to be conserved in topoisomerase IV across the Bacillus genus of bacteria (including, but not limited to, B. anthracis, B. subtilis, B. cereus, B. halodurans, B. thuringiensis, B. amyloliquefaciens, and B. safensis), the residue is not conserved in gyrase or in topoisomerase IV from other genera. However, the equivalent residue is usually an aromatic or hydrophobic amino acid. Consequently, while the results seen with B. anthracis GrlA^V96A topoisomerase IV may be translatable across genera, further studies will be required to assess this possibility. Regardless, two important generalized conclusions can be drawn from the present study. First, even though mutations in the residues that anchor the water-metal ion bridge appear to be responsible for the most common and clinically relevant forms of quinolone resistance (1, 3–6, 24–32), other resistance mechanisms that result from alternative mutations in bacterial type II topoisomerases exist. Second, the fact that the V96A mutation did not alter the water-metal ion bridge strongly suggests that changes in topoisomerase IV-quinolone interactions that accompany mutations in the serine or acidic residue can be attributed specifically to the loss of the bridge anchors rather than to a more general effect on protein structure.