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Antimicrobial Agents and Chemotherapy, May 2009, p. 2110-2119, Vol. 53, No. 5
0066-4804/09/$08.00+0     doi:10.1128/AAC.01440-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

In Vivo and In Vitro Patterns of the Activity of Simocyclinone D8, an Angucyclinone Antibiotic from Streptomyces antibioticus{triangledown} ,{dagger}

Lisa M. Oppegard,1 Bree L. Hamann,2 Kathryn R. Streck,1 Keith C. Ellis,3,4 Hans-Peter Fiedler,5 Arkady B. Khodursky,2 and Hiroshi Hiasa1*

Department of Pharmacology, University of Minnesota Medical School—Twin Cities, Minneapolis, Minnesota 55455,1 Department of Biochemistry, Molecular Biology, and Biophysics and Biotechnology Institute, University of Minnesota, St. Paul, Minnesota 55108,2 Department of Medicinal Chemistry and Institute for Therapeutic Discovery and Development, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55414,3 Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia 23298,4 Mikrobiologisches Institut, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany5

Received 28 October 2008/ Returned for modification 8 December 2008/ Accepted 1 March 2009


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ABSTRACT
 
Simocyclinone D8 (SD8) exhibits antibiotic activity against gram-positive bacteria but not against gram-negative bacteria. The molecular basis of the cytotoxicity of SD8 is not fully understood, although SD8 has been shown to inhibit the supercoiling activity of Escherichia coli gyrase. To understand the mechanism of SD8, we have employed biochemical assays to directly measure the sensitivities of E. coli and Staphylococcus aureus type II topoisomerases to SD8 and microarray analysis to monitor the cellular responses to SD8 treatment. SD8 is a potent inhibitor of either E. coli or S. aureus gyrase. In contrast, SD8 exhibits only a moderate inhibitory effect on S. aureus topoisomerase IV, and E. coli topoisomerase IV is virtually insensitive to SD8. The antimicrobial effect of SD8 against E. coli has become evident in the absence of the AcrB multidrug efflux pump. As expected, SD8 treatment exhibits the signature responses to the loss of supercoiling activity in E. coli: upregulation of gyrase genes and downregulation of the topoisomerase I gene. Unlike quinolone treatment, however, SD8 treatment does not induce the SOS response. These results suggest that DNA gyrase is the target of SD8 in both gram-positive and gram-negative bacteria and that the lack of the antibacterial effect against gram-negative bacteria is due, in part, to the activity of the AcrB efflux pump.


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INTRODUCTION
 
All type II topoisomerases, except Sulfolobus shibatae topoisomerase VI, are well conserved and belong to a large protein family, the type IIA topoisomerases (4, 45). They are essential for DNA replication and chromosome segregation, as well as for the maintenance of chromosome structure and DNA superhelicity. Although these enzymes share common biochemical properties, each enzyme exhibits a distinct catalytic preference, which reflects its specialized function in vivo (4, 45). For instance, DNA gyrase is the only enzyme that can introduce negative supercoils into DNA, whereas topoisomerase IV (Topo IV) is an efficient decatenase that is responsible for segregating daughter chromosomes. DNA gyrase and Topo IV consist of GyrA and GyrB subunits and ParC and ParE subunits, respectively. GyrA and ParC subunits are responsible for catalyzing the strand breakage and religation reactions, whereas GyrB and ParE subunits bind and hydrolyze ATP. The active forms of gyrase and Topo IV are an {alpha}2β2 tetramer (4, 45).

Both DNA gyrase and Topo IV are the cellular targets of the quinolone and coumarin antibacterial drugs (6, 24). The quinolones, such as norfloxacin and ciprofloxacin, are poisons of gyrase and Topo IV, which can trap either gyrase or Topo IV in a topoisomerase-drug-DNA ternary complex. In contrast, the coumarins, such as novobiocin and coumermycin A1, are competitive inhibitors of the ATPase activity. Interestingly, gyrase has been shown to be the primary target of quinolone drugs in Escherichia coli, whereas Topo IV becomes the primary target in Staphylococcus aureus and Streptococcus pneumoniae (6, 7, 22, 24, 28). These observations have suggested that gyrase and Topo IV are the primary targets in gram-negative and gram-positive bacteria, respectively. It has been demonstrated, however, that each quinolone drug has a preferred target and the target selection can be altered by changes in quinolone structure (28, 29). Thus, it remains unclear exactly what determines the primary target of a quinolone drug in cells.

Simocyclinone D8 (SD8) belongs to a new class of angucyclinone antibiotics isolated from Streptomyces antibioticus Tü 6040 (15, 16, 38, 42). SD8 contains a halogenated aminocoumarin moiety (Fig. 1), a unique feature of the coumarin drugs. However, in addition to the coumarin moiety, SD8 contains an angucyclic polyketide core, a deoxyhexose, and a tetraene side chain. An agar plate diffusion assay and a broth dilution method have shown that SD8 exhibits antibiotic activity against gram-positive bacteria, such as S. aureus, Bacillus brevis, Bacillus subtilis, and Streptomyces viridochromogenes, and little to no activity against gram-negative bacteria, such as E. coli, Proteus mirabilis, and Pseudomonas fluorescens (38).


Figure 1
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  		FIG. 1. Structure of SD8. Two functional groups, the aminocoumarin and the glycone, are shown. The calculated molecular mass of SD8 is 932.2169, whereas the molecular masses, determined by mass spectrometry, of the original and current preparations of SD8 are 932.2134 and 932.2139, respectively. Me, methyl.

Despite the potential importance of SD8, only one study has been conducted on the mechanism of SD8 (8). Although the coumarin drugs and SD8 share an aminocoumarin moiety, unlike the coumarins, SD8 does not inhibit the ATPase activity of gyrase. Instead, it inhibits the supercoiling activity of E. coli gyrase by interacting with the GyrA subunit and preventing gyrase from binding to DNA (8). However, the effects of SD8 on topoisomerases from gram-positive bacteria have not been examined. In this study, we conducted a comprehensive analysis of the activity of SD8 to understand the mechanism of its action in both gram-positive and gram-negative bacteria. We decided to use two distinct approaches, biochemical assays and microarray analysis, to achieve our objective. Biochemical assays showed that SD8 was a potent inhibitor of both E. coli gyrase and S. aureus gyrase but a much less potent inhibitor of Topo IVs. We took advantage of our finding that a bacteriostatic effect of SD8 against E. coli became evident in a {Delta}acrB strain and monitored the transcriptional response to SD8 treatment in E. coli. SD8 treatment led to the signature response to the loss of supercoiling activity. Unlike quinolone treatment, however, SD8 treatment did not induce the SOS response. Thus, in vivo and in vitro patterns of the activity of SD8 suggested that DNA gyrase is the target of SD8 in both gram-positive and gram-negative bacteria and that the activity of the AcrB multidrug efflux pump is, at least in part, responsible for the ineffectiveness of SD8 against gram-negative bacteria.


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MATERIALS AND METHODS
 
DNAs and proteins. Negatively supercoiled pBR322 DNA was purchased from New England Biolabs, and relaxed DNA was prepared by incubating the negatively supercoiled DNA with human topoisomerase I (Topogen). Kinetoplast DNA was purchased from Topogen. The subunits of E. coli gyrase, E. coli Topo IV, S. aureus gyrase, and S. aureus Topo IV were expressed and purified, and the active enzymes were reconstituted as described previously (11-14, 30).

Fermentation, isolation, and purification of SD8. The fermentation of S. antibioticus Tü 6040 was carried out at the University of Minnesota Biotechnology Resources Center following previously described procedures (38). We found that the choice of carbon and nitrogen sources was critical to the production of SD8. Production of SD8 was monitored by analytical reversed-phase high-performance liquid chromatography (HPLC) (analytical HPLC with a photodiode array detector; Waters Alliance) on a C18 column (5 µm; 4.6 by 150 mm; Restek) using acetonitrile and 0.1% formic acid as the mobile phase. Separation was achieved using a linear gradient from 50:50 acetonitrile-0.1% formic acid to 60:40 acetonitrile-0.1% formic acid over 30 min.

After completion of the fermentation, cells were separated from the broth by passing the culture through a 150 US mesh sieve. Packed cells (665 g) were processed as described previously (38). The combined methanol extracts (wet with water) were concentrated to a dark orange crude solid (20.2 g) on a rotary evaporator. HPLC analysis of this crude extract showed SD8 to be the least polar constituent (comparison to HPLC of the original preparation by H.-P. Fiedler and his colleagues). HPLC analysis of an ethyl acetate extract of the fermentation broth, as well as a lyophilized sample, showed that the broth did not contain SD8.

The crude natural product was purified using catch-and-release solid-phase extraction on diol-modified silica gel. Attempts to purify SD8 on normal silica gel using standard chromatography failed with a range of solvents. A ~5-g sample of the crude product was dry loaded (50 g of diol-modified silica) onto a 400-g diol-modified silica gel column prepared with 100% dichloromethane. Dry loading of the sample was imperative given that the crude material is only sparingly soluble in dichloromethane and had to be loaded in methanol. Solid-phase extraction purification of the crude product on diol-modified silica gel at 1.25% (5 g crude product on 400 g matrix) allowed elution of SD8 as the first yellow band off the column with 1% methanol-dichloromethane. The fractions from this first band were combined, and analysis by HPLC and nuclear magnetic resonance showed them to contain only SD8.

Extensive analytical chemistry and biological activity testing was performed on this "current" preparation of SD8 to ensure that it is identical to the "original" preparation of SD8 that was isolated from S. antibioticus Tü 6040. Details of these experiments and their results are included in the supplemental material.

Supercoiling assay. Standard reaction mixtures (20 µl) contained 50 mM Tris-HCl (pH 8.0 at 23°C), 10 mM MgCl2, either 100 mM (for E. coli gyrase) or 600 mM (for S. aureus gyrase) potassium glutamate (KGlu), 10 mM dithiothreitol, 50 µg/ml bovine serum albumin, 1 mM ATP, 0.3 µg of the relaxed DNA, either 10 fmol (as a tetramer) of E. coli gyrase or 50 fmol (as a tetramer) of S. aureus gyrase, and various concentrations of either SD8 or ciprofloxacin (a generous gift from the Bayer Corporation). Reaction mixtures were incubated at 37°C for 15 min and terminated by adding EDTA to 25 mM and further incubating at 37°C for 5 min. The DNA products were analyzed by electrophoresis through vertical 1.2% SeaKem ME agarose (Lonza) gels (14 by 10 by 0.3 cm) at 2 V/cm for 15 h in a running buffer of 50 mM Tris-HCl (pH 7.9 at 23°C), 40 mM sodium acetate, and 1 mM EDTA (TAE buffer). Gels were stained with ethidium bromide (EtBr), then photographed, and quantified using an Eagle Eye II system (Stratagene).

Decatenation assay. Reaction mixtures (20 µl) contained 50 mM Tris-HCl (pH 8.0 at 23°C), 10 mM MgCl2, either 100 mM (for E. coli Topo IV) or 400 mM (for S. aureus Topo IV) KGlu, 10 mM dithiothreitol, 50 µg/ml bovine serum albumin, 1 mM ATP, 0.3 µg of kinetoplast DNA, 50 fmol (as a tetramer) of either E. coli Topo IV or S. aureus Topo IV, and various concentrations of either SD8 or ciprofloxacin. Reaction mixtures were incubated at 37°C for 15 min and terminated by adding EDTA to 25 mM and further incubating at 37°C for 5 min. The DNA products were analyzed, and the gels were photographed and quantified as described in the preceding paragraph.

Two-step DNA cleavage assay. Reaction mixtures (20 µl) contained 50 mM Tris-HCl (pH 8.0 at 23°C), 10 mM MgCl2, the indicated concentrations of KGlu (only when indicated in the figure legends), 10 mM dithiothreitol, 50 µg/ml bovine serum albumin, 1 mM ATP, 5 µg/ml tRNA, 0.3 µg of either the supercoiled or relaxed DNA (as indicated in the figure legends), and the indicated amounts (as a tetramer) of various topoisomerases. The indicated concentrations of either SD8 or ciprofloxacin were incubated in the first stage for 5 min at 37°C. Then, the indicated concentrations of either ciprofloxacin or SD8 were added, and the reaction mixtures were incubated during the second stage for 10 min at 37°C. Sodium dodecyl sulfate was added to a concentration of 1%, and the reaction mixtures were further incubated at 37°C for 5 min. EDTA and proteinase K were then added to 25 mM and 100 µg/ml, respectively, and the incubation was continued for an additional 15 min at 37°C. The DNA products were purified by extraction with phenol-chloroform-isoamyl alcohol (25:24:1, vol/vol) and then analyzed by electrophoresis through vertical 1.2% agarose gels at 2 V/cm for 15 h in TAE buffer that contained 0.5 µg/ml EtBr (unless otherwise indicated). After destaining in water, gels were photographed and quantified using an Eagle Eye II system.

SD8 treatment of E. coli. E. coli MG1655 (wild-type) cells were grown to mid-exponential phase in 10 ml of LB medium in Erlenmeyer flasks at 37°C. The cells were then reinoculated into 96-well plates at a dilution in LB medium such that cells were at a concentration giving an optical density at 600 nm (OD600) of 0.1. Various concentrations of SD8 (0 µM [dimethyl sulfoxide {DMSO}, the solvent for the drug], 5 µM, 10 µM, 20 µM, and 40 µM) were added to the wells. The OD600 of the culture was measured every 4 minutes (with a 30-s shaking period before measurement) over the course of 3 hours at 37°C in a Wallac 1420 plate reader (Perkin Elmer). In a separate assay, cells were grown in LB medium up to exponential phase and reinoculated into Erlenmeyer flasks containing 10 ml of fresh LB medium, at which point drugs of various concentrations (identical to the concentrations listed above) were added to the medium, and the cells were grown for 2 hours at 37°C, then diluted, and plated on LB agar plates containing no antibiotic. The plates were incubated overnight at 37°C, and the number of CFU was determined the following day.

The E. coli {Delta}acrB strain, a direct mutant of the wild-type MG1655 strain, was obtained from the Keio collection (1). E. coli {Delta}acrB cells were treated in a manner similar to that described above, except that the drug concentrations used were from 10 nM to 40 µM for the growth assay and the cells used for viability counts were grown with higher concentrations of drug (0 µM [DMSO], 20 µM, and 80 µM) for 2 hours before diluting and plating on LB agar plates.

Microarray and real-time PCR (RT-PCR) analyses. Relative mRNA abundances between treated and nontreated cells, or a mutant and the wild type, were compared using E. coli whole-genome DNA microarrays containing 99% of all annotated open reading frames and the stable RNA genes. The protocols for slide preparation, RNA purification, reverse transcription with the Cy dyes, hybridization, and image scanning were described previously (20).

The RNA samples were extracted from the cultures grown in triplicate from single isolates to an OD600 of 0.5 to 0.6 using the hot-phenol method (20). After treatment with 20 µM SD8 or an equivalent volume of DMSO as a control for 2 hours, cells were fixed with a 10% solution of ethanol and phenol (95%/5%, vol/vol), and total RNA was extracted using an RNeasy minikit (Qiagen).

Portions of the control and treated sample RNA were reverse transcribed using Superscript II reverse transcriptase (Invitrogen) and labeled for microarray hybridization using Cy3 and Cy5 dyes (Amersham Biosciences), and the dyes were swapped for one of the replicates. Arrays were hybridized for 6 h at 65°C, scanned, and then analyzed using the GenePix Pro 3.0 software. Intensities in both channels were smoothed using the lowess method, and dye- and array-specific noise was removed using the analysis of variance error model (19). In pairwise comparisons, differentially expressed genes were identified at a false discovery rate of less than 5% using the SAM package (44).

The remaining RNA was reverse transcribed to cDNA using Superscript II, and the RT-PCR for RNA quantification was carried out using Sybr green kit and ABI Prism 7900 (Applied Biosystems) according to the manufacturer's protocol. The absolute change in gene expression was calculated based on the method described by Livak and Schmittgen (23). The primers used for RT-PCR were as follows: topA forward, 5'-CGGCCCGATCGTTGAGTGTGA-3'; topA reverse, 5'-GTGGTGCCACTTCGCCGTTAC-3'; gyrA forward, 5'-GTTCGCGGTATTCGCTTAG-3'; gyrA reverse, 5'-GCGACTTGGTTGGGTATTC-3'; gyrB forward, 5'-AACGAACTGCTGGCAGAATAC-3'; gyrB reverse, 5'-TAAGTCGAGCGCACCTTTAC-3'; 16S rRNA forward: TGGCAAGCTTGAGTCTCGTA-3'; and 16S rRNA reverse, 5'-ACCTGAGCGTCAGTCTTCGT-3'.

Microarrays and the RT-PCR analyses for {Delta}acrB cells were performed in a similar fashion, except that cells were treated with either 5 µM of D8 or an equivalent volume (25 µl) of DMSO for 2 hours.

Visualization of nucleoids using DAPI staining. A 10-ml culture of {Delta}acrB cells was grown to mid-exponential phase in LB medium at 37°C, then split into two cultures, one treated with 20 µM SD8 and the other with DMSO (the solvent for the drug) for 30 min at 37°C. Cells were harvested and washed with 1x phosphate-buffered saline. 4',6-Diamidino-2-phenylindole (DAPI) (Promega) was added to a concentration of 5 µg/ml, and the cells were incubated at room temperature for 10 min. After the cells were washed twice with 1x phosphate-buffered saline, they were placed under the glass coverslips coated with poly-L-lysine solution and visualized using a BH-2 Olympus fluorescence/differential interference contrast microscope.


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RESULTS
 
In vitro pattern of SD8 activity. SD8 inhibits E. coli DNA gyrase in vitro (8) but has no antibacterial effect against E. coli (38). At the same time, SD8 inhibits S. aureus growth via an unknown mechanism (38). To develop a consistent picture of SD8's antibacterial effects, we set out to determine SD8 activity against representative gram-negative and gram-positive topoisomerases. We first investigated which topoisomerases are more likely to be targeted by SD8 on the basis of its inhibitory effects on E. coli and S. aureus type II topoisomerases.

(i) SD8 inhibits the supercoiling activity of S. aureus gyrase. As described previously (2, 14, 40), S. aureus topoisomerases require high concentrations of KGlu for their optimal catalytic activities. We found that the optimal KGlu concentrations for the supercoiling activity of S. aureus gyrase and the decatenation activity of S. aureus Topo IV were 600 to 800 mM and 400 mM, respectively (14; data not shown). Almost no activity of either S. aureus enzyme was detected under the conditions (50 to 100 mM KGlu) used in the catalytic assays for E. coli topoisomerases. Accordingly, the supercoiling assay for S. aureus gyrase was performed in the presence of 600 mM KGlu. Under these conditions, 50 fmol of S. aureus gyrase was capable of completely supercoiling 100 fmol of the relaxed DNA. The supercoiling activity of S. aureus gyrase was measured in the presence of various concentrations of either SD8 or ciprofloxacin (Fig. 2A). Unlike ciprofloxacin, SD8 was a potent inhibitor of S. aureus gyrase (50% inhibitory concentration [IC50] of 1.45 µM; Table 1), suggesting that gyrase is an effective target of SD8 in S. aureus.


Figure 2
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  		FIG. 2. SD8 is a potent inhibitor of S. aureus gyrase. The supercoiling reaction mixtures containing the relaxed DNA, either 50 fmol of S. aureus (Sa) gyrase (A) or 10 fmol of E. coli (Ec) gyrase (B), and the indicated concentrations of either SD8 or ciprofloxacin (Cipro) were incubated, and the DNA products were analyzed as described in Materials and Methods. Lanes 1 and 2 contain controls with gyrase and drug present (+) or absent (–). The arrows in panels A and B show that gyrase was present in lanes 3 to 13.


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  		TABLE 1. Drug sensitivities of S. aureus and E. coli topoisomerasesa

It required only 10 fmol of E. coli gyrase to completely supercoil 100 fmol of the relaxed DNA under our standard reaction conditions (100 mM KGlu). Therefore, the supercoiling assay for E. coli gyrase was performed using 10 fmol of E. coli gyrase (Fig. 2B). The IC50 of SD8 for E. coli gyrase was 0.41 µM, whereas that of ciprofloxacin was 2.35 µM (Table 1). Thus, SD8 was a more potent inhibitor of E. coli gyrase than ciprofloxacin was.

We also employed a two-step DNA cleavage assay to assess the differences, if any, in the SD8 sensitivity between S. aureus gyrase and E. coli gyrase (Fig. 3). This assay allowed us to measure the ability of SD8 to block the binding of gyrase to DNA as the inhibition of ciprofloxacin-induced covalent topoisomerase-DNA complex formation. On the basis of the comparable effects of SD8 on the supercoiling activities of S. aureus gyrase and E. coli gyrase (Table 1), we had expected to observe results with S. aureus gyrase at levels of SD8 similar to those seen with E. coli gyrase (Fig. 3A). Instead, significantly higher concentrations of SD8 were required for the inhibition of ciprofloxacin-induced covalent S. aureus gyrase-DNA complex formation (Fig. 3B). One possible explanation was that the high concentrations of KGlu present in the supercoiling assay for S. aureus gyrase could affect the apparent SD8 sensitivity of S. aureus gyrase by changing the conformation of S. aureus gyrase and/or the interactions among SD8, S. aureus gyrase, and DNA. To test this possibility, the two-step DNA cleavage assay was performed in the presence of a high concentration of KGlu (Fig. 3C). To accommodate the inhibitory effect of KGlu on the stimulation of the cleavage activity of bacterial type II topoisomerases by ciprofloxacin (2, 14, 32), 400 fmol (instead of 50 fmol) of S. aureus gyrase and 400 mM KGlu (we could not detect the DNA cleavage activity in the presence of 600 mM KGlu) were used in this assay. The presence of 400 mM KGlu significantly increased (about fourfold) the ability of SD8 to inhibit the formation of ciprofloxacin-induced covalent S. aureus gyrase-DNA complexes (Fig. 3C). These results suggest that SD8 can inhibit the supercoiling activity of S. aureus gyrase by interfering with its DNA binding, as was shown with E. coli gyrase (8).


Figure 3
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  		FIG. 3. The presence of KGlu affects the apparent SD8 sensitivity of S. aureus gyrase in the DNA cleavage assay. The two-step DNA cleavage assay was performed as described in Materials and Methods. (A) For E. coli (Ec) gyrase, 50 fmol of E. coli gyrase, the supercoiled DNA, and the indicated concentrations of SD8 were first incubated, and then 10 µM of ciprofloxacin (Cipro) was added to the reaction mixtures. (B and C) For S. aureus (Sa) gyrase, 400 fmol of S. aureus gyrase, the supercoiled DNA, and the indicated concentrations of SD8 were first incubated in the absence (B) or presence (C) of 400 mM KGlu, and then 50 µM of ciprofloxacin was added to the reaction mixtures. No EtBr was included in the TAE buffer. Lanes 1 and 2 contain controls with gyrase and drug present (+) or absent (– and 0). The arrows in panels A, B, and C show that gyrase was present in lanes 3 to 9 or 10.

(ii) Topo IVs are poor targets of SD8. Because of the high degree of similarity between gyrases and Topo IVs, many gyrase inhibitors also inhibit Topo IV, although the sensitivities of gyrase and Topo IV from a particular bacterium to each drug are usually different (6, 7, 22, 24, 28). Thus, we examined whether SD8 could also inhibit the decatenation activity of either S. aureus Topo IV or E. coli Topo IV.

Again, because of the differences in optimal salt concentrations, the decatenation assays for S. aureus Topo IV and E. coli Topo IV were performed in the presence of 400 mM and 100 mM KGlu, respectively, using 50 fmol of either S. aureus Topo IV or E. coli Topo IV (Fig. 4). SD8 exhibited a moderate inhibitory effect on S. aureus Topo IV, but E. coli Topo IV was essentially insensitive to SD8 (Table 1). The IC50 of SD8 for E. coli Topo IV (270 µM) was almost 20-fold higher than that for S. aureus Topo IV (14.5 µM) and almost 8-fold higher than the IC50 of ciprofloxacin for E. coli Topo IV (34 µM). On the basis of the differences in SD8 sensitivity between gyrases and Topo IVs, gyrase appeared to be the target of SD8 in both S. aureus and E. coli.


Figure 4
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  		FIG. 4. Effects of SD8 on the decatenation activity of Topo IV. The decatenation reaction mixtures containing kinetoplast DNA, 50 fmol of either S. aureus (Sa) Topo IV (A) or E. coli (Ec) Topo IV (B), and the indicated concentrations of either SD8 or ciprofloxacin (Cipro) were incubated, and the DNA products were analyzed as described in Materials and Methods. Lanes 1 and 2 contain controls with Topo IV and drug present (+) or absent (– and 0). The arrows in panels A and B show that Topo IV was present in lanes 3 to 12.

The two-step DNA cleavage assay for S. aureus Topo IV was also performed in the absence (see Fig. S3A in the supplemental material) or presence of 400 mM KGlu (see Fig. S3B in the supplemental material). Again, the IC50 of SD8 was reduced about fourfold by the presence of 400 mM KGlu. The IC50 for E. coli Topo IV was 200 to 400 µM (see Fig. S3C in the supplemental material), which was close to the value seen in the catalytic assay (Table 1 and Fig. 4).

In vivo pattern of SD8 activity. The data presented above essentially ruled out Topo IV as an SD8 target in E. coli. They also suggested that the inhibitory effect of SD8 against E. coli gyrase and S. aureus gyrase was comparable and that it was better than the antisupercoiling activity of ciprofloxacin against either enzyme. If the in vitro effective concentrations are any indication, then the enzymatic activities of E. coli gyrase and S. aureus gyrase in vivo should be as susceptible to SD8 as they are to ciprofloxacin. Thus, the effect of SD8 on S. aureus is mediated, at least in part, by targeting DNA gyrase. On the other hand, the lack of SD8 antibacterial activity against E. coli could be due to the low effective concentration of the drug in vivo, the tolerance of the specific bacterial system to the inhibition of supercoiling activity (as can be inferred from a study by Jensen et al. [17]), or a combination of both factors.

(i) The AcrB multidrug efflux pump affects the efficacy of SD8 against E. coli. SD8 did not affect the growth of the wild-type MG1655 strain at any concentration up to 80 µM (Fig. 5A and data not shown). It has been shown that E. coli type II topoisomerases can be inhibited in vivo by coumarin antibiotics in mutants with deficient efflux (10, 21). To test whether efficient efflux also protects the E. coli target(s) from SD8, we examined the effect of SD8 on growth of a {Delta}acrB mutant. The AcrB protein is one of the resistance-nodulation-division family of multidrug efflux pumps (26, 27, 43). SD8 inhibited bacterial growth of the {Delta}acrB strain at a concentration as low as 5 µM (Fig. 5B). SD8 treatment of {Delta}acrB cells also resulted in the elongation of the cells (see Fig. S4 in the supplemental material). Despite clear bacteriostatic effects across a range of concentrations, SD8 treatment did not result in more than a 50% reduction in viability counts, even at 80 µM (data not shown). These results showed that the resistance of wild-type E. coli to SD8 was, at least in part, due to efflux of the drug from the cell. In the absence of AcrB-mediated efflux, SD8 effectively inhibited bacterial growth at concentrations comparable to the effective concentration of novobiocin (data not shown), and as with novobiocin, the effect of SD8 was largely bacteriostatic.


Figure 5
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  		FIG. 5. Effect of SD8 on growth of wild-type and {Delta}acrB strains. Absorbance (Abs) over time at 600 nm of wild-type (A) and {Delta}acrB (B) cultures treated with various concentrations of SD8 recorded in a 96-well format. The error bars represent 1 standard deviation of measurements done in triplicate. The cells were treated with SD8 (D8) concentrations ranging from 10 nM to 50 µM; however, only results from 1 µM to 50 µM are depicted, as there was little difference in the growth pattern of cells treated with <1 µM SD8.

(ii) The SD8 treatment causes the loss of supercoiling activity, but not DNA damage, in E. coli. The results presented above point toward the existence of an intracellular target(s) for SD8. Because the drug does not fully inhibit bacterial growth in liquid medium or on plates, it was not feasible to set up a positive genetic screen to identify the target(s). On the other hand, it has been repeatedly demonstrated that genome-wide transcriptional profiles can reliably capture regulatory patterns associated with molecular mechanisms of drug activity (5, 9, 18, 31, 43). Therefore, we proceeded to assess the nature of the SD8 target by examining the regulatory consequences of the drug treatment on the transcriptional activity of the entire genome. An "SD8 expression profile" was constructed from the set of genes most significantly affected by SD8. When we compared the SD8 profile with the expression profiles, for the same set of genes, from almost 200 conditions of normal growth, growth arrest, and killing by antibacterial drugs and conditional mutations (37), we found that the SD8 expression profile was most similar to the profiles that were observed under conditions of gyrase inhibition, either by a mutation or by norfloxacin or novobiocin treatment (Fig. 6). The probability of observing such similarities by chance, estimated by bootstrapping, was less than 1 in 1,000.


Figure 6
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  		FIG. 6. Transcriptional profile of SD8 treatment is most similar to the profile of gyrase inhibition. For the set of genes significantly affected by SD8 treatment, the Pearson correlation coefficients between the vector obtained in SD8 treatment and each of the 198 microarray conditions were calculated and plotted in descending order.

In 30 min of SD8 treatment, twice as many genes were significantly upregulated as were downregulated (132 versus 64 with a 95th percentile false discovery rate of 5%; see Table S2 in the supplemental material). The most downregulated gene (topA) encodes for topoisomerase I. Additionally, eight genes were downregulated at least twofold. The products of three of these genes, topA, hns, and fis, are known to be involved in removal of negative supercoils, either directly by the reaction of relaxation, as with topoisomerase I, or by constraining free supercoils, as with H-NS and FIS (3). The most downregulated genes also included cspA, a positive regulator of hns.

Of the 22 most upregulated genes (twofold or more), the products of 5 genes are associated with the control of initiation of chromosomal DNA replication, including the gyrA and gyrB genes, dnaA, mioC, and gidA. Importantly, the upregulation of DNA gyrase genes gyrA and gyrB was consistent with the downregulation of the topA gene, demonstrating that SD8 treatment activated the supercoiling-dependent regulatory loop, in which reduction in the supercoiling activity of DNA gyrase leads to downregulation of topA transcription and upregulation of transcription of the gyrase genes (25). The changes in the transcript levels of topA and the gyrase genes were confirmed by the RT-PCR experiments. The changes in the gyrase and topA transcripts in E. coli {Delta}acrB cells treated with SD8 were 0.31-fold ± 0.14-fold for topA, 8.21-fold ± 2.76-fold for gyrA, and 5.55-fold ± 5.55-fold for gyrB compared to a solvent-treated control.

More formal analysis of gene sets revealed that two classes of genes were overrepresented among the genes significantly upregulated by SD8, genes encoding surface antigens (enterobacterial common antigen and the O antigen of LPS) (P value corrected for multiplicity of measurements < 10–3) and genes encoding DNA replication proteins (P < 0.02). Both sets of genes were affected to a similar extent by SD8 and novobiocin treatment (Fig. 7), whereas norfloxacin had a smaller effect on these sets of genes (data not shown). We carried out a similar analysis with the characteristic profiles of norfloxacin and novobiocin treatments. The transcriptional signature of the norfloxacin effect was the induction of the SOS response (adjusted P value < 10–11), induction of the tricarboxylic acid (TCA) cycle genes (P < 10–18), and downregulation of genes encoding components of the citrate-dependent iron transport system (P < 10–7) (18, 37). We found that the downregulation profiles of the iron transport cluster were similar in norfloxacin and SD8 treatments (Fig. 8). However, SD8 treatment did not result in significant induction of the SOS and TCA genes compared to norfloxacin treatment (Fig. 8). The transcriptional signature of the novobiocin effect was induction of RpoH targets (P < 10–8), induction of relaxation-sensitive genes (P < 10–5), and downregulation of supercoiling-sensitive genes (P < 10–10) (31, 37). In SD8 treatment, relaxation-sensitive genes as a group were similarly affected, but not the RpoH and supercoiling-sensitive genes (Fig. 9). It must be pointed out that similar differences can be seen between the effects of norfloxacin and novobiocin.


Figure 7
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  		FIG. 7. Surface antigen and DNA replication genes are similarly upregulated by SD8 and novobiocin. Pairwise correlations between profiles (expressed as one-dimensional vectors of log-transformed ratios of transcript abundances) of genes encoding DNA replication proteins and of genes encoding surface antigens. The reference treatments are indicated along the x axes. In Fig. 7 to 9, only the genes that were identified as significantly affected at a median false discovery rate of 5% or less are labeled.


Figure 8
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  		FIG. 8. The pattern of SD8 activity shares limited similarity with transcriptional patterns of the norfloxacin effect. The characteristic profiles of norfloxacin treatment, the induction of the SOS response, upregulation of the TCA cycle genes, and downregulation of genes encoding components of the citrate-dependent iron transport system, were compared with those of SD8 treatment. Pairwise correlations between profiles of gene classes are shown as described in the legend to Fig. 7.


Figure 9
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  		FIG. 9. The pattern of SD8 activity shares limited similarity with transcriptional patterns of the novobiocin effect. The transcriptional signatures of the novobiocin effect, the induction of RpoH targets, upregulation of relaxation-sensitive genes, and downregulation of supercoiling-sensitive genes, were compared with those of the SD8 treatment. Pairwise correlations between profiles of gene classes are shown as described in the legend to Fig. 7.

(iii) Effect of SD8 on the state of the bacterial nucleoid. The data presented above showed that SD8 affected the state of DNA supercoiling in vivo, likely by inhibiting the supercoiling activity of gyrase. Since the level of superhelicity is an essential attribute of the bacterial nucleoid, which contributes to chromosome organization (34) and packing (39), we hypothesized that the inhibition of supercoiling by SD8 in vivo should result in at least partial unraveling of the nucleoid. This unraveling could be visualized by fluorescence microscopy as characteristic changes in the nucleoid morphology. We examined the morphology of the E. coli nucleoid following SD8 treatment of {Delta}acrB cells using DAPI staining. SD8 treatment resulted in detectable changes in the morphology of the nucleoid, such as a noticeably larger area occupied by the nucleoids, as well as the unusual shape of the nucleoid contour (Fig. 10). Such an outcome, which may be indicative of reduced DNA scaffolding by gyrase, can be explained by the inhibition of gyrase biding to DNA by SD8 (8). Consistent with these observations, we found that SD8 interfered with the cleavage activity of gyrase in vitro (see Fig. S5 in the supplemental material). This may also explain the lack of the DNA damage response (Fig. 8).


Figure 10
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  		FIG. 10. Effect of SD8 on nucleoid morphology. Exponentially grown {Delta}acrB cells were treated with either SD8 or DMSO, the solvent of SD8, stained with DAPI, and visualized by fluorescence (A, B, E, and F) and phase-contrast (C, D, G, and H) microscopy.


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DISCUSSION
 
DNA gyrase and Topo IV are essential enzymes that display distinct cellular functions (4, 45). Gyrase is the only topoisomerase that introduces negative supercoils into DNA, whereas Topo IV is responsible for decatenation of sister chromosomes. The importance of bacterial type II topoisomerases is underscored by the fact that both gyrase and Topo IV are the cellular targets of clinically important antibacterial agents, the quinolones and the coumarins (6, 24). The quinolone drugs poison gyrase and Topo IV, whereas the coumarins inhibit their ATPase activities. Interestingly, each quinolone drug selects one of two topoisomerases as its primary target in vivo, and the primary target changes depending on the drug, as well as the bacterium (6, 7, 22, 24, 28). For instance, ciprofloxacin targets gyrase in E. coli and Topo IV in S. aureus. In our catalytic assays (Table 1), the primary targets, E. coli gyrase and S. aureus Topo IV, are at least fivefold more sensitive to ciprofloxacin than the secondary targets, E. coli Topo IV and S. aureus gyrase.

It has been shown that SD8 inhibits E. coli gyrase by a novel mechanism, by binding to the catalytic domain of the GyrA subunit and preventing gyrase from binding to DNA (8). SD8, however, exhibits antibiotic activity against gram-positive, but not gram-negative, bacteria (38). We examined the effects of SD8 on the catalytic activities of S. aureus topoisomerases in order to determine the target of SD8 in gram-positive bacteria. We found that SD8 could inhibit the supercoiling activity of S. aureus gyrase, as well as that of E. coli gyrase (Table 1). In addition, we found that, as was the case with E. coli gyrase (8), SD8 could interfere with the binding of S. aureus gyrase to DNA (Fig. 3). In contrast, SD8 had only a modest inhibitory effect on S. aureus Topo IV, and E. coli Topo IV was not sensitive to SD8. Thus, gyrase seems to be the target of SD8 in both gram-positive and gram-negative bacteria, suggesting that the differences in the susceptibilities of gram-positive and gram-negative bacteria to SD8 are not due to large differences in the sensitivities of topoisomerases to SD8.

DNA gyrases and Topo IVs share extensive homologies, and their differences in susceptibilities to various inhibitors are determined by subtle differences in the amino acid residues of the drug binding pockets (4, 6, 24, 45). Although the binding pocket of SD8 has yet to be determined, it appears that SD8 binds to the GyrA subunit through a bipartite interaction (A. Maxwell, personal communication). Such an interaction might explain the differences in the susceptibilities of gyrase and Topo IV to SD8. For instance, although SD8 interacts with the GyrA subunit at two binding sites, SD8 might be able to bind to the ParC subunit only at one of the two binding sites. Of course, the determination of the SD8 binding pockets on gyrase and Topo IV is required to understand the molecular basis of the different susceptibilities of gyrase and Topo IV to SD8.

The DNA cleavage assays for S. aureus topoisomerases revealed an interesting effect of KGlu on the SD8 sensitivity (Fig. 3). S. aureus gyrase can bind and cleave DNA in the absence of KGlu but requires high concentrations of KGlu to catalyze the supercoiling reaction (14). The concentrations of SD8 required to inhibit the formation of ciprofloxacin-induced S. aureus gyrase-DNA complexes in the absence of KGlu were significantly higher than those in the presence of KGlu. Thus, KGlu affected the apparent SD8 sensitivity of S. aureus gyrase. This could be due to the accessibility of SD8 to its binding pocket in different conformations of the topoisomerase. It is possible that SD8 can interact with S. aureus gyrase effectively when S. aureus gyrase is in an active conformation for supercoiling (in the presence of high concentrations of KGlu), but not when S. aureus gyrase is in an inactive conformation for supercoiling (in the absence of KGlu).

Genome-wide transcriptional profiles can reliably capture regulatory patterns associated with molecular mechanisms of drug activity (5, 9, 18, 31, 43). The transcriptional response of E. coli to SD8 treatment mimics the profiles of gyrase inhibition. It also has certain unique characteristics that suggest that SD8-treated cells are not as stressed (limited DNA damage response, no heat shock response, and general stress response) as cells treated with either norfloxacin or novobiocin. It is possible that these differences are due to the fact that SD8 does not reach sufficiently high concentration in the cell to produce those secondary effects. However, because the effects of norfloxacin and novobiocin are also different and because the intracellular SD8 concentration appears to be high enough to elicit certain characteristic responses, it is also quite possible that the observed transcriptional variations are due to SD8's unique mechanism of gyrase inhibition, which does not involve conversion of gyrase into a poison. These results also suggest that the main target for SD8 in vivo is indeed gyrase.

There are many possible reasons why SD8 is effective against gram-positive bacteria, but not against gram-negative bacteria. It is possible, although unlikely, that a modest inhibition of the S. aureus Topo IV activity by SD8 could contribute to the effectiveness of SD8 against S. aureus. However, since SD8 was capable of inhibiting both S. aureus gyrase and E. coli gyrase, the lack of inhibition of E. coli gyrase is highly unlikely to be the reason why SD8 is ineffective against E. coli. Multidrug efflux pumps, especially those belonging to the resistance-nodulation-division family, play an important role in establishing the resistance of gram-negative bacteria to a number of agents (33, 35). The acrB deletion affected the sensitivity of E. coli to SD8 (Fig. 5). Thus, the AcrB multidrug efflux pump is partially responsible for the ineffectiveness of SD8 against gram-negative bacteria.

Antibiotics have been life-saving interventions for decades. Infections of gram-positive bacteria, particularly methicillin-resistant S. aureus, are often considered life threatening, and drug development tends to focus on gram-positive pathogens. However, the recent emergence of drug-resistant gram-negative bacteria presents an equally, if not more, important challenge to public health (36, 41). Unfortunately, the development of drug-resistant bacterial pathogens seems to occur faster than the development of new antibacterial drugs by pharmaceutical companies. One approach to develop a drug that is effective against gram-negative pathogens is the co-optation of drugs that are active against gram-positive bacteria but not against gram-negative bacteria. This process of co-optation would involve creating artificial conditions where gram-negative bacteria could become susceptible to the drugs, investigating mechanisms of action, and tailoring chemical structures of the drugs to overcome nonspecific defensive mechanisms of gram-negative bacteria if such targets can be found. This work presents a case study, using SD8 as a model drug, of the first two steps. Efforts are under way to identify chemical modifications that improve the potency of SD8 and to broaden its spectrum.


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ACKNOWLEDGMENTS
 
We thank Gunda Georg for her support and encouragement, Anthony Maxwell for his critical comments on the manuscript, Fred Schendel of the University of Minnesota Biotechnology Resources Center for his expertise and services, and Dipen Sangurdekar for his help in generating a transcriptional heat map of various gyrase-inhibiting conditions and summarizing genes into GenProtEC classifications. Microscopy was performed at the University of Minnesota-College of Biological Sciences, Imaging Center.

This work was supported in part by a Faculty Research Development Grant from the University of Minnesota Academic Heath Center (to H.H.), a Pilot Project Grant from the Specialized Program in Research Excellence grant P20 CA101955 (to H.H.), funds from the University of Minnesota Medical School (to H.H.), National Institutes of Health grant GM66098 (to A.B.K.), Biotechnology Training Grant (to B.L.H.), and funds from a University of Minnesota McKnight Presidential Endowment in Medicinal Chemistry (to K.C.E. via Gunda Georg).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pharmacology, University of Minnesota Medical School—Twin Cities, 6-120 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455. Phone: (612) 626-3101. Fax: (612) 625-8408. E-mail: hiasa001{at}umn.edu Back

{triangledown} Published ahead of print on 9 March 2009. Back

{dagger} Supplemental material for this article may be found at http://aac.asm.org/. Back


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Antimicrobial Agents and Chemotherapy, May 2009, p. 2110-2119, Vol. 53, No. 5
0066-4804/09/$08.00+0     doi:10.1128/AAC.01440-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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