Dual Targeting of DNA Gyrase and Topoisomerase IV: Target Interactions of Garenoxacin (BMS-284756, T-3811ME), a New Desfluoroquinolone

İnce, Dilek; Zhang, Xiamei; Silver, Leah Christine; Hooper, David C.

doi:10.1128/aac.46.11.3370-3380.2002

Cited by 75 publications

(70 citation statements)

References 59 publications

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“…Thus, in order to obtain the expression of the resistance phenotype, which could only be selected on sparfloxacin or moxifloxacin, the PCR products which harbored the different mutations in topoisomerase IV were introduced into TR6, a derivative of S. pneumoniae R6 harboring a GyrA Ser81Phe mutation (50). Interestingly in GrlB of Staphylococcus aureus, an Asp33Tyr mutation at a position equivalent to position 37 in ParE of S. pneumoniae was recently found to increase by twofold the MICs of FQs in the absence of other mutations in GyrA (24). This result would indicate that for S. aureus and in contrast to S. pneumoniae, a mutation in the N terminus of the ATPase domain of topoisomerase IV can be expressed in the absence of a GyrA mutation.…”

Section: Vol 185 2003 Atp-bound Conformation Of Topoisomerase IV 6143mentioning

confidence: 82%

ATP-Bound Conformation of Topoisomerase IV: a Possible Target for Quinolones in Streptococcus pneumoniae

et al. 2003

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Topoisomerase IV, a C 2 E 2 tetramer, is involved in the topological changes of DNA during replication. This enzyme is the target of antibacterial compounds, such as the coumarins, which target the ATP binding site in the ParE subunit, and the quinolones, which bind, outside the active site, to the quinolone resistance-determining region (QRDR). After site-directed and random mutagenesis, we found some mutations in the ATP binding site of ParE near the dimeric interface and outside the QRDR that conferred quinolone resistance to Streptococcus pneumoniae, a bacterial pathogen. Modeling of the N-terminal, 43-kDa ParE domain of S. pneumoniae revealed that the most frequent mutations affected conserved residues, among them His43 and His103, which are involved in the hydrogen bond network supporting ATP hydrolysis, and Met31, at the dimeric interface. All mutants showed a particular phenotype of resistance to fluoroquinolones and an increase in susceptibility to novobiocin. All mutations in ParE resulted in resistance only when associated with a mutation in the QRDR of the GyrA subunit. Our models of the closed and open conformations of the active site indicate that quinolones preferentially target topoisomerase IV of S. pneumoniae in its ATP-bound closed conformation.Topoisomerase IV and DNA gyrase are bacterial type II topoisomerases, which modify DNA topology during the replication process by unlinking DNA and facilitating chromosome segregation (59). Topoisomerase IV forms a C 2 E 2 tetramer involved in segregation of the chromosome at cell division (1, 30, 59). The ParC subunit contains the site of topoisomerization catalyzing the double-stranded DNA break (30, 53), while the ParE subunit catalyzes the hydrolysis of ATP, providing the free energy necessary for these reactions (2, 6, 11). The ParE and ParC subunits share extensive sequence homology with, respectively, the GyrB and GyrA subunits of the DNA gyrase A 2 B 2 tetramer, which catalyzes negative DNA supercoiling during the initiation and elongation processes of DNA replication (16,53).Both topoisomerases are the targets of antibacterial molecules, such as the quinolones and the coumarins. The coumarins inhibit supercoiling and enzyme turnover by preventing the binding and hydrolysis of ATP (45). The quinolones form a ternary complex with the topoisomerases in the presence of DNA, resulting in lethal double-stranded DNA breaks (11). Enzymatic studies and binding assays have also shown that the quinolones can form a complex with the topoisomerases before binding DNA (15, 29); however, some binding data indicate the occurrence of a specific and higher level of binding to the enzyme-DNA complex rather than to the enzyme alone (43,56). These families of molecules, in particular, the fluoroquinolones (FQs), are under continuous development as resistance to antibiotics in pathogenic bacteria has dramatically increased during the last decade (20,22,49).

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Section: Vol 185 2003 Atp-bound Conformation Of Topoisomerase IV 6143mentioning

confidence: 82%

ATP-Bound Conformation of Topoisomerase IV: a Possible Target for Quinolones in Streptococcus pneumoniae

et al. 2003

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“…The MICs of premafloxacin and ciprofloxacin were fourfold greater for mutant P18 than for wild-type strain ISP794 (Table 3); this difference in MICs was similar to the differences observed for other first-step mutants with parEC mutations selected with premafloxacin (20). The MICs of novobiocin (which was used to screen for some parE mutations [11,12,20,21,23,24]) and nalidixic acid (which was used to screen for some gyrA mutations [23]) were not different for the mutant and the wild type. Because other premafloxacin-selected, first-step mutants of S. aureus had had mutations in parE or parC (20), we first sequenced the quinolone resistance-determining regions of the parE and parC structural genes of mutant P18.…”

Section: Resultsmentioning

confidence: 98%

“…Thus, we speculate that S. aureus tolerates reduced levels of topoisomerase IV and that this tolerance may account for the broader range of quinolone resistance mutations found for this drug target compared to the mutations found for DNA gyrase. In addition to the mutation in P18, we found a range of quinolone resistance mutations outside the classical quinolone resistance-determining regions of ParC and ParE (20)(21)(22)(23)(24), and purified topoisomerase IV reconstituted with some of the mutant subunits exhibited reduced catalytic function (X. Zhang and D. Hooper, unpublished observations). Although DNA gyrase and topoisomerase IV are both essential enzymes and there are lethal null mutations in the genes encoding subunits of these enzymes, the cell may tolerate incomplete reductions in the amounts of the two enzymes differently.…”

Section: Discussionmentioning

confidence: 99%

“…Experiments were repeated several times and were performed with different cell lysates to ensure that the differences seen were not due to chance differences in loading. The methods used for overexpression and purification of histidine-tagged ParE and GyrB proteins have been described previously (23). Purified ParE protein was used as a positive control, and purified GyrB was used to show that there was no cross-reactivity between anti-ParE antibodies and GyrB.…”

Section: Methodsmentioning

confidence: 99%

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Quinolone Resistance Due to Reduced Target Enzyme Expression

İnce

Hooper

2003

J Bacteriol

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We report for the first time low-level quinolone resistance mediated by decreased expression of topoisomerase IV in Staphylococcus aureus. A single-step mutant of wild-type S. aureus strain ISP794, P18 selected by using twice the MIC of premafloxacin, had four-and four-to eightfold greater MICs of premafloxacin and ciprofloxacin, respectively, than the wild type. Sequencing of parEC and gyrBA with their promoter regions revealed a point mutation (G3A) 13 bp upstream of the start codon of parE. Genetic linkage studies showed that there was a high level of correlation between the mutation and the resistance phenotype, and allelic exchange confirmed the contribution of the mutation to resistance. Decreased expression of ParE and decreased steady-state levels of parEC transcripts in P18 and in resistant allelic exchange mutants were observed. The steady-state levels of gyrBA and topB transcripts were increased in P18 but not in two resistant allelic exchange mutants, and sequencing upstream of either gene did not reveal a difference between ISP794 and P18. The steady-state levels of topA transcripts were similar in the various strains. Growth competition experiments performed at 30, 37, and 41°C with a susceptible allelic exchange strain and a resistant allelic exchange strain suggested that loss of fitness was associated with reduced levels of ParE at 41°C. However, P18 had a growth advantage over ISP794 at all temperatures, suggesting that a compensatory mechanism was associated with the increased levels of gyrBA and topB transcripts. Thus, reduced levels of ParE appear to be compatible with cell survival, although there may be a fitness cost during rapid cell multiplication, which might be overcome by compensatory mechanisms without reversion of the resistance phenotype.

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“…Fluoroquinolones act on DNA gyrase and topoisomerase IV to inhibit bacterial DNA replication (12). Mutations in gyrA and gyrB encoding the subunits of DNA gyrase (21,23,26) and grlA and grlB encoding the subunits of DNA topoisomerase IV of S. aureus (13,38) lead to quinolone resistance in gram-positive and gram-negative bacteria. One of the most intriguing mechanisms underlying resistance to fluoroquinolones as well as a range of other antimicrobial agents involves the extrusion of a variety of structurally unrelated compounds due to active efflux by membrane pumps (19,51).…”

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confidence: 99%

Characterization of NorR Protein, a Multifunctional Regulator of norA Expression in Staphylococcus aureus

2003

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We characterized a Staphylococcus aureus norA gene expression regulator, NorR, initially identified from its binding to the norA promoter. The norR gene was 444 bp in length, located ϳ7 kb upstream from the norA gene, and encoded a predicted 17.6-kDa protein. Overexpression of norR in wild-type S. aureus strain ISP794 led to a fourfold decrease in sensitivity to quinolones and ethidium bromide and an increase in the level of norA transcripts, suggesting that NorR acts as a positive regulator of norA expression. Overexpression of norR in sarA and agr mutants did not alter quinolone sensitivity or levels of norA transcription, indicating that the presence of these two global regulatory systems is necessary for NorR to affect the expression of norA. Insertion and disruption of norR in ISP794 increased resistance to quinolones by 4-to 16-fold but had no effect on norA transcription, suggesting that NorR acts as a repressor for another unidentified efflux pump or pumps. These mutants also exhibited an exaggerated clumping phenotype in liquid media, which was complemented fully by a plasmid-encoded norR gene. Collectively, these results indicate that NorR is a multifunctional regulator, affecting cell surface properties as well as the expression of NorA and likely other multidrug resistance efflux pumps.

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Dual Targeting of DNA Gyrase and Topoisomerase IV: Target Interactions of Garenoxacin (BMS-284756, T-3811ME), a New Desfluoroquinolone

Cited by 75 publications

References 59 publications

ATP-Bound Conformation of Topoisomerase IV: a Possible Target for Quinolones in Streptococcus pneumoniae

ATP-Bound Conformation of Topoisomerase IV: a Possible Target for Quinolones in Streptococcus pneumoniae

Quinolone Resistance Due to Reduced Target Enzyme Expression

Characterization of NorR Protein, a Multifunctional Regulator of norA Expression in Staphylococcus aureus

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