Fluoroquinolones Inhibit Preferentially<i>Streptococcus pneumoniae</i>DNA Topoisomerase IV Than DNA Gyrase Native Proteins

Fernández-Moreira, Esteban; Balas, Delia; González, Isabel del Bosque; Campa, Adela G. de la

doi:10.1089/mdr.2000.6.259

Cited by 27 publications

(23 citation statements)

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“…Under these conditions the WT and H103Y mutant ParE had identical specific activities either in the decatenation assay (3 ϫ 10 5 U/mg) or in the relaxation assay (4 ϫ 10 4 U/mg). These activities were within the range reported for the WT topoisomerase IV of S. pneumoniae (5,20) and, as described previously, the relaxation activity was even about 11-fold lower than the decatenation activity for the ParE mutant topoisomerase IV (8,11,20). Since the specific topoisomerase IV activities of the homologs containing WT ParE and H103Y mutant ParE were determined in the presence of an excess of ATP, this could have hidden more-subtle alterations in ATP binding.…”

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Contribution of the ATP Binding Site of ParE to Susceptibility to Novobiocin and Quinolones in Streptococcus pneumoniae

et al. 2005

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In Streptococcus pneumoniae, an H103Y substitution in the ATP binding site of the ParE subunit of topoisomerase IV was shown to confer quinolone resistance and hypersensitivity to novobiocin when associated with an S84F change in the A subunit of DNA gyrase. We reconstituted in vitro the wild-type topoisomerase IV and its ParE mutant. The ParE mutant enzyme showed a decreased activity for decatenation at subsaturating ATP levels and was more sensitive to inhibition by novobiocin but was as sensitive to quinolones. These results show that the ParE alteration H103Y alone is not responsible for quinolone resistance and agree with the assumption that it facilitates the open conformation of the ATP binding site that would lead to novobiocin hypersensitivity and to a higher requirement of ATP.Topoisomerase IV and DNA gyrase are bacterial type II topoisomerases that modify DNA topology during the replication process by unlinking DNA and facilitating chromosome segregation (33). DNA gyrase, encoded by gyrA and gyrB, forms an A 2 B 2 tetramer that removes the positive supercoils introduced during DNA replication and helps to maintain the steady-state balance of negative supercoiling of the bacterial DNA (30). Topoisomerase IV, encoded by parC and parE, forms a C 2 E 2 tetramer. Its primary role is the decatenation of daughter chromosomes following DNA replication, and other functions are DNA relaxation and double-strand DNA breakage (1,6,14,33). Both enzymes are the target of two antibiotic families: the quinolones, such as ciprofloxacin, sparfloxacin, and grepafloxacin, and the coumarins, such as novobiocin. The quinolones form a ternary complex with topoisomerases in the presence of DNA, resulting in double-stranded breaks (4). The coumarins inhibit ATP-dependent functions of these enzymes by preventing binding and hydrolysis of ATP (27).In Streptococcus pneumoniae, it has been shown that the primary and the secondary targets of quinolones are either the gyrase or the topoisomerase IV, depending upon the molecule (21, 29). Mutants resistant to quinolones most frequently harbor mutations in the quinolone resistance-determining regions (QRDR) of GyrA and ParC and less frequently in that of ParE (7). The level of quinolone resistance increases when a mutation is present in the secondary target in addition to one in the primary target (21, 29). In S. pneumoniae, in vitro-selected resistance to novobiocin is associated with the S127L substitution in the GyrB subunit (17). This position is located upstream from and close to positions 136 and 164 described in the ATP binding domain of GyrB in Escherichia coli and also involved in coumarin resistance (3). The substitution in ParE of E. coli at a position equivalent to that of position 136 in GyrB resulted in a decreased inhibition of topoisomerase IV by novobiocin (6).In previous studies (11, 26), we showed in S. pneumoniae that the H103Y substitution in the N terminus of the ParE subunit of the topoisomerase IV resulted in an original phenotype of resistance only if it was as...

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Contribution of the ATP Binding Site of ParE to Susceptibility to Novobiocin and Quinolones in Streptococcus pneumoniae

et al. 2005

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“…The drug concentrations causing 50% inhibition of kinetoplast DNA decatenation by topoisomerase IV were 10 to 20 M for both drugs (data not shown). Other studies have reported similar findings (4,14,17,22). In DNA cleavage assays for gyrase and topoisomerase IV, both drugs caused 25% linearization of input DNA at 80 and 2.5 M, respectively.…”

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confidence: 73%

Grepafloxacin, a Dimethyl Derivative of Ciprofloxacin, Acts Preferentially through Gyrase in Streptococcus pneumoniae : Role of the C-5 Group in Target Specificity

Morris

Pan

Fisher

2002

Antimicrob Agents Chemother

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Grepafloxacin, a 5-methyl-7-piperazinyl-3-methyl analogue of ciprofloxacin, was used to obtain stepwiseselected mutants of Streptococcus pneumoniae 7785. Analysis of the quinolone resistance-determining regions of the gyrA, gyrB, parC, and parE genes in these mutants revealed that gyrA mutations preceded those in parC. Given that ciprofloxacin (5-H,7-piperazinyl) and AM-1121 (5-H,7-piperazinyl-3-methyl) both act through topoisomerase IV, we conclude that the 5-methyl group of grepafloxacin favors gyrase in S. pneumoniae.In recent years, fluoroquinolones such as levofloxacin, grepafloxacin, gatifloxacin, moxifloxacin, and sparfloxacin have been introduced for the treatment of pneumonia and other diseases due to Streptococcus pneumoniae, a major gram-positive pathogen (2, 26). By interfering with DNA gyrase and topoisomerase IV, two essential enzymes that function through a double-stranded DNA break (3,7,8,13,31), these agents are also effective against S. pneumoniae isolates that exhibit decreased susceptibility to penicillins and macrolides (29).The precise mode of action of a quinolone against S. pneumoniae depends on its molecular structure (20,23). One group of quinolones, whose archetype is ciprofloxacin and which includes levofloxacin, norfloxacin, pefloxacin, and trovafloxacin, selects first-step mutants altered in the quinolone resistancedetermining region (QRDR) of parC or parE, indicating that topoisomerase IV is the intracellular target (5,9,11,16,18,24,28). A second group includes sparfloxacin and NSFQ-105 and selects first-step gyrA QRDR mutants, implying that gyrase is the preferred target (1,5,20,24). Clinafloxacin, gatifloxacin, gemifloxacin, and moxifloxacin select gyrA mutants at a rather low frequency (6, 10, 21) and seem to act through both enzymes, so they are called dual-targeting agents. Efforts to understand drug targeting have indicated a role for the C-7 and C-8 substituents (1, 6), but little is known about the effects of C-5 substitution. We realized that grepafloxacin, a simple 5-methyl-7-piperazinyl-3Ј-methyl derivative of ciprofloxacin (Fig. 1), could be informative. Previous work has shown that gyrA or parC mutations in S. pneumoniae raised the MIC of grepafloxacin some two-to fourfold, but no definite target assignment was made (30). Here we examine grepafloxacin action in S. pneumoniae by using defined mutants, by characterizing stepwise-selected mutants, and by using recombinant enzymes.(Part of this study was presented at the 39th InterscienceConference on Antimicrobial Agents and Chemotherapy, San Francisco, Calif., 26 to 29 September 1999.) Approximately 5 ϫ 10 9 CFU of susceptible isolate 7785 (18) was spread on brain heart infusion plates containing 10% horse blood and grepafloxacin at concentrations of 0.5 and 1 g/ml, i.e., one and two times the MIC for the parent strain (Fig. 2). Mutants appeared after 48 h of aerobic incubation at 37°C. Mutants 1G1 to 1G15 and 1G16 to 1G25 from the two independent drug challenges were studied further. Second-step mutants 2G1 to 2G12 were...

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“…Despite the functional similarities between topoisomerase IV (topo IV) and gyrase, their susceptibility to FQs varies across bacterial species (14). In S. pneumoniae, the primary target for LVX is topo IV (15)(16)(17)(18), while gyrase is the primary target for moxifloxacin (19). Type II topoisomerases maintain DNA topology and solve the topological problems associated with DNA replication, transcription, and recombination (20).…”

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The Fluoroquinolone Levofloxacin Triggers the Transcriptional Activation of Iron Transport Genes That Contribute to Cell Death in Streptococcus pneumoniae

Ferrándiz

Campa

2014

Antimicrob Agents Chemother

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We studied the transcriptomic response of Streptococcus pneumoniae to levofloxacin (LVX) under conditions inhibiting topoisomerase IV but not gyrase. Although a complex transcriptomic response was observed, the most outstanding result was the upregulation of the genes of the fatDCEB operon, involved in iron (Fe 2؉ and Fe 3؉ ) uptake, which were the only genes varying under every condition tested. Although the inhibition of topoisomerase IV by levofloxacin did not have a detectable effect in the level of global supercoiling, increases in general supercoiling and fatD transcription were observed after topoisomerase I inhibition, while the opposite was observed after gyrase inhibition with novobiocin. Since fatDCEB is located in a topological chromosomal domain downregulated by DNA relaxation, we studied the transcription of a copy of the 422-bp (including the P fat promoter) region located upstream of fatDCEB fused to the cat reporter inserted into the chromosome 106 kb away from its native position: P fat fatD was upregulated in the presence of LVX in its native location, whereas no change was observed in the P fat cat construction. Results suggest that topological changes are indeed involved in P fat fatDCE transcription. Upregulation of fatDCEB would lead to an increase of intracellular iron and, in turn, to the activation of the Fenton reaction and the increase of reactive oxygen species. In accordance, we observed an attenuation of levofloxacin lethality in iron-deficient media and in a strain lacking the gene coding for SpxB, the main source of hydrogen peroxide. In addition, we observed an increase of reactive oxygen species that contributed to levofloxacin lethality.

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Fluoroquinolones Inhibit PreferentiallyStreptococcus pneumoniaeDNA Topoisomerase IV Than DNA Gyrase Native Proteins

Cited by 27 publications

References 37 publications

Contribution of the ATP Binding Site of ParE to Susceptibility to Novobiocin and Quinolones in Streptococcus pneumoniae

Contribution of the ATP Binding Site of ParE to Susceptibility to Novobiocin and Quinolones in Streptococcus pneumoniae

Grepafloxacin, a Dimethyl Derivative of Ciprofloxacin, Acts Preferentially through Gyrase in Streptococcus pneumoniae : Role of the C-5 Group in Target Specificity

The Fluoroquinolone Levofloxacin Triggers the Transcriptional Activation of Iron Transport Genes That Contribute to Cell Death in Streptococcus pneumoniae

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