Mutation in the DNA Gyrase A Gene of Escherichia coli That Expands the Quinolone Resistance-Determining Region
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ABSTRACT In three Escherichia coli mutants, a change (Ala-51 to Val) in the gyrase A protein outside the standard quinolone resistance-determining region (QRDR) lowered the level of quinolone susceptibility more than changes at amino acids 67, 82, 84, and 106 did. Revision of the QRDR to include amino acid 51 is indicated.Keywords:
Quinolone
Quinolone
Topoisomerase IV
Pathogenic bacteria
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Inhibitory effects of five quinolones against DNA gyrases purified from four quinolone-resistant clinical isolates of Pseudomonas aeruginosa and the quinolone-susceptible strain PAO1 were examined. All of the quinolone-resistant strains tested were found to be DNA gyrase mutants. The 50% inhibitory concentrations (IC50s) of the quinolones for these DNA gyrases roughly correlated with their MICs. Interestingly, gyrase inhibition by DU-6859a was found to be significantly less affected by these mutations that inhibition by other currently available quinolones. To assess the enhanced activity shown by DU-6859a, the effects of quinolones with altered substituents at the N-1, C-7, and C-8 positions of the quinolone ring of DU-6859a were tested. Measurement of MICs for four DNA gyrase mutants and IC50s for their purified DNA gyrases showed that removal of the C-8 chlorine of DU-6859a significantly increased MICs and IC50s for DNA gyrase mutants. However, no deleterious effects were observed when either the fluorine on the cyclopropyl substituent at the N-1 position or the cyclopropyl ring at the C-7 substituent was removed. Moreover, removal of the C-8 chlorine also increased the MIC for 19 of 20 quinolone-resistant clinical isolates. Our results led to the conclusion that DU-6859a is much more active against quinolone-resistant clinical isolates of P. aeruginosa than other currently available quinolones, probably because of its strong inhibitory effects against mutant quinolone-resistant DNA gyrases, and that the C-8 chlorine is necessary for these potent effects.
Quinolone
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Spontaneous quinolone-resistant mutants of MP050, a quinolone-susceptible clinical strain of Serratia marcescens, were isolated on nutrient agar containing 0.5 microgram of ciprofloxacin per ml. One mutant, designated MP051, was selected for further study. Quinolone MICs for MP051 were 4- to 16-fold higher than those for MP050; nonquinolone MICs were unchanged. The DNA gyrase isolated from MP051 was 24-fold less sensitive to inhibition of supercoiling by ciprofloxacin than the DNA gyrase isolated from MP050 was. Inhibition studies on reconstituted combinations of heterologous gyrase subunits showed that the decreased inhibition was dependent on the A subunit of DNA gyrase from MP051. Further evidence that this decreased inhibition was due to a gyrA mutation was provided by analysis of Escherichia coli gyrA gene expression in S. marcescens heterodiploids containing pNJR3-2, a broad-host-range gyrA gene probe. Quinolone susceptibilities of MP051 heterodiploids containing the wild-type E. coli gyrA gene decreased to those of MP050, while quinolone susceptibilities of MP050 containing the same plasmid were unchanged. These results indicate that spontaneous quinolone resistance in MP051 was due to a mutation in gyrA.
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Recent studies concerning the mechanism of action of quinolones against DNA gyrase are reviewed. DNA gyrase is an essential bacterial enzyme known to be a primary target of quinolone agents. Quinolone-resistant alleles of both the gyrA and gyrB genes of DNA gyrase have been sequenced, and domains that affect the action of quinolones have been identified within the amino terminus of the gyrase A peptide and the midportion of the gyrase B peptide. In addition, an ATP-induced structural transition of DNA complexed with DNA gyrase was shown to be blocked by norfloxacin, but the means by which quinolones effect this change and the molecular site of quinolone binding remain unclear. Studies of structure-activity relationships of the quinolone molecule have been expanded and have included effects of quinolones on DNA gyrase. Stereochemical effects at positions I and 7 have been found. Substitutions at position 7 that improve potency against gram-positive bacteria have also been identified. Novel mono- and three-ring structures and an isothiazolo substitution at position 3 have broadened the range of structures known to have activity. Studies of bacterial killing by quinolones have revealed additional correlations with markers of DNA damage and additional alterations in bacteria and growth conditions that affect bacterial killing. The exact events responsible for quinolone-mediated lethality, however, remain undefined.
Quinolone
Mechanism of Action
Mode of Action
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Quinolone
Topoisomerase IV
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Bacterial DNA gyrase is a type II topoisomerase that can introduce negative supercoils to DNA substrates and is a clinically-relevant target for the development of new antibacterials. DNA gyrase is one of the primary targets of quinolones, broad-spectrum antibacterial agents and are used as a first-line drug for various types of infections. However, currently used quinolones are becoming less effective due to drug resistance. Common resistance comes in the form of mutation in enzyme targets, with this type being the most clinically relevant. Additional mechanisms, conducive to quinolone resistance, are arbitrated by chromosomal mutations and/or plasmid-gene uptake that can alter quinolone cellular concentration and interaction with the target, or affect drug metabolism. Significant synthetic strategies have been employed to modify the quinolone scaffold and/or develop novel quinolones to overcome the resistance problem. This review discusses the development of quinolone antibiotics targeting DNA gyrase to overcome bacterial resistance and reduce toxicity. Moreover, structural activity relationship (SAR) data included in this review could be useful for the development of future generations of quinolone antibiotics.
Quinolone
Topoisomerase IV
Resistome
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ABSTRACT DNA gyrase is a prokaryotic type II topoisomerase and a major target of quinolone antibacterials. The majority of mutations conferring resistance to quinolones arise within the quinolone resistance-determining region of GyrA close to the active site (Tyr 122 ) where DNA is bound and cleaved. However, some quinolone resistance mutations are known to exist in GyrB. Present structural data suggest that these residues lie a considerable distance from the quinolone resistance-determining region, and it is not obvious how they affect quinolone action. We have made and purified two such mutant proteins, GyrB(Asp 426 →Asn) and GyrB(Lys 447 →Glu), and characterized them in vitro. We found that the two proteins behave similarly to GyrA quinolone-resistant proteins. We showed that the mutations exert their effect by decreasing the amount of quinolone bound to a gyrase-DNA complex. We suggest that the GyrB residues form part of a quinolone-binding pocket that includes DNA and the quinolone resistance-determining region in GyrA and that large conformational changes during the catalytic cycle of the enzyme allow these regions to come into close proximity.
Quinolone
Topoisomerase IV
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The Qnr pentapeptide repeat proteins interact with DNA gyrase and protect it from quinolone inhibition. The two external loops, particularly the larger loop B, of Qnr proteins are essential for quinolone protection of DNA gyrase. The specific QnrB1 interaction sites on DNA gyrase are not known. In this study, we investigated the interaction between GyrA and QnrB1 using site-specific photo-cross-linking of QnrB1 loop B combined with mass spectrometry. We found that amino acid residues 286 to 298 on the tower domain of GyrA interact with QnrB1 and play a key role in QnrB1 protection of gyrase from quinolone inhibition. Alanine replacement of arginine at residue 293 and a small deletion of amino acids 286 to 289 of GyrA resulted in a decrease in the QnrB1-mediated increase in quinolone MICs and also abolished the QnrB1 protection of purified DNA gyrase from ciprofloxacin inhibition.
Quinolone
Pentapeptide repeat
Novobiocin
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Since their discovery over 5 decades ago, quinolone antibiotics have found enormous success as broad spectrum agents that exert their activity through dual inhibition of bacterial DNA gyrase and topoisomerase IV. Increasing rates of resistance, driven largely by target-based mutations in the GyrA/ParC quinolone resistance determining region, have eroded the utility and threaten the future use of this vital class of antibiotics. Herein we describe the discovery and optimization of a series of 4-(aminomethyl)quinolin-2(1H)-ones, exemplified by 34, that inhibit bacterial DNA gyrase and topoisomerase IV and display potent activity against ciprofloxacin-resistant Gram-negative pathogens. X-ray crystallography reveals that 34 occupies the classical quinolone binding site in the topoisomerase IV-DNA cleavage complex but does not form significant contacts with residues in the quinolone resistance determining region.
Topoisomerase IV
Quinolone
Topoisomerase inhibitor
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OBJECTIVE To study the mutation in DNA gyrase A of Escherichia coli resistante to quinolone antibiotics. METHODS The gene of gyrA DNA in three quinolone resistant and one quinolone susceptible isolates of E.coli ATCC25922 was amplified by PCR and then analysed by single strand conformatiom polymorphism(SSCP) and nucleotide sequencing. RESULTS DNA sequence analysis of gyrA gene revealed three mutations resulting in amino acid change: Ser 83→Leu,Asp 87→Asn and His 184→Asn. Ser 83→Leu and Asp 87→Asn were located in the quinolone resistance determining region, His 184→Asn was a newly discovered substitution in gyrA of E.coli. The results of PCR SSCP were in concordance with results of mucleotide sequencing. CONCLUSIONS The mutation in DNA gyrase A of E.coli was implicated in resistance to quinolone.
Quinolone
Single-strand conformation polymorphism
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