scholarly journals Role of the Extended α4 Domain of Staphylococcus aureus Gyrase A Protein in Determining Low Sensitivity to Quinolones

2006 ◽  
Vol 50 (2) ◽  
pp. 600-606 ◽  
Author(s):  
Jacob Strahilevitz ◽  
Ari Robicsek ◽  
David C. Hooper
Keyword(s):  
Staphylococcus Aureus ◽  
Acquired Resistance ◽  
Dna Gyrase ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
E Coli ◽  
Gyrase A ◽  
Low Sensitivity ◽  
Emergence Of Resistance

ABSTRACT Fluoroquinolones target two bacterial type II topoisomerases, DNA gyrase and topoisomerase IV. Acquired resistance to quinolones occurs stepwise, with the first mutation occurring in the more sensitive target enzyme. To limit the emergence of resistance, quinolones should ideally possess dual activities against the two enzymes. For reasons that are as yet unclear, Staphylococcus aureus gyrase is less sensitive to quinolones than topoisomerase IV, counter to its greater sensitivity in Escherichia coli, thereby limiting the use of quinolones for the treatment of staphylococcal infections. Mutations in the α4-helix domain of the GyrA subunit of gyrase are important in determining quinolone resistance. We replaced an extended region encompassing the α4 domain in the E. coli GyrA protein with its homolog in S. aureus and tested for its ability to complement a thermosensitive gyrase and its catalytic and noncatalytic properties. Purified gyrase reconstituted with chimeric GyrA was more resistant to ciprofloxacin than wild-type gyrase at both inhibition of catalytic activity and stimulation of cleavage complexes, and this difference was more apparent in the presence of K+-glutamate. The chimeric GyrA subunit was able to complement thermosensitive gyrase, similar to wild-type GyrA. Without supplemental K+-glutamate the MICs of ciprofloxacin for thermosensitive E. coli complemented with chimeric DNA gyrase were equal to those for E. coli complemented with wild-type gyrase but were twofold higher in the presence of K+-glutamate. Our findings suggest that the extended α4 domain of S. aureus GyrA is responsible, at least in part, for the increased resistance of S. aureus gyrase to quinolones and that this effect is modulated by K+-glutamate.

scholarly journals Differential behaviors of Staphylococcus aureus and Escherichia coli type II DNA topoisomerases.

1996 ◽  
Vol 40 (12) ◽  
pp. 2714-2720 ◽  
Author(s):  
F Blanche ◽  
B Cameron ◽  
F X Bernard ◽  
L Maton ◽  
B Manse ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Strong Support ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Type Ii ◽  
Topoisomerase Iv ◽  
E Coli ◽  
Dna Relaxation

Staphylococcus aureus gyrA and gyrB genes encoding DNA gyrase subunits were cloned and coexpressed in Escherichia coli under the control of the T7 promoter-T7 RNA polymerase system, leading to soluble gyrase which was purified to homogeneity. Purified gyrase was catalytically indistinguishable from the gyrase purified from S. aureus and did not contain detectable amounts of topoisomerases from the E. coli host. Topoisomerase IV subunits GrlA and GrlB from S. aureus were also expressed in E. coli and were separately purified to apparent homogeneity. Topoisomerase IV, which was reconstituted by mixing equimolar amounts of GrlA and GrlB, had both ATP-dependent decatenation and DNA relaxation activities in vitro. This enzyme was more sensitive than gyrase to inhibition by typical fluoroquinolone antimicrobial agents such as ciprofloxacin or sparfloxacin, adding strong support to genetic studies which indicate that topoisomerase IV is the primary target of fluoroquinolones in S. aureus. The results obtained with ofloxacin suggest that this fluoroquinolone could also primarily target gyrase. No cleavable complex could be detected with S. aureus gyrase upon incubation with ciprofloxacin or sparfloxacin at concentrations which fully inhibit DNA supercoiling. This suggests that these drugs do not stabilize the open DNA-gyrase complex, at least under standard in vitro incubation conditions, but are more likely to interfere primarily with the DNA breakage step, contrary to what has been reported with E. coli gyrase. Both S. aureus gyrase-catalyzed DNA supercoiling and S. aureus topoisomerase IV-catalyzed decatenation were dramatically stimulated by potassium glutamate or aspartate (500- and 50-fold by 700 and 350 mM glutamate, respectively), whereas topoisomerase IV-dependent DNA relaxation was inhibited 3-fold by 350 mM glutamate. The relevance of the effect of dicarboxylic amino acids on the activities of type II topoisomerases is discussed with regard to the intracellular osmolite composition of S. aureus.

scholarly journals Cloning, Expression, and Enzymatic Characterization of Pseudomonas aeruginosaTopoisomerase IV

1999 ◽  
Vol 43 (3) ◽  
pp. 530-536 ◽  
Author(s):  
Takaaki Akasaka ◽  
Yoshikuni Onodera ◽  
Mayumi Tanaka ◽  
Kenichi Sato
Keyword(s):  
Dna Gyrase ◽  
Fusion System ◽  
Enzymatic Characterization ◽  
Wild Type ◽  
Inhibitory Effects ◽  
Topoisomerase Iv ◽  
Upstream Gene ◽  
E Coli ◽  
New Quinolones

ABSTRACT The topoisomerase IV subunit A gene, parC homolog, has been cloned and sequenced from Pseudomonas aeruginosa PAO1, with cDNA encoding the N-terminal region of Escherichia coli parC used as a probe. The homolog and its upstream gene were presumed to be parC and parE through sequence homology with the parC and parE genes of other organisms. The deduced amino acid sequence of ParC and ParE showed 33 and 32% identity with that of the P. aeruginosa DNA gyrase subunits, GyrA and GyrB, respectively, and 69 and 75% identity with that of E. coli ParC and ParE, respectively. The putative ParC and ParE proteins were overexpressed and separately purified by use of a fusion system with a maltose-binding protein, and their enzymatic properties were examined. The reconstituted enzyme had ATP-dependent decatenation activity, which is the main catalytic activity of bacterial topoisomerase IV, and relaxing activities but had no supercoiling activity. So, the cloned genes were identified asP. aeruginosa topoisomerase IV genes. The inhibitory effects of quinolones on the activities of topoisomerase IV and DNA gyrase were compared. The 50% inhibitory concentrations of quinolones for the decatenation activity of topoisomerase IV were from five to eight times higher than those for the supercoiling activities ofP. aeruginosa DNA gyrase. These results confirmed that topoisomerase IV is less sensitive to fluoroquinolones than is DNA gyrase and may be a secondary target of new quinolones in wild-typeP. aeruginosa.

Molecular Cloning of the gyrA andgyrB Genes of Bacteroides fragilis Encoding DNA Gyrase

1999 ◽  
Vol 43 (10) ◽  
pp. 2423-2429 ◽  
Author(s):  
Yoshikuni Onodera ◽  
Kenichi Sato
Keyword(s):  
Hot Spot ◽  
Bacteroides Fragilis ◽  
Dna Gyrase ◽  
E Coli ◽  
Important Target ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Gyrase A ◽  
Selection For

ABSTRACT The genes encoding the DNA gyrase A and B subunits ofBacteroides fragilis were cloned and sequenced. ThegyrA and gyrB genes code for proteins of 845 and 653 amino acids, respectively. These proteins were expressed inEscherichia coli, and the combination of GyrA and GyrB exhibited ATP-dependent supercoiling activity. To analyze the role of DNA gyrase in quinolone resistance of B. fragilis, we isolated mutant strains by stepwise selection for resistance to increasing concentrations of levofloxacin. We analyzed the resistant mutants and showed that Ser-82 of GyrA, equivalent to resistance hot spot Ser-83 of GyrA in E. coli, was in each case replaced with Phe. These results suggest that DNA gyrase is an important target for quinolones in B. fragilis.

scholarly journals Alterations in the DNA topoisomerase IV grlA gene responsible for quinolone resistance in Staphylococcus aureus.

1996 ◽  
Vol 40 (5) ◽  
pp. 1157-1163 ◽  
Author(s):  
J Yamagishi ◽  
T Kojima ◽  
Y Oyamada ◽  
K Fujimoto ◽  
H Hattori ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Gene Dosage ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Wild Type Allele ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Dna Topoisomerase ◽  
Transformation Experiment ◽  
Dna Fragment

A 4.2-kb DNA fragment conferring quinolone resistance was cloned from a quinolone-resistant clinical isolate of Staphylococcus aureus and was shown to possess a part of the grlB gene and a mutated grlA gene. S-80-->F and E-84-->K mutations in the grlA gene product were responsible for the quinolone resistance. The mutated grlA genes responsible for quinolone resistance were dominant over the wild-type allele, irrespective of gene dosage in a transformation experiment with the grlA gene alone. However, dominance by mutated grlA genes depended on gene dosage when bacteria were transformed with the grlA and grlB genes in combination. Quinolone-resistant gyrA mutants were easily isolated from a strain, S. aureus RN4220, carrying a plasmid with the mutated grlA gene, though this was not the case for other S. aureus strains lacking the plasmid. The elimination of this plasmid from such quinolone-resistant gyrA mutants resulted in marked increases in quinolone susceptibility. These results suggest that both DNA gyrase and DNA topoisomerase IV may be targets of quinolones and that the quinolone susceptibility of organisms may be determined by which of these enzymes is most quinolone sensitive.

scholarly journals In Vitro Activities of Novel Nonfluorinated Quinolones PGE 9262932 and PGE 9509924 against Clinical Isolates of Staphylococcus aureus and Streptococcus pneumoniae with Defined Mutations in DNA Gyrase and Topoisomerase IV

2002 ◽  
Vol 46 (6) ◽  
pp. 1651-1657 ◽  
Author(s):  
Mark E. Jones ◽  
Ian A. Critchley ◽  
James A. Karlowsky ◽  
Renée S. Blosser-Middleton ◽  
Franz-Josef Schmitz ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Streptococcus Pneumoniae ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Clinical Isolates ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Selection Of

ABSTRACT Two 8-methoxy nonfluorinated quinolones (NFQs), PGE 9262932 and PGE 9509924, were tested against contemporary clinical isolates of Staphylococcus aureus (n = 122) and Streptococcus pneumoniae (n = 69) with genetically defined quinolone resistance-determining regions (QRDRs). For S. aureus isolates with wild-type (WT) sequences at the QRDRs, the NFQs demonstrated activities 4- to 32-fold more potent (MICs at which 90% of isolates are inhibited [MIC90s], 0.03 μg/ml) than those of moxifloxacin (MIC90, 0.12 μg/ml), gatifloxacin (MIC90, 0.25 μg/ml), levofloxacin (MIC90, 0.25 μg/ml), and ciprofloxacin (MIC90, 1 μg/ml). Against S. pneumoniae isolates with WT sequences at gyrA and parC, the NFQs PGE 9262932 (MIC90, 0.03 μg/ml) and PGE 9509924 (MIC90, 0.12 μg/ml) were 8- to 64-fold and 2- to 16-fold more potent, respectively, than moxifloxacin (MIC90, 0.25 μg/ml), gatifloxacin (MIC90, 0.5 μg/ml), levofloxacin (MIC90, 2 μg/ml), and ciprofloxacin (MIC90, 2 μg/ml). The MICs of all agents were elevated for S. aureus isolates with alterations in GyrA (Glu88Lys or Ser84Leu) and GrlA (Ser80Phe) and S. pneumoniae isolates with alterations in GyrA (Ser81Phe or Ser81Tyr) and ParC (Ser79Phe or Lys137Asn). Fluoroquinolone MICs for S. aureus strains with double alterations in GyrA combined with double alterations in GrlA were ≥32 μg/ml, whereas the MICs of the NFQs for strains with these double alterations were 4 to 8 μg/ml. The PGE 9262932 and PGE 9509924 MICs for the S. pneumoniae isolates did not exceed 0.5 and 1 μg/ml, respectively, even for isolates with GyrA (Ser81Phe) and ParC (Ser79Phe) alterations, for which levofloxacin MICs were >16 μg/ml. No difference in the frequency of selection of mutations (<10−8 at four times the MIC) in wild-type or first-step mutant isolates of S. aureus or S. pneumoniae was detected for the two NFQs. On the basis of their in vitro activities, these NFQ agents show potential for the treatment of infections caused by isolates resistant to currently available fluoroquinolones.

scholarly journals Mutations in Topoisomerase IV and DNA Gyrase of Staphylococcus aureus: Novel Pleiotropic Effects on Quinolone and Coumarin Activity

1998 ◽  
Vol 42 (1) ◽  
pp. 121-128 ◽  
Author(s):  
Bénédicte Fournier ◽  
David C. Hooper
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Gyrase ◽  
Single Step ◽  
Pleiotropic Effects ◽  
Fluoroquinolone Resistance ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Topoisomerase Iv ◽  
Gene Encoding ◽  
Double Mutation

ABSTRACT Previous studies have shown that topoisomerase IV and DNA gyrase interact with quinolones and coumarins in different ways. The MICs of coumarins (novobiocin and coumermycin) for MT5, a Staphylococcus aureus nov mutant, are higher than those for wild-type strains. Sequencing the gyrB gene encoding one subunit of the DNA gyrase revealed the presence of a double mutation likely to be responsible for this resistance: at codon 102 (Ile to Ser) and at codon 144 (Arg to Ile). For single-step flqA mutant MT5224c9, previously selected on ciprofloxacin, the fluoroquinolone MIC was higher and the coumarin MIC was lower than those for its parent, MT5. Sequencing the grlB andgrlA genes of topoisomerase IV of MT5224c9 showed a single Asn-470-to-Asp mutation in GrlB. Genetic outcrosses by transformation with chromosomal DNA and introduction of plasmids carrying either the wild-type or the mutated grlB gene indicated that this mutation causes both increased MICs of fluoroquinolones and decreased MICs of coumarins and that the mutant grlBallele is codominant for both phenotypes with multicopy alleles. Integration of these plasmids into the chromosome confirmed the codominance of fluoroquinolone resistance, butgrlB + appeared dominant over grlB(Asp-470) for coumarin resistance. Finally, the gyrA(Leu-84) mutation previously described as silent for fluoroquinolone resistance increased the MIC of nalidixic acid, a nonfluorinated quinolone. Combining the grlA (Phe-80) and grlB(Asp-470) mutations with this gyrA mutation also had differing effects. The findings indicate that alterations in topoisomerases may have pleiotropic effects on different classes of inhibitors as well as on inhibitors within the same class. A full understanding of drug action and resistance at the molecular level must take into account both inhibitor structure-activity relationships and the effects of different classes of topoisomerase mutants.

scholarly journals Hybrid Inhibitors of DNA Gyrase A and B: Design, Synthesis and Evaluation

Pharmaceutics ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 6
Author(s):  
Martina Durcik ◽  
Žiga Skok ◽  
Janez Ilaš ◽  
Nace Zidar ◽  
Anamarija Zega ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Dna Gyrase ◽  
Topoisomerase Iv ◽  
Design Synthesis ◽  
Bacterial Dna ◽  
E Coli ◽  
Catalytic Inhibitor ◽  
Catalytic Sites ◽  
Competitive Inhibitors ◽  
Gyrase A

The discovery of multi-targeting ligands of bacterial enzymes is an important strategy to combat rapidly spreading antimicrobial resistance. Bacterial DNA gyrase and topoisomerase IV are validated targets for the development of antibiotics. They can be inhibited at their catalytic sites or at their ATP binding sites. Here we present the design of new hybrids between the catalytic inhibitor ciprofloxacin and ATP-competitive inhibitors that show low nanomolar inhibition of DNA gyrase and antibacterial activity against Gram-negative pathogens. The most potent hybrid 3a has MICs of 0.5 µg/mL against Klebsiella pneumoniae, 4 µg/mL against Enterobacter cloacae, and 2 µg/mL against Escherichia coli. In addition, inhibition of mutant E. coli strains shows that these hybrid inhibitors interact with both subunits of DNA gyrase (GyrA, GyrB), and that binding to both of these sites contributes to their antibacterial activity.

scholarly journals The Anti-Methicillin-Resistant Staphylococcus aureus Quinolone WCK 771 Has Potent Activity against Sequentially Selected Mutants, Has a Narrow Mutant Selection Window against Quinolone-Resistant Staphylococcus aureus, and Preferentially Targets DNA Gyrase

2006 ◽  
Vol 50 (11) ◽  
pp. 3568-3579 ◽  
Author(s):  
Sachin S. Bhagwat ◽  
Lakshmi A. Mundkur ◽  
Shrikant V. Gupte ◽  
Mahesh V. Patel ◽  
Habil F. Khorakiwala
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Gyrase ◽  
Primary Target ◽  
Topoisomerase Iv ◽  
Mutant Selection ◽  
Low Concentrations ◽  
Selection Window ◽  
High Level ◽  
The Impact

ABSTRACT WCK 771 is a broad-spectrum fluoroquinolone with enhanced activity against quinolone-resistant staphylococci. To understand the impact of the target-level interactions of WCK 771 on its antistaphylococcal pharmacodynamic properties, we determined the MICs for genetically defined mutants and studied the mutant prevention concentrations (MPCs), the frequency of mutation, and the cidality against the wild type and double mutants. There was a twofold increase in the MICs of WCK 771 for single gyrA mutants, indicating that DNA gyrase is its primary target. All first- and second-step mutants selected by WCK 771 revealed gyrA and grlA mutations, respectively. The MICs of WCK 771 and clinafloxacin were found to be superior to those of other quinolones against strains with double and triple mutations. WCK 771 was also cidal for high-density double mutants at low concentrations. WCK 771 and clinafloxacin showed narrow mutant selection windows compared to those of the other quinolones. Against a panel of 50 high-level quinolone-resistant clinical isolates of staphylococci (ciprofloxacin MIC ≥ 16 μg/ml), the WCK 771 MPCs were ≤2 μg/ml for 68% of the strains and ≤4 μg/ml for 28% of the strains. Our results demonstrate that gyrA is the primary target of WCK 771 and that it has pharmacodynamic properties remarkably different from those of quinolones with dual targets (garenoxacin and moxifloxacin) and topoisomerase IV-specific quinolones (trovafloxacin). WCK 771 displayed an activity profile comparable to that of clinafloxacin, a dual-acting quinolone with a high affinity to DNA gyrase. Overall, the findings signify the key role of DNA gyrase in determining the optimal antistaphylococcal features of quinolones.

scholarly journals Antibacterial Activity of Gatifloxacin (AM-1155, CG5501, BMS-206584), a Newly Developed Fluoroquinolone, against Sequentially Acquired Quinolone-Resistant Mutants and thenorA Transformant of Staphylococcus aureus

1998 ◽  
Vol 42 (8) ◽  
pp. 1917-1922 ◽  
Author(s):  
Hideyuki Fukuda ◽  
Satoshi Hori ◽  
Keiichi Hiramatsu
Keyword(s):  
Staphylococcus Aureus ◽  
Low Frequency ◽  
Dna Gyrase ◽  
Second Step ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Sequential Selection ◽  
In The Wild ◽  
Fourth Step ◽  
Resistant Mutants

ABSTRACT Alternate mutations in the grlA and gyrAgenes were observed through the first- to fourth-step mutants which were obtained from four Staphylococcus aureus strains by sequential selection with several fluoroquinolones. The increases in the MICs of gatifloxacin accompanying those mutational steps suggest that primary targets of gatifloxacin in the wild type and the first-, second-, and third-step mutants are wild-type topoisomerase IV (topo IV), wild-type DNA gyrase, singly mutated topo IV, and singly mutated DNA gyrase, respectively. Gatifloxacin had activity equal to that of tosufloxacin and activity more potent than those of norfloxacin, ofloxacin, ciprofloxacin, and sparfloxacin against the second-step mutants (grlA gyrA; gatifloxacin MIC range, 1.56 to 3.13 μg/ml) and had the most potent activity against the third-step mutants (grlA gyrA grlA; gatifloxacin MIC range, 1.56 to 6.25 μg/ml), suggesting that gatifloxacin possesses the most potent inhibitory activity against singly mutated topo IV and singly mutated DNA gyrase among the quinolones tested. Moreover, gatifloxacin selected resistant mutants from wild-type and the second-step mutants at a low frequency. Gatifloxacin possessed potent activity (MIC, 0.39 μg/ml) against the NorA-overproducing strain S. aureus NY12, thenorA transformant, which was slightly lower than that against the parent strain SA113. The increases in the MICs of the quinolones tested against NY12 were negatively correlated with the hydrophobicity of the quinolones (correlation coefficient, −0.93;P < 0.01). Therefore, this slight decrease in the activity of gatifloxacin is attributable to its high hydrophobicity. Those properties of gatifloxacin likely explain its good activity against quinolone-resistant clinical isolates of S. aureusharboring the grlA, gyrA, and/ornorA mutations.

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