Targeting of DNA gyrase in Streptococcus pneumoniae by sparfloxacin: selective targeting of gyrase or topoisomerase IV by quinolones.

1997 ◽  
Vol 41 (2) ◽  
pp. 471-474 ◽  
Author(s):  
X S Pan ◽  
L M Fisher
Keyword(s):  
Escherichia Coli ◽  
Streptococcus Pneumoniae ◽  
Hot Spot ◽  
Dna Gyrase ◽  
Second Step ◽  
Topoisomerase Iv ◽  
Dna Topoisomerase ◽  
Selective Targeting

gyrA and parC mutations have been identified inn Streptococcus pneumoniae mutants stepwise selected for resistance to sparfloxacin, an antipneumococcal fluoroquinolone. GyrA mutations (at the position equivalent to resistance hot spot Ser-83 in Escherichia coli GyrA) were found in all 17 first-step mutants examined and preceded DNA topoisomerase IV parC mutations (at Ser-79 or Glu-83), which appeared only in second-step mutants. The targeting of gyrase by sparfloxacin in S. pneumoniae but of topoisomerase IV by ciprofloxacin indicates that target preference can be altered by changes in quinolone structure.

scholarly journals Quinolone-resistant mutants of escherichia coli DNA topoisomerase IV parC gene.

1996 ◽  
Vol 40 (3) ◽  
pp. 710-714 ◽  
Author(s):  
Y Kumagai ◽  
J I Kato ◽  
K Hoshino ◽  
T Akasaka ◽  
K Sato ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Gyrase ◽  
Mutant Gene ◽  
Missense Mutations ◽  
Topoisomerase Iv ◽  
Dna Topoisomerase ◽  
E Coli ◽  
A Subunit ◽  
Resistant Strains ◽  
Mutant Genes

Escherichia coli quinolone-resistant strains with mutations of the parC gene, which codes for a subunit of topoisomerase IV, were isolated from a quinolone-resistant gyrA mutant of DNA gyrase. Quinolone-resistant parC mutants were also identified among the quinolone-resistant clinical strains. The parC mutants became susceptible to quinolones by introduction of a parC+ plasmid. Introduction of the multicopy plasmids carrying the quinolone-resistant parC mutant gene resulted in an increase in MICs of quinolones for the parC+ and quinolone-resistant gyrA strain. Nucleotide sequences of the quinolone-resistant parC mutant genes were determined, and missense mutations at position Gly-78, Ser-80, or Glu-84, corresponding to those in the quinolone-resistance-determining region of DNA gyrase, were identified. These results indicate that topoisomerase IV is a target of quinolones in E. coli and suggest that the susceptibility of E. coli cells to quinolones is determined by sensitivity of the targets, DNA gyrase and topoisomerase IV.

scholarly journals Transferable Quinolone Resistance in Vibrio cholerae

2009 ◽  
Vol 54 (2) ◽  
pp. 799-803 ◽  
Author(s):  
Hong Bin Kim ◽  
Minghua Wang ◽  
Sabeena Ahmed ◽  
Chi Hye Park ◽  
Regina C. LaRocque ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Vibrio Cholerae ◽  
Dna Gyrase ◽  
Multidrug Resistant ◽  
Quinolone Resistance ◽  
Topoisomerase Iv ◽  
Dna Topoisomerase ◽  
A Subunit ◽  
Resistant Strains ◽  
Over Time

ABSTRACT Ciprofloxacin was introduced for treatment of patients with cholera in Bangladesh because of resistance to other agents, but its utility has been compromised by the decreasing ciprofloxacin susceptibility of Vibrio cholerae over time. We correlated levels of susceptibility and temporal patterns with the occurrence of mutation in gyrA, which encodes a subunit of DNA gyrase, followed by mutation in parC, which encodes a subunit of DNA topoisomerase IV. We found that ciprofloxacin activity was more recently further compromised in strains containing qnrVC3, which encodes a pentapeptide repeat protein of the Qnr subfamily, members of which protect topoisomerases from quinolone action. We show that qnrVC3 confers transferable low-level quinolone resistance and is present within a member of the SXT integrating conjugative element family found commonly on the chromosomes of multidrug-resistant strains of V. cholerae and on the chromosomes of Escherichia coli transconjugants constructed in the laboratory. Thus, progressive increases in quinolone resistance in V. cholerae are linked to cumulative mutations in quinolone targets and most recently to a qnr gene on a mobile multidrug resistance element, resulting in further challenges for the antimicrobial therapy of cholera.

scholarly journals Potent Antipneumococcal Activity of Gemifloxacin Is Associated with Dual Targeting of Gyrase and Topoisomerase IV, an In Vivo Target Preference for Gyrase, and Enhanced Stabilization of Cleavable Complexes In Vitro

2000 ◽  
Vol 44 (11) ◽  
pp. 3112-3117 ◽  
Author(s):  
Victoria J. Heaton ◽  
Jane E. Ambler ◽  
L. Mark Fisher
Keyword(s):  
Hot Spot ◽  
Dna Gyrase ◽  
Second Step ◽  
Wild Type ◽  
Resistance Mutations ◽  
Topoisomerase Iv ◽  
Dual Targeting ◽  
Resistant Mutants

ABSTRACT We investigated the roles of DNA gyrase and topoisomerase IV in determining the susceptibility of Streptococcus pneumoniaeto gemifloxacin, a novel fluoroquinolone which is under development as an antipneumococcal drug. Gemifloxacin displayed potent activity against S. pneumoniae 7785 (MIC, 0.06 μg/ml) compared with ciprofloxacin (MIC, 1 to 2 μg/ml). Complementary genetic and biochemical approaches revealed the following. (i) The gemifloxacin MICs for isogenic 7785 mutants bearing either parC orgyrA quinolone resistance mutations were marginally higher than wild type at 0.12 to 0.25 μg/ml, whereas the presence of both mutations increased the MIC to 0.5 to 1 μg/ml. These data suggest that both gyrase and topoisomerase IV contribute significantly as gemifloxacin targets in vivo. (ii) Gemifloxacin selected first-stepgyrA mutants of S. pneumoniae 7785 (gemifloxacin MICs, 0.25 μg/ml) encoding Ser-81 to Phe or Tyr, or Glu-85 to Lys mutations. These mutants were cross resistant to sparfloxacin (which targets gyrase) but not to ciprofloxacin (which targets topoisomerase IV). Second-step mutants (gemifloxacin MICs, 1 μg/ml) exhibited an alteration in parC resulting in changes of ParC hot spot Ser-79 to Phe or Tyr. Thus, gyrase appears to be the preferential in vivo target. (iii) Gemifloxacin was at least 10- to 20-fold more effective than ciprofloxacin in stabilizing a cleavable complex (the cytotoxic lesion) with either S. pneumoniaegyrase or topoisomerase IV enzyme in vitro. These data suggest that gemifloxacin is an enhanced affinity fluoroquinolone that acts against gyrase and topoisomerase IV in S. pneumoniae, with gyrase the preferred in vivo target. The marked potency of gemifloxacin against wild type and quinolone-resistant mutants may accrue from greater stabilization of cleavable complexes with the target enzymes.

scholarly journals Contribution of Topoisomerase IV and DNA Gyrase Mutations in Streptococcus pneumoniae to Resistance to Novel Fluoroquinolones

1999 ◽  
Vol 43 (8) ◽  
pp. 2000-2004 ◽  
Author(s):  
Ekaterina Pestova ◽  
Rebecca Beyer ◽  
Nicholas P. Cianciotto ◽  
Gary A. Noskin ◽  
Lance R. Peterson
Keyword(s):  
Streptococcus Pneumoniae ◽  
Molecular Basis ◽  
Inhibitory Concentration ◽  
Dna Gyrase ◽  
Structural Genes ◽  
Primary Target ◽  
Topoisomerase Iv ◽  
Dna Topoisomerase ◽  
Enhanced Activity

ABSTRACT In this study, we assessed the activity of ciprofloxacin, levofloxacin, sparfloxacin, and trovafloxacin against clinical isolates of Streptococcus pneumoniae that were resistant to the less-recently developed fluoroquinolones by using defined amino acid substitutions in DNA gyrase and topoisomerase IV. The molecular basis for resistance was assessed by using mutants selected with trovafloxacin, ciprofloxacin, and levofloxacin in vitro. This demonstrated that the primary target of trovafloxacin in S. pneumoniae is the ParC subunit of DNA topoisomerase IV, similar to most other fluoroquinolones. However, first-step mutants bearing the Ser79→Phe/Tyr substitution in topoisomerase IV subunit ParC were susceptible to trovafloxacin with a minimum inhibitory concentration of 0.25 μg/ml, and mutations in the structural genes for both topoisomerase IV subunit ParC (parC) and the DNA gyrase subunit (gyrA) were required to achieve levels of resistance above the breakpoint. The data also suggest that enhanced activity of trovafloxacin against pneumococci is due to a combination of factors that may include reduced efflux of this agent and an enhanced activity against both DNA gyrase and topoisomerase IV.

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 Streptococcus pneumoniae DNA Gyrase and Topoisomerase IV: Overexpression, Purification, and Differential Inhibition by Fluoroquinolones

1999 ◽  
Vol 43 (5) ◽  
pp. 1129-1136 ◽  
Author(s):  
Xiao-Su Pan ◽  
L. Mark Fisher
Keyword(s):  
Streptococcus Pneumoniae ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Topoisomerase Iv ◽  
T7 Promoter ◽  
Bacterial Killing ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Comparable Activity

ABSTRACT Streptococcus pneumoniae gyrA and gyrBgenes specifying the DNA gyrase subunits have been cloned into pET plasmid vectors under the control of an inducible T7 promoter and have been separately expressed in Escherichia coli. Soluble 97-kDa GyrA and 72-kDa GyrB proteins bearing polyhistidine tags at their respective C-terminal and N-terminal ends were purified to apparent homogeneity by one-step nickel chelate column chromatography and were free of host E. coli topoisomerase activity. Equimolar amounts of the gyrase subunits reconstituted ATP-dependent DNA supercoiling with comparable activity to gyrase of E. coli and Staphylococcus aureus. In parallel, S. pneumoniae topoisomerase IV ParC and ParE subunits were similarly expressed in E. coli, purified to near homogeneity as 93- and 73-kDa proteins, and shown to generate efficient ATP-dependent DNA relaxation and DNA decatenation activities. Using the purified enzymes, we examined the inhibitory effects of three paradigm fluoroquinolones—ciprofloxacin, sparfloxacin, and clinafloxacin—which previous genetic studies with S. pneumoniae suggested act preferentially through topoisomerase IV, through gyrase, and through both enzymes, respectively. Surprisingly, all three quinolones were more active in inhibiting purified topoisomerase IV than gyrase, with clinafloxacin showing the greatest inhibitory potency. Moreover, the tested agents were at least 25-fold more effective in stabilizing a cleavable complex (the relevant cytotoxic lesion) with topoisomerase IV than with gyrase, with clinafloxacin some 10- to 32-fold more potent against either enzyme, in line with its superior activity againstS. pneumoniae. The uniform target preference of the three fluoroquinolones for topoisomerase IV in vitro is in apparent contrast to the genetic data. We interpret these results in terms of a model for bacterial killing by quinolones in which cellular factors can modulate the effects of target affinity to determine the cytotoxic pathway.

scholarly journals Quinolones: Mechanism, Lethality and Their Contributions to Antibiotic Resistance

Molecules ◽  
2020 ◽  
Vol 25 (23) ◽  
pp. 5662
Author(s):  
Natassja G. Bush ◽  
Isabel Diez-Santos ◽  
Lauren R. Abbott ◽  
Anthony Maxwell
Keyword(s):  
Cell Death ◽  
Antibiotic Resistance ◽  
Dna Gyrase ◽  
Mechanisms Of Action ◽  
Quinolone Resistance ◽  
Topoisomerase Iv ◽  
Bacterial Enzymes ◽  
Dna Topoisomerase ◽  
Quinolone Antibiotics ◽  
Dna Complex

Fluoroquinolones (FQs) are arguably among the most successful antibiotics of recent times. They have enjoyed over 30 years of clinical usage and become essential tools in the armoury of clinical treatments. FQs target the bacterial enzymes DNA gyrase and DNA topoisomerase IV, where they stabilise a covalent enzyme-DNA complex in which the DNA is cleaved in both strands. This leads to cell death and turns out to be a very effective way of killing bacteria. However, resistance to FQs is increasingly problematic, and alternative compounds are urgently needed. Here, we review the mechanisms of action of FQs and discuss the potential pathways leading to cell death. We also discuss quinolone resistance and how quinolone treatment can lead to resistance to non-quinolone antibiotics.

Activities of Trovafloxacin Compared with Those of Other Fluoroquinolones against Purified Topoisomerases and gyrA and grlA Mutants ofStaphylococcus aureus

1999 ◽  
Vol 43 (8) ◽  
pp. 1845-1855 ◽  
Author(s):  
Thomas D. Gootz ◽  
Richard P. Zaniewski ◽  
Suzanne L. Haskell ◽  
Frank S. Kaczmarek ◽  
Alison E. Maurice
Keyword(s):  
Dna Cleavage ◽  
Dna Gyrase ◽  
The Other ◽  
Second Step ◽  
Ofstaphylococcus Aureus ◽  
Topoisomerase Iv ◽  
Direct Plating ◽  
Clinical Strains ◽  
Plating Method ◽  
Susceptibility Breakpoint

ABSTRACT Frequencies of mutation to resistance with trovafloxacin and four other quinolones were determined with quinolone-susceptibleStaphylococcus aureus RN4220 by a direct plating method. First-step mutants were selected less frequently with trovafloxacin (1.1 × 10−10 at 2 to 4× the MIC) than with levofloxacin or ciprofloxacin (3.0 × 10−7 to 3.0 × 10−8 at 2 to 4× the MIC). Mutants with a change in GrlA (Ser80→Phe or Tyr) were most commonly selected with trovafloxacin, ciprofloxacin, levofloxacin, or pefloxacin. First-step mutants were difficult to select with sparfloxacin; however, second-step mutants with mutations in gyrA were easily selected when a preexisting mutation in grlA was present. Against 29 S. aureus clinical isolates with known mutations in gyrA and/or grlA, trovafloxacin was the most active quinolone tested (MIC at which 50% of isolates are inhibited [MIC50] and MIC90, 1 and 4 μg/ml, respectively); in comparison, MIC50s and MIC90s were 32 and 128, 16 and 32, 8 and 32, and 128 and 256 μg/ml for ciprofloxacin, sparfloxacin, levofloxacin, and pefloxacin, respectively. Strains with a mutation in grlA only were generally susceptible to all of the quinolones tested. For mutants with changes in both grlA and gyrA MICs were higher and were generally above the susceptibility breakpoint for ciprofloxacin, sparfloxacin, levofloxacin, and pefloxacin. Addition of reserpine (20 μg/ml) lowered the MICs only of ciprofloxacin fourfold or more for 18 of 29 clinical strains. Topoisomerase IV and DNA gyrase genes were cloned from S. aureus RN4220 and from two mutants with changes in GrlA (Ser80→Phe and Glu84→Lys). The enzymes were overexpressed in Escherichia coli GI724, purified, and used in DNA catalytic and cleavage assays that measured the relative potency of each quinolone. Trovafloxacin was at least five times more potent than ciprofloxacin, sparfloxacin, levofloxacin, or pefloxacin in stimulating topoisomerase IV-mediated DNA cleavage. While all of the quinolones were less potent in cleavage assays with the altered topoisomerase IV, trovafloxacin retained its greater potency relative to those of the other quinolones tested. The greater intrinsic potency of trovafloxacin against the lethal topoisomerase IV target in S. aureus contributes to its improved potency against clinical strains of S. aureus that are resistant to other quinolones.

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