Unique Biological Properties and Molecular Mechanism of 5,6-Bridged Quinolones

ABSTRACT We have characterized an early series of 5,6-bridged dioxinoquinolones which behaved strikingly different from typical quinolones. The 5,6-bridged dioxinoquinolones inhibited Escherichia coli DNA gyrase supercoiling activity but, unlike typical quinolones, failed to stimulate gyrase-dependent cleavable complex formation. Analogous unsubstituted compounds stimulated cleavable complex formation but were considerably less potent than the corresponding 5,6-bridged compounds. Consistent with a previous report (M. Antoine et al., Chim. Ther. 7:434-443, 1972) and contrary to established quinolone SAR trends, a compound with an N-1 methyl substitution (PGE-8367769) was more potent than its analog with an N-1 ethyl substitution (PGE-6596491). PGE-8367769 was shown to antagonize ciprofloxacin-mediated cleavable complex formation in a dose-dependent manner, suggesting an interaction with the gyrase-DNA complex that overlaps that of ciprofloxacin. Resistance to PGE-8367769 in E. coli was found to arise through missense mutations in gyrA, implicating DNA gyrase as the primary antibacterial target. Notably, only 1 of 15 distinct mutations selected on PGE-8367769 (D87G) has previously been implicated in quinolone resistance in E. coli. The remaining 14 mutations (E16V, G31V, R38L, G40A, Y50D, V70A, A84V, I89L, M135T, G173S, T180I, F217C, P218T, and F513C) have not been previously reported, and most were located outside of the traditional quinolone resistance-determining region. These novel GyrA mutations decreased sensitivity to 5,6-bridged dioxinoquinolones by four- to eightfold, whereas they did not confer resistance to other quinolones such as ciprofloxacin, clinafloxacin, or nalidixic acid. These results demonstrate that the 5,6-bridged quinolones act via a mechanism that is related to but qualitatively different from that of typical quinolones.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.3.710 ◽

1996 ◽

Vol 40 (3) ◽

pp. 710-714 ◽

Cited By ~ 53

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.

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Quinolones: Mechanism, Lethality and Their Contributions to Antibiotic Resistance

Molecules ◽

10.3390/molecules25235662 ◽

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.

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Full Capacity of Recombinant Escherichia coliHeat-Stable Enterotoxin Fusion Proteins for Extracellular Secretion, Antigenicity, Disulfide Bond Formation, and Activity

Infection and Immunity ◽

10.1128/iai.68.7.4064-4074.2000 ◽

2000 ◽

Vol 68 (7) ◽

pp. 4064-4074 ◽

Cited By ~ 16

Author(s):

Isabelle Batisson ◽

Maurice Der Vartanian ◽

Brigitte Gaillard-Martinie ◽

Michel Contrepois

Keyword(s):

Disulfide Bonds ◽

Secretory Pathway ◽

Biological Properties ◽

Leader Peptide ◽

Dependent Manner ◽

Reporter Protein ◽

E Coli ◽

Heat Stable

ABSTRACT We have successfully used the major subunit ClpG ofEscherichia coli CS31A fimbriae as an antigenic and immunogenic exposure-delivery vector for various heterologous peptides varying in nature and length. However, the ability of ClpG as a carrier to maintain in vitro and in vivo the native biological properties of passenger peptide has not yet been reported. To address this possibility, we genetically fused peptides containing all or part of the E. coli human heat-stable enterotoxin (STh) sequence to the amino or carboxyl ends of ClpG. Using antibodies to the ClpG and STh portions for detecting the hybrids; AMS (4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate), a potent free thiol-trapping reagent, for determining the redox state of STh in the fusion; and the suckling mouse assay for enterotoxicity, we demonstrated that all ClpG-STh proteins were secreted in vitro and in vivo outside the E. coli cells in a heat-stable active oxidized (disulfide-bonded) form. Indeed, in contrast to many earlier studies, blocking the natural NH2 or COOH extremities of STh had, in all cases, no drastic effect on cell release and toxin activity. Only antigenicity of STh C-terminally extended with ClpG was strongly affected in a conformation-dependent manner. These results suggest that the STh activity was not altered by the chimeric structure, and therefore that, like the natural toxin, STh in the fusion had a spatial structure flexible enough to be compatible with secretion and enterotoxicity (folding and STh receptor recognition). Our study also indicates that disulfide bonds were essential for enterotoxicity but not for release, that spontaneous oxidation by molecular oxygen occurred in vitro in the medium, and that the E. coli cell-bound toxin activity in vivo resulted from an effective export processing of hybrids and not cell lysis. None of the ClpG-STh subunits formed hybrid CS31A-STh fimbriae at the cell surface ofE. coli, and a strong decrease in the toxin activity was observed in the absence of CS31A helper proteins. In fact, chimeras translocated across the outer membrane as a free folded monomer once they were guided into the periplasm by the ClpG leader peptide through the CS31A-dependent secretory pathway. In summary, ClpG appears highly attractive as a carrier reporter protein for basic and applied research through the engineering of E. coli for culture supernatant delivery of an active cysteine-containing protein, such as the heat-stable enterotoxin.

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Molecular Basis for the Differential Quinolone Susceptibility of Mycobacterial DNA Gyrase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01958-13 ◽

2014 ◽

Vol 58 (4) ◽

pp. 2013-2020 ◽

Cited By ~ 14

Author(s):

Rupesh Kumar ◽

Bhavani Shankar Madhumathi ◽

Valakunja Nagaraja

Keyword(s):

Differential Susceptibility ◽

Dna Gyrase ◽

Drug Binding ◽

Double Strand Breaks ◽

Type Ii ◽

Content Type ◽

Strand Breaks ◽

E Coli ◽

Dna Complex ◽

Negative Supercoils

ABSTRACTDNA gyrase is a type II topoisomerase that catalyzes the introduction of negative supercoils in the genomes of eubacteria. Fluoroquinolones (FQs), successful as drugs clinically, target the enzyme to trap the gyrase-DNA complex, leading to the accumulation of double-strand breaks in the genome. Mycobacteria are less susceptible to commonly used FQs. However, an 8-methoxy-substituted FQ, moxifloxacin (MFX), is a potent antimycobacterial, and a higher susceptibility of mycobacterial gyrase to MFX has been demonstrated. Although several models explain the mechanism of FQ action and gyrase-DNA-FQ interaction, the basis for the differential susceptibility of mycobacterial gyrase to various FQs is not understood. We have addressed the basis of the differential susceptibility of the gyrase and revisited the mode of action of FQs. We demonstrate that FQs bind bothEscherichia coliandMycobacterium tuberculosisgyrases in the absence of DNA and that the addition of DNA enhances the drug binding. The FQs bind primarily to the GyrA subunit of mycobacterial gyrase, while inE. coliholoenzyme is the target. The binding of MFX to GyrA ofM. tuberculosiscorrelates with its effectiveness as a better inhibitor of the enzyme and its efficacy in cell killing.

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Mutagenesis in the α3α4 GyrA Helix and in the Toprim Domain of GyrB Refines the Contribution of Mycobacterium tuberculosis DNA Gyrase to Intrinsic Resistance to Quinolones

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01380-07 ◽

2008 ◽

Vol 52 (8) ◽

pp. 2909-2914 ◽

Cited By ~ 13

Author(s):

Stéphanie Matrat ◽

Alexandra Aubry ◽

Claudine Mayer ◽

Vincent Jarlier ◽

Emmanuelle Cambau

Keyword(s):

Escherichia Coli ◽

Mycobacterium Tuberculosis ◽

Primary Structure ◽

Three Dimensional ◽

Dna Gyrase ◽

Quinolone Resistance ◽

Dimensional Model ◽

Three Dimensional Model ◽

Intrinsic Resistance ◽

E Coli

ABSTRACT The replacement of M74 in GyrA, A83 in GyrA, and R447 in GyrB of Mycobacterium tuberculosis gyrase by their Escherichia coli homologs resulted in active enzymes as quinolone susceptible as the E. coli gyrase. This demonstrates that the primary structure of gyrase determines intrinsic quinolone resistance and was supported by a three-dimensional model of N-terminal GyrA.

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Heparin Synergistically Enhances Interleukin-11 Signaling through Upregulation of the Map Kinase Pathway.

Blood ◽

10.1182/blood.v106.11.2632.2632 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2632-2632

Author(s):

Raghav Rajgopal ◽

Martin Butcher ◽

Kimberly J. Walton ◽

Stephen G. Shaughnessy

Keyword(s):

Complex Formation ◽

Map Kinase ◽

Osteoclast Formation ◽

Dependent Manner ◽

Serine Kinase ◽

Mitogen Activated Protein ◽

Map Kinase Pathway ◽

Dna Complex ◽

Kinase Pathway ◽

Interleukin 11

Abstract Using an animal model of heparin-induced osteoporosis we previously demonstrated that heparin causes bone loss, in part, by increasing osteoclast number and activity. In an effort to account for these findings, we examined the effect of heparin on osteoclast formation in vitro. We found that while heparin alone had no effect, it was able to synergistically enhance Interleukin-11 (IL-11) - induced STAT3 (Signal Transducer and Activator of Transcription) activation and thus, increase both gp130 expression and osteoclast formation. In the present study, we examine the effect of various serine kinase inhibitors on heparin’s ability to synergistically enhance both STAT3 activation and gp130 expression. As assessed by electrophoretic mobility shift assays (EMSAs), treatment of murine calvaria cells with heparin was found to increase IL-11-induced STAT3-DNA complex formation by 2 fold. Inhibition of the c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein (MAP) kinase, or the phosphatidylinositol 3-kinase (PI3-K) pathway had no effect on heparin’s ability to promote STAT3-DNA complex formation. In contrast, the mitogen-activated protein kinase kinase (MEK) inhibitor, PD098059, completely abolished the ability of heparin to act synergistically with IL-11. Similar results were obtained when northern blot analysis was used to assess heparin’s ability to enhance IL-11-induced gp130 expression. In an attempt to resolve the mechanism by which this was occurring, we examined the ability of heparin to influence STAT3 Ser727 phosphorylation in the presence or absence of IL-11. Heparin alone was found to have no effect on Ser727 phosphorylation, nor did heparin alter the phosphorylation status of Ser727 in the presence of IL-11. Heparin was, however, found to increase ERK1/2 (extracellular signal regulated kinase) activation in both a time and dose-dependent manner. When taken together, these findings suggest that heparin enhances IL-11-induced STAT3 activation and thus, gp130 expression, by a mechanism that is independent of STAT3 Ser727 phosphorylation, but which involves upregulation of the MAP Kinase pathway.

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Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.10.2321 ◽

1996 ◽

Vol 40 (10) ◽

pp. 2321-2326 ◽

Cited By ~ 302

Author(s):

X S Pan ◽

J Ambler ◽

S Mehtar ◽

L M Fisher

Keyword(s):

Streptococcus Pneumoniae ◽

Dna Gyrase ◽

Quinolone Resistance ◽

Clinical Isolates ◽

Resistant Isolate ◽

Topoisomerase Iv ◽

E Coli ◽

Low Level ◽

Drug Concentrations ◽

Level Resistance

Ciprofloxacin-resistant mutants of Streptococcus pneumoniae 7785 were generated by stepwise selection at increasing drug concentrations. Sequence analysis of PCR products from the strains was used to examine the quinolone resistance-determining regions of the GyrA and GyrB proteins of DNA gyrase and the analogous regions of the ParC and ParE subunits of DNA topoisomerase IV. First-step mutants exhibiting low-level resistance had no detectable changes in their topoisomerase quinolone resistance-determining regions, suggesting altered permeation or another novel resistance mechanism. Nine of 10 second-step mutants exhibited an alteration in ParC at Ser-79 to Tyr or Phe or at Ala-84 to Thr. Third- and fourth-step mutants displaying high-level ciprofloxacin resistance were found to have, in addition to the ParC alteration, a change in GyrA at residues equivalent to Escherichia coli GyrA resistance hot spots Ser-83 and Asp-87 or in GyrB at Asp-435 to Asn, equivalent to E. coli Asp-426, part of a highly conserved EGDSA motif in GyrB. No ParE changes were observed. Complementary analysis of two S. pneumoniae clinical isolates displaying low-level resistance to ciprofloxacin revealed a ParC change at Ser-79 to Phe or Arg-95 to Cys but no changes in GyrA, GyrB, or ParE. A highly resistant isolate, in addition to a ParC mutation, had a GyrA alteration at the residue equivalent to E. coli Asp-87. Thus, in both laboratory strains and clinical isolates, ParC mutations preceded those in GyrA, suggesting that topoisomerase IV is a primary topoisomerase target and gyrase is a secondary target for ciprofloxacin in S. pneumoniae.

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The Extracellular Domain of Human High Affinity Copper Transporter (hNdCTR1), Synthesized by E. coli Cells, Chelates Silver and Copper Ions in Vivo

10.20944/preprints201708.0096.v1 ◽

2017 ◽

Author(s):

Tatiana P. Sankova ◽

Iurii A. Orlov ◽

Andrey N. Saveliev ◽

Demid A. Kirilenko ◽

Polina S. Babich ◽

...

Keyword(s):

Fusion Protein ◽

Metal Binding ◽

Copper Ions ◽

Silver Ions ◽

Biological Properties ◽

Dependent Manner ◽

Copper Transporter ◽

E Coli ◽

High Affinity

There is much interest in effective copper chelators to correct copper dyshomeostasis in neurodegenerative and oncological diseases. In this study, a recombinant fusion protein for expression in E. coli cells was constructed from glutathione-S-transferase (GST) and the N-terminal domain (ectodomain) of human high affinity copper transporter CTR1 (hNdCTR1), which has three metal-bound motifs. Several biological properties of the GST-hNdCTR1 fusion protein were assessed. It was demonstrated that in cells, the protein was prone to oligomerization, formed inclusion bodies and displayed no toxicity. Treatment of E. coli cells with copper and silver ions reduced cell viability in a dose- and time-dependent manner. Cells expressing GST-hNdCTR1 protein demonstrated resistance to the metal treatments. These cells accumulated silver ions and formed nanoparticles that contained AgCl and Ag0. In this bacterial population, filamentous bacteria with length about 10 μm were often observed. The possibility for the fusion protein carrying extracellular metal binding motifs to integrate into the cell’s copper metabolism and its chelating properties are discussed.

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Rationalizing the pH-Activity Response for Escherichia Coli’s Glycinamide Ribonucleotidetransformylase Through Computational Methods

10.26434/chemrxiv.7985618.v1 ◽

2019 ◽

Author(s):

Adrian Roitberg ◽

Pancham Lal Gupta

Keyword(s):

Transfer Reaction ◽

Catalytic Mechanism ◽

Ph Dependence ◽

Ph Effects ◽

Dependent Manner ◽

E Coli ◽

Gar Tfase ◽

Activity Curve ◽

Ph Dependent ◽

Formyl Transfer

<div>Human Glycinamide ribonucleotide transformylase (GAR Tfase), a regulatory enzyme in the de novo purine biosynthesis pathway, has been established as an anti-cancer target. GAR Tfase catalyzes the formyl transfer reaction from the folate cofactor to the GAR ligand. In the present work, we study E. coli GAR Tfase, which has high sequence similarity with the human GAR Tfase with most functional residues conserved. E. coli GAR Tfase exhibits structural changes and the binding of ligands that varies with pH which leads to change the rate of the formyl transfer reaction in a pH-dependent manner. Thus, the inclusion of pH becomes essential for the study of its catalytic mechanism. Experimentally, the pH-dependence of the kinetic parameter kcat is measured to evaluate the pH-range of enzymatic activity. However, insufficient information about residues governing the pH-effects on the catalytic activity leads to ambiguous assignments of the general acid and base catalysts and consequently its catalytic mechanism. In the present work, we use pH-replica exchange molecular dynamics (pH-REMD) simulations to study the effects of pH on E. coli GAR Tfase enzyme. We identify the titratable residues governing the pH-dependent conformational changes in the system. Furthermore, we filter out the protonation states which are essential in maintaining the structural integrity, keeping the ligands bound and assisting the catalysis. We reproduce the experimental pH-activity curve by computing the population of key protonation states. Moreover, we provide a detailed description of residues governing the acidic and basic limbs of the pH-activity curve.</div>

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