Quinolone-resistant gyrase mutants demonstrate decreased susceptibility to triclosan

Objectives: Cross-resistance between antibiotics and biocides is a potentially important driver of MDR. A relationship between susceptibility of Salmonella to quinolones and triclosan has been observed. This study aimed to: (i) investigate the mechanism underpinning this; (ii) determine whether the phenotype is conserved in Escherichia coli; and (iii) evaluate the potential for triclosan to select for quinolone resistance. Methods: WT E. coli, Salmonella enterica serovar Typhimurium and gyrA mutants were used. These were characterized by determining antimicrobial susceptibility, DNA gyrase activity and sensitivity to inhibition. Expression of stress response pathways (SOS, RpoS, RpoN and RpoH) was measured, as was the fitness of mutants. The potential for triclosan to select for quinolone resistance was determined. Results: All gyrase mutants showed increased triclosan MICs and altered supercoiling activity. There was no evidence for direct interaction between triclosan and gyrase. Identical substitutions in GyrA had different impacts on supercoiling in the two species. For both, there was a correlation between altered supercoiling and expression of stress responses. This was more marked in E. coli, where an Asp87Gly GyrA mutant demonstrated greatly increased fitness in the presence of triclosan. Exposure of parental strains to low concentrations of triclosan did not select for quinolone resistance. Conclusions: Our data suggest gyrA mutants are less susceptible to triclosan due to up-regulation of stress responses. The impact of gyrA mutation differs between E. coli and Salmonella. The impacts of gyrA mutation beyond quinolone resistance have implications for the fitness and selection of gyrA mutants in the presence of non-quinolone antimicrobials

Structural and mechanistic analysis of ATPase inhibitors targeting mycobacterial DNA gyrase

Objectives To evaluate the efficacy of two novel compounds against mycobacteria and determine the molecular basis of their action on DNA gyrase using structural and mechanistic approaches. Methods Redx03863 and Redx04739 were tested in antibacterial assays, and also against their target, DNA gyrase, using DNA supercoiling and ATPase assays. X-ray crystallography was used to determine the structure of the gyrase B protein ATPase sub-domain from Mycobacterium smegmatis complexed with the aminocoumarin drug novobiocin, and structures of the same domain from Mycobacterium thermoresistibile complexed with novobiocin, and also with Redx03863. Results Both compounds, Redx03863 and Redx04739, were active against selected Gram-positive and Gram-negative species, with Redx03863 being the more potent, and Redx04739 showing selectivity against M. smegmatis. Both compounds were potent inhibitors of the supercoiling and ATPase reactions of DNA gyrase, but did not appreciably affect the ATP-independent relaxation reaction. The structure of Redx03863 bound to the gyrase B protein ATPase sub-domain from M. thermoresistibile shows that it binds at a site adjacent to the ATP- and novobiocin-binding sites. We found that most of the mutations that we made in the Redx03863-binding pocket, based on the structure, rendered gyrase inactive. Conclusions Redx03863 and Redx04739 inhibit gyrase by preventing the binding of ATP. The fact that the Redx03863-binding pocket is distinct from that of novobiocin, coupled with the lack of activity of resistant mutants, suggests that such compounds could have potential to be further exploited as antibiotics

Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides.

In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, we overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species

The dimer state of GyrB is an active form: implications for the initial complex assembly and processive strand passage

In a previous study, we presented the dimer structure of DNA gyrase B′ domain (GyrB C-terminal domain) from Mycobacterium tuberculosis and proposed a ‘sluice-like’ model for T-segment transport. However, the role of the dimer structure is still not well understood. Cross-linking and analytical ultracentrifugation experiments showed that the dimer structure exists both in the B′ protein and in the full-length GyrB in solution. The cross-linked dimer of GyrB bound GyrA very weakly, but bound dsDNA with a much higher affinity than that of the monomer state. Using cross-linking and far-western analyses, the dimer state of GyrB was found to be involved in the ternary GyrA–GyrB–DNA complex. The results of mutational studies reveal that the dimer structure represents a state before DNA cleavage. Additionally, these results suggest that the dimer might also be present between the cleavage and reunion steps during processive transport

Application of a novel microtitre plate-based assay for the discovery of new inhibitors of DNA gyrase and DNA topoisomerase VI.

DNA topoisomerases are highly exploited targets for antimicrobial drugs. The spread of antibiotic resistance represents a significant threat to public health and necessitates the discovery of inhibitors that target topoisomerases in novel ways. However, the traditional assays for topoisomerase activity are not suitable for the high-throughput approaches necessary for drug discovery. In this study we validate a novel assay for screening topoisomerase inhibitors. A library of 960 compounds was screened against Escherichia coli DNA gyrase and archaeal Methanosarcina mazei DNA topoisomerase VI. Several novel inhibitors were identified for both enzymes, and subsequently characterised in vitro and in vivo. Inhibitors from the M. mazei topoisomerase VI screen were tested for their ability to inhibit Arabidopsis topoisomerase VI in planta. The data from this work present new options for antibiotic drug discovery and provide insight into the mechanism of topoisomerase VI

Structural and Biochemical Analysis of the Pentapeptide Repeat Protein EfsQnr, a Potent DNA Gyrase Inhibitor▿ †

The chromosomally encoded Qnr homolog protein from Enterococcus faecalis (EfsQnr), when expressed, confers to its host a decreased susceptibility to quinolones and consists mainly of tandem repeats, which is consistent with belonging to the pentapeptide repeat family of proteins (PRPs). EfsQnr was cloned with an N-terminal 6× His tag and purified to homogeneity. EfsQnr partially protected DNA gyrase from fluoroquinolone inhibition at concentrations as low as 20 nM. EfsQnr inhibited the ATP-dependent supercoiling activity of DNA gyrase with a 50% inhibitory concentration (IC50) of 1.2 μM, while no significant inhibition of ATP-independent relaxation activity was observed. EfsQnr was cytotoxic when overexpressed in Escherichia coli, resulting in the clumping of cells and a loss of viability. The X-ray crystal structure of EfsQnr was determined to 1.6-Å resolution. EfsQnr exhibits the right-handed quadrilateral beta-helical fold typical of PRPs, with features more analogous to MfpA (mycobacterium fluoroquinolone resistance pentapeptide) than to the PRPs commonly found in cyanobacteria

A rapid high-resolution method for resolving DNA topoisomers

Abstract Objective Agarose gel electrophoresis has been the mainstay technique for the analysis of DNA samples of moderate size. In addition to separating linear DNA molecules, it can also resolve different topological forms of plasmid DNAs, an application useful for the analysis of the reactions of DNA topoisomerases. However, gel electrophoresis is an intrinsically low-throughput technique and suffers from other potential disadvantages. We describe the application of the QIAxcel Advanced System, a high-throughput capillary electrophoresis system, to separate DNA topoisomers, and compare this technique with gel electrophoresis. Results We prepared a range of topoisomers of plasmids pBR322 and pUC19, and a 339 bp DNA minicircle, and compared their separation by gel electrophoresis and the QIAxcel System. We found superior resolution with the QIAxcel System, and that quantitative analysis of topoisomer distributions was straightforward. We show that the QIAxcel system has advantages in terms of speed, resolution and cost, and can be applied to DNA circles of various sizes. It can readily be adapted for use in compound screening against topoisomerase targets

<i>In vitro</i> characterisation of DNA gyrase screen hits.

<p>A. Determination of the IC₅₀ for mitoxantrone in a supercoiling assay with 1 unit of gyrase (12 nM); 100 μM ciprofloxacin (Cip.) was used as a positive control for inhibition. The positions of relaxed (Rel.) and negatively supercoiled (SC) DNA are indicated. B. Determination of the IC₅₀ for suramin. C. Assaying the abilities of mitoxantrone and suramin to induce gyrase-mediated DNA cleavage. The reactions were carried out in the absence of ATP. Ciprofloxacin was used as a positive control for gyrase-mediated cleavage. The position of linear DNA (Lin.) is indicated. D. Suramin-induced protection of DNA from Ca²⁺-induced, gyrase-mediated cleavage. E. Inhibition of gyrase binding to a 147 bp DNA fragment by suramin.</p

Inhibition of <i>Arabidopsis</i> hypocotyl extension by hexylresorcinol.

<p>All plants were grown for 5 days in the dark. A. Average length of seedlings grown on 20, 30, 40 50, 80 or 100 µM hexylresorcinol. Error bars represent the standard deviation of the samples. B. Percentage germination of seedlings grown on 20, 30, 40 50, 80 or 100 µM hexylresorcinol.</p