The role of DNA gyrase in illegitimate recombination

DNA, due to its double-helical structure, is subject to changes in topology due to the nature of transcription and replication. To overcome this, cells have processes and enzymes that ameliorate these changes. One such group of enzymes are the DNA topoisomerases, which are responsible for the maintenance of DNA topology. Despite this important role, these enzymes participate in illegitimate recombination (IR), which is genetic recombination between regions of DNA that share little or no homology. This can result in chromosomal rearrangements and is often a consequence of DNA-damaging agents. A consequence of topoisomerase-induced IR is thought to be therapy-related acute myeloid leukaemia (tAML). Analogously, there is evidence that exposure to sublethal concentrations of ciprofloxacin, a topoisomerase inhibitor, can cause resistance to non-quinolone antibiotics. This may work by a similar mechanism as that proposed for t-AML. This project centres around the examination of DNA gyrase-mediated IR focussing on the proposed subunit-exchange model. Using Blue-Native PAGE, I set up an assay to examine subunit exchange in topoisomerases. I have also characterised previously identified gyrase hyper-recombination mutations, known to increase the frequency of IR. Furthermore, I have investigated quinolone-induced antibiotic resistance and what the mechanism is. Here, I show that DNA gyrase can undergo subunit exchange, and that this seems to occur within higherorder oligomers of the enzyme, which have not been investigated before. Biochemical characterisation of the hyper-recombination mutations shows that they impair DNA gyrase activity which, in vivo, may have downstream consequences that may lead to IR. Using an in vivo assay where E. coli is treated with subinhibitory levels of quinolones, I have seen resistance to other non-quinolone antibiotics. This is not seen when other antibiotics, including other topoisomerase inhibitors, are tested. Whole genome sequencing has revealed point mutations that explain the resistances seen, however other larger chromosomal modifications have been observed as well

Expression Atlas update: gene and protein expression in multiple species.

The EMBL-EBI Expression Atlas is an added value knowledge base that enables researchers to answer the question of where (tissue, organism part, developmental stage, cell type) and under which conditions (disease, treatment, gender, etc) a gene or protein of interest is expressed. Expression Atlas brings together data from >4500 expression studies from >65 different species, across different conditions and tissues. It makes these data freely available in an easy to visualise form, after expert curation to accurately represent the intended experimental design, re-analysed via standardised pipelines that rely on open-source community developed tools. Each study's metadata are annotated using ontologies. The data are re-analyzed with the aim of reproducing the original conclusions of the underlying experiments. Expression Atlas is currently divided into Bulk Expression Atlas and Single Cell Expression Atlas. Expression Atlas contains data from differential studies (microarray and bulk RNA-Seq) and baseline studies (bulk RNA-Seq and proteomics), whereas Single Cell Expression Atlas is currently dedicated to Single Cell RNA-Sequencing (scRNA-Seq) studies. The resource has been in continuous development since 2009 and it is available at https://www.ebi.ac.uk/gxa

Quinolones: Mechanism, Lethality and Their Contributions to Antibiotic Resistance

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

Predictive modeling targets thymidylate synthase ThyX in Mycobacterium tuberculosis

International audienceThere is an urgent need to identify new treatments for tuberculosis (TB), a major infectious disease caused by Mycobacterium tuberculosis (Mtb), which results in 1.5 million deaths each year. We have targeted two essential enzymes in this organism that are promising for antibacterial therapy and reported to be inhibited by naphthoquinones. ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the human enzyme. DNA gyrase is a DNA topoisomerase present in bacteria and plants but not animals. The current study set out to understand the structure-activity relationships of these targets in Mtb using a combination of cheminformatics and in vitro screening. Here, we report the identification of new Mtb ThyX inhibitors, 2-chloro-3-(4-methanesulfonylpiperazin-1-yl)-1,4-dihydronaphthalene-1,4-dione) and idebenone, which show modest whole-cell activity and appear to act, at least in part, by targeting ThyX in Mtb

Lead selection and characterization of antitubercular compounds using the Nested Chemical Library

Discovering new drugs to treat tuberculosis more efficiently and to overcome multidrug resistance is a world health priority. To find novel antitubercular agents several approaches have been used in various institutions worldwide, including target-based approaches against several validated mycobacterial enzymes and phenotypic screens. We screened more than 17,000 compounds from Vichem's Nested Chemical Library(TM) using an integrated strategy involving whole cell-based assays with Corynebacterium glutamicum and Mycobacterium tuberculosis, and target-based assays with protein kinases PknA, PknB and PknG as well as other targets such as PimA and bacterial topoisomerases simultaneously. With the help of the target-based approach we have found very potent hits inhibiting the selected target enzymes, but good minimal inhibitory concentrations (MIC) against M. tuberculosis were not achieved. Focussing on the whole cell-based approach several potent hits were found which displayed minimal inhibitory concentrations (MIC) against M. tuberculosis below 10 mu M and were non-mutagenic, non-cytotoxic and the targets of some of the hits were also identified. The most active hits represented various scaffolds. Medicinal chemistry-based lead optimization was performed applying various strategies and, as a consequence, a series of novel potent compounds were synthesized. These efforts resulted in some effective potential antitubercular lead compounds which were confirmed in phenotypic assays. (C) 2015 Elsevier Ltd. All rights reserved