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A high-throughput, homogeneous, bioluminescent assay for Pseudomonas aeruginosa gyrase inhibitors and other DNA-damaging agents.

ABSTRACT: A homogeneous, sensitive, cellular bioluminescent high-throughput screen was developed for inhibitors of gyrase and other DNA-damaging agents in Pseudomonas aeruginosa. The screen is based on a Photorhabdus luminescens luciferase operon transcriptional fusion to a promoter that responds to DNA damage caused by reduced gyrase levels and fluoroquinolone inhibition. This reporter strain is sensitive to levels of ciprofloxacin as low as one-fourth minimum inhibitory concentration (MIC) with Z' scores greater than 0.5, indicating the assay is suitable for high-throughput screening. This screen combines the benefits of a whole-cell assay with a sensitivity and target specificity superior to those of traditional cell-based screens for inhibitors of viability or growth. In duplicate pilot screens of 2000 known bioactive compounds, 13 compounds generated reproducible signals >50% of that of the control (ciprofloxacin at one-half MIC) using bioluminescence readings after 7 h of incubation. Ten are fluoroquinolones known to cause accumulation of cleaved DNA-enzyme complexes in bacterial cells; the other 3 are known to create DNA adducts. Therefore, all 13 hits inhibit DNA synthesis but by a variety of different DNA-damaging mechanisms. This convenient, inexpensive screen will be useful for rapidly identifying DNA gyrase inhibitors and other DNA-damaging agents, which may lead to potent new antibacterials.

SUBMITTER: Moir DT

PROVIDER: S-EPMC2561246 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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A high-throughput, homogeneous, bioluminescent assay for Pseudomonas aeruginosa gyrase inhibitors and other DNA-damaging agents.

Moir Donald T DT Ming Di Opperman Timothy T Schweizer Herbert P HP Bowlin Terry L TL

Journal of biomolecular screening 20070720 6

A homogeneous, sensitive, cellular bioluminescent high-throughput screen was developed for inhibitors of gyrase and other DNA-damaging agents in Pseudomonas aeruginosa. The screen is based on a Photorhabdus luminescens luciferase operon transcriptional fusion to a promoter that responds to DNA damage caused by reduced gyrase levels and fluoroquinolone inhibition. This reporter strain is sensitive to levels of ciprofloxacin as low as one-fourth minimum inhibitory concentration (MIC) with Z' score ...[more]

PMID: 17644773

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Project description:The design of novel antibiotics relies on a profound understanding of their mechanism of action. While it has been shown that cellular effects of antibiotics cluster according to their molecular targets, we investigated whether compounds binding to different sites of the same target can be differentiated by their transcriptome or metabolome signatures. The effects of three fluoroquinolones, two aminocoumarins, and two cystobactamids, all inhibiting bacterial gyrase, on Pseudomonas aeruginosa at subinhibitory concentrations could be distinguished clearly by RNA sequencing as well as metabolomics. We observed a strong (2.8- to 212-fold) induction of autolysis-triggering pyocins in all gyrase inhibitors, which correlated with extracellular DNA (eDNA) release. Gyrase B-binding aminocoumarins induced the most pronounced changes, including a strong downregulation of phenazine and rhamnolipid virulence factors. Cystobactamids led to a downregulation of a glucose catabolism pathway. The study implies that clustering cellular mechanisms of action according to the primary target needs to take class-dependent variances into account. IMPORTANCE Novel antibiotics are urgently needed to tackle the growing worldwide problem of antimicrobial resistance. Bacterial pathogens possess few privileged targets for a successful therapy: the majority of existing antibiotics as well as current candidates in development target the complex bacterial machinery for cell wall synthesis, protein synthesis, or DNA replication. An important mechanistic question addressed by this study is whether inhibiting such a complex target at different sites with different compounds has similar or differentiated cellular consequences. Using transcriptomics and metabolomics, we demonstrate that three different classes of gyrase inhibitors can be distinguished by their molecular signatures in P. aeruginosa. We describe the cellular effects of a promising, recently identified gyrase inhibitor class, the cystobactamids, in comparison to those of the established gyrase A-binding fluoroquinolones and the gyrase B-binding aminocoumarins. The study results have implications for mode-of-action discovery approaches based on target-specific reference compounds, as they highlight the intraclass variability of cellular compound effects.

Dataset Information

A high-throughput, homogeneous, bioluminescent assay for Pseudomonas aeruginosa gyrase inhibitors and other DNA-damaging agents.

Publications

A high-throughput, homogeneous, bioluminescent assay for Pseudomonas aeruginosa gyrase inhibitors and other DNA-damaging agents.

Similar Datasets

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