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. (Journal of Biomolecular Screening 2007:855-864)

Discovery of Bacterial Fatty Acid Synthase Type II Inhibitors Using a Novel Cellular Bioluminescent Reporter Assay

“…The response of the P PA2030-lux reporter strain to incubation with BAB was unexpected and may indicate a degree of nonspecificity for BAB. Conversely, the non-FASII reporters did not respond to TLM or PYP, but the reporter for gyrase inhibitors (P PA0614 -lux) did respond to ciprofloxacin as described previously (18). In summary, the luminescent signals generated by the FASII reporter strains were selectively induced in response to FASII inhibitors that target the initiation stage (AccC) or the elongation cycle (FabB) of the FASII pathway.…”

“…Finally, to demonstrate that the reporter strain responses are highly selective for FASII inhibitors and promoters, we evaluated two FASII inhibitor-sensitive reporter strains and four reporter strains designed to respond to inhibitors of other targets (18,19,27) for sensitivity to five inhibitors of FASII and non-FASII targets ( Fig. 3C).…”

“…To simulate reduced FASII pathway flow, we constructed P. aeruginosa strains in which the expression of FASII genes could be reduced to levels that inhibit normal growth. Since most genes encoding the FASII pathway are essential (35), these strains were constructed by deleting the essential gene in the presence of a P lac -regulated complementing copy carried on the replicating plasmid pUCP24GW (18,19), enabling the expression of the FASII genes to be regulated by IPTG. Accordingly, we generated P. aeruginosa strains with regulated expression of accD, which encodes a subunit of the FASII initiation activity acetyl-CoA carboxylase, and fabG, which encodes ␤-ketoacyl-acyl carrier protein (ACP) reductase, the first reduction step in the FASII elongation cycle.…”

“…For the accC gene, the pEX18ApGW vector carried a markerless, in-frame deletion that was flanked by about 1 kb of homologous DNA on each side. These constructs were used successfully to create merodiploids in P. aeruginosa strains PAO-LAC and PAO397, which were then resolved in the presence of a complementing copy of the appropriate wild-type or mutant gene on the extrachromosomally replicating plasmid pUCP24GW to create lac-regulated complemented deletions as described previously (18). The pUCP24 vector carried the lacI q gene for the accC and accD deletions but not for the fabG deletion, because complementation appeared to be inadequate to support growth in the presence of multiple copies of lacI q .…”

“…The resulting cellular screens of this study proved to be more sensitive to FASII inhibition than are growth assays, and they select for inhibitors that can penetrate the P. aeruginosa cell. In this study, we optimized and applied one of the FASII screens to over 100,000 diverse compounds and identified a novel series of compounds that generate highly significant luminescent responses in several FASII reporter strains but not in reporter strains designed to respond to inhibition of other targets (18,19). To verify the ability of these new reporter screens to identify novel FASII inhibitors, we used molecular genetic tools and biochemical assays to identify the molecular target of the most potent hit compound series.…”