Multicopy suppressors for novel antibacterial compounds reveal targets and drug efflux susceptibility.
Gene dosage has frequently been exploited to select for genetic interactions between a particular mutant and clones from a random genomic library at high copy. We report here the first use of multicopy suppression as a forward genetic method to determine cellular targets and potential resistance mechanisms for novel antibacterial compounds identified through high-throughput screening. A screen of 8640 small molecules for growth inhibition of a hyperpermeable strain of Escherichia coli led to the identification of 49 leads for suppressor selection from clones harboring an E. coli genomic library. The majority of suppressors were found to encode the multidrug efflux pump AcrB, indicating that those compounds were substrates for efflux. Two leads, which produced clones containing the gene folA, encoding dihydrofolate reductase (DHFR), proved to target DHFR in vivo and were competitive inhibitors in vitro.
Department of Biochemistry, Antimicrobial Research Centre, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada.
Xiaoming
X
Li
Michela
M
Zolli-Juran
Jonathan D
JD
Cechetto
Denis M
DM
Daigle
Gerard D
GD
Wright
Eric D
ED
Brown
D000900:Anti-Bacterial Agents / Q000008:administration & dosage* / Q000737:chemistry; D016503:Drug Delivery Systems / Q000379:methods*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D018628:Gene Dosage; D016680:Genome, Bacterial; D015698:Genomic Library; D006131:Growth Inhibitors / Q000008:administration & dosage* / Q000737:chemistry; D008826:Microbial Sensitivity Tests / Q000706:statistics & numerical data; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000502:physiology
D000900:Anti-Bacterial Agents; D006131:Growth Inhibitors; D013762:Tetrahydrofolate Dehydrogenase