title: A pathway-specific cell based screening system to detect bacterial cell wall inhibitors.
abstract: A pathway-specific cell-based screen is described to detect compounds that inhibit the biosynthesis of the cell wall of bacteria. The basis for detection is the discovery that the beta-lactamase gene from Citrobacterfreundii, cloned into Escherichia coli, is induced when cells are exposed to known cell wall inhibitors, and not just beta-lactam-based antibiotics. In a wild type host, cell wall inhibitors such as moenomycin, vancomycin, and ramoplanin, which are excluded by the outer membrane, only induce at high concentrations. However, these compounds, as well as fosfomycin, cycloserine, and cefoxitin, induce at concentrations at or below the MIC of a host carrying the envA-mutation, which causes a defect in the outer membrane. As additional proof that induction of beta-lactamase is the direct result of cell wall inhibition, a host strain carrying a temperature-sensitive mutation in the murG gene, whose product converts the cell wall intermediate Lipid I, to Lipid II, also induced beta-lactamase at the restrictive temperature. A protocol is described for screening samples in high-throughput mode.
authors.affiliation: Millennium Pharmaceuticals, Inc, Cambridge, MA, USA.
authors.forename: Dongyu
authors.initials: D
authors.lastname: Sun
authors.forename: Seth
authors.initials: S
authors.lastname: Cohen
authors.forename: Nagraj
authors.initials: N
authors.lastname: Mani
authors.forename: Chris
authors.initials: C
authors.lastname: Murphy
authors.forename: David M
authors.initials: DM
authors.lastname: Rothstein
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002473:Cell Wall / Q000187:drug effects* / Q000378:metabolism; D016971:Citrobacter freundii / Q000201:enzymology / Q000235:genetics; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008826:Microbial Sensitivity Tests; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001618:beta-Lactamases