title: Identification of antimicrobial targets using a comprehensive genomic approach.
abstract: Regulated antisense RNA enables the construction of a defined set of conditional growth-defective/lethal strains. In this study, we expanded the regulated antisense RNA interference technology and developed a high-throughput screening strategy to identify the potential drug targets of novel antimicrobials. To prove this concept, the specific antisense sublibrary of different essential open reading frames were pooled in the presence of an inducer, and treated with or without sublethal levels of mupirocin, triclosan, or gentamicin. Antisense RNA-expressing strains that were sensitized for increased susceptibility to the antibiotics were selectively detected via DNA subtractive hybridization, microarray, and whole-cell analyses. No strain was identified as supersensitive to gentamicin because there was no target-specific antisense strain in this sublibrary. In contrast, strains expressing antisense to isoleucine tRNA synthetase (ileS) and enoyl-[acyl-carrier-protein] reductase (fabI) were specifically identified as having increased susceptibility to mupirocin and triclosan, respectively. These results demonstrated that ileS and fabI antisense strains showed significant increases of susceptibility only to their specific inhibitors. This data demonstrates that a regulated antisense RNA expression library provides an effective tool to assist in the identification of potential targets for novel antibacterial agents.
authors.affiliation: Department of Microbiology, GlaxoSmithKline Pharmaceuticals Research and Development, Collegeville, PA 19462, USA.
authors.forename: Dezhong
authors.initials: D
authors.lastname: Yin
authors.forename: Brian
authors.initials: B
authors.lastname: Fox
authors.forename: Michael L
authors.initials: ML
authors.lastname: Lonetto
authors.forename: Mark R
authors.initials: MR
authors.lastname: Etherton
authors.forename: David J
authors.initials: DJ
authors.lastname: Payne
authors.forename: David J
authors.initials: DJ
authors.lastname: Holmes
authors.forename: Martin
authors.initials: M
authors.lastname: Rosenberg
authors.forename: Yinduo
authors.initials: Y
authors.lastname: Ji
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D015342:DNA Probes; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D023281:Genomics; D007533:Isoleucine-tRNA Ligase / Q000235:genetics; D008826:Microbial Sensitivity Tests; D008957:Models, Genetic; D020411:Oligonucleotide Array Sequence Analysis; D016366:Open Reading Frames; D010088:Oxidoreductases / Q000235:genetics; D016372:RNA, Antisense / Q000235:genetics*; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D015342:DNA Probes; D016372:RNA, Antisense; D010088:Oxidoreductases; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D007533:Isoleucine-tRNA Ligase