Validation of antibacterial mechanism of action using regulated antisense RNA expression in Staphylococcus aureus

Yinduo Ji, Dezhong Yin, Brian Fox, David J. Holmes, David Payne, Martin Rosenberg

Research output: Contribution to journalArticlepeer-review

68 Scopus citations

Abstract

Validation of antibiotic mode of action in whole bacterial cells is a key step for antibiotic drug discovery. In this study, one potential drug target, enoyl-acyl carrier protein reductase (FabI), an essential enzyme in the fatty acid biosynthesis pathway, was used to evaluate the feasibility of using a regulated antisense RNA interference approach to determine antibiotic mode of action. Antisense isogenic strains expressing antisense RNA to fabI were created using a tetracycline-regulated vector in Staphylococcus aureus. We demonstrated that down-regulation of FabI expression by induction of fabI antisense RNA induces a conditional lethal phenotype. In contrast, partial down-regulation gives a viable cell with a significant increase in sensitivity to FabI-specific inhibitors (i.e., a sensitized phenotype). More importantly, the mode of action for novel FabI inhibitors has been confirmed using this genetic approach in whole cell assay. These results indicate that controlled antisense technology provides a robust tool for defining and tracking the mode of action of novel antibacterial agents.

Original languageEnglish (US)
Pages (from-to)177-184
Number of pages8
JournalFEMS Microbiology Letters
Volume231
Issue number2
DOIs
StatePublished - Feb 16 2004

Keywords

  • Antisense RNA
  • FabI inhibitor
  • Mode of action
  • Staphylococcus aureus

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