Identification of antimicrobial targets using a comprehensive genomic approach

Area of Science:

• Molecular Biology
• Antimicrobial Drug Discovery
• Genetics

Background:

• Regulated antisense RNA technology allows for conditional control of gene expression, enabling the creation of growth-defective/lethal strains.
• Identifying novel antimicrobial drug targets is crucial for combating antibiotic resistance.

Purpose of the Study:

• To expand regulated antisense RNA interference technology for high-throughput screening of potential antimicrobial drug targets.
• To validate the identification of specific gene targets for existing antibiotics.

Main Methods:

• Construction of a specific antisense RNA sublibrary targeting essential open reading frames.
• Treatment of pooled antisense strains with sublethal levels of mupirocin, triclosan, and gentamicin.
• Selective detection of sensitized strains using DNA subtractive hybridization, microarray, and whole-cell analyses.

Main Results:

• No strains showed supersensitivity to gentamicin, as no target-specific antisense strain was included for this antibiotic.
• Antisense strains targeting isoleucine tRNA synthetase (ileS) and enoyl-[acyl-carrier-protein] reductase (fabI) were identified.
• The ileS and fabI antisense strains exhibited increased susceptibility specifically to mupirocin and triclosan, respectively.

Conclusions:

• Regulated antisense RNA expression libraries provide an effective tool for identifying potential targets of novel antibacterial agents.
• The study successfully demonstrated the utility of this screening strategy in validating known drug-target interactions.