Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux...

Area of Science:

• Microbiology
• Genomics
• Molecular Biology

Background:

• Multidrug efflux pumps are crucial for drug resistance in Gram-negative pathogens.
• Identifying these pumps and their regulators in bacteria like Acinetobacter baumannii is challenging.
• Existing methods struggle to comprehensively identify all functional efflux systems and regulatory elements.

Purpose of the Study:

• To develop and validate a high-throughput screening method (TraDISort) for identifying multidrug efflux pumps and regulators.
• To apply TraDISort to the nosocomial pathogen Acinetobacter baumannii to identify major ethidium efflux systems.
• To discover novel regulators controlling antimicrobial efflux pump expression.

Main Methods:

• Developed TraDISort, combining transposon insertion sequencing (TraDIS) with fluorescence-activated cell sorting (FACS).
• Created a dense library of over 100,000 transposon insertion mutants in Acinetobacter baumannii.
• Enriched mutants accumulating ethidium bromide using FACS, followed by TraDIS to identify insertion locations.

Main Results:

• Identified AdeABC, AdeIJK, and AmvA as the major ethidium efflux pumps in Acinetobacter baumannii.
• Discovered a novel transcriptional regulator controlling the expression of the amvA efflux pump.
• TraDISort also identified genes involved in cell division, morphology, and aggregation.

Conclusions:

• TraDISort is an effective and innovative method for identifying major multidrug efflux pumps and their regulators.
• This approach significantly advances the study of antimicrobial resistance mechanisms in bacterial pathogens.
• TraDISort has broad applications in characterizing efflux pump inhibitors and understanding bacterial physiology.