High-Throughput Flow Cytometry Screening of Multidrug Efflux Systems

Area of Science:

• Microbiology
• Biochemistry
• Drug Discovery

Background:

• Resistance Nodulation Division (RND) proteins are crucial inner membrane transporters in Gram-negative bacteria.
• RND transporters facilitate substrate transport and are implicated in antibiotic resistance, virulence, and colonization.
• Targeting RND transporters is a promising strategy for antimicrobial development, but novel efflux pump inhibitors (EPIs) are scarce.

Purpose of the Study:

• To develop and validate a high-throughput efflux profiling and discovery strategy for RND model systems.
• To identify novel EPIs against RND transporters in pathogenic Gram-negative bacteria.
• To establish a platform for characterizing RND-mediated efflux in clinically relevant pathogens.

Main Methods:

• A flow cytometric assay utilizing fluorescein diacetate (FDA) to measure dye accumulation in Escherichia coli, Franscisella tularensis, and Burkholderia pseudomallei.
• High-throughput screening of chemical libraries to identify compounds that inhibit RND transporter function.
• Validation of identified EPIs through growth inhibition and antibiotic potentiation assays in a Biosafety Level-3 environment.

Main Results:

• The FDA dye accumulation assay successfully identified known EPIs and RND deletion strains by increased fluorochrome retention.
• The assay demonstrated utility in evaluating dye-substrate efflux and screening for novel EPIs.
• The combined approach effectively characterized efflux activity in pathogenic Gram-negative bacteria.

Conclusions:

• Flow cytometry provides a powerful tool for characterizing RND-mediated efflux in Gram-negative pathogens.
• The developed assay platform facilitates the discovery of novel EPIs, offering potential for new antibacterial agents.
• This strategy enhances the pipeline for developing RND-targeted antimicrobials to combat resistance.