EKO-36: Construction of an efflux platform to investigate the substrate profiles and functions of bacterial drug efflux pumps

Efflux pumps are a significant challenge for the development of new antibacterial agents. Overcoming efflux requires an in-depth understanding of efflux pump functions, substrate specificities, and the development of inhibitors. However, the complexities of drug efflux systems have limited such studies. To address these challenges, we report the generation of Efflux KnockOut-36 (EKO-36), a highly susceptible Escherichia coli strain lacking 36 efflux pumps. We demonstrate the utility of this strain by constructing an efflux platform comprising pumps forming tripartite complexes with the outer membrane channel TolC. This platform was profiled against a curated diverse compound collection, which enabled us to define physicochemical properties that contribute to transport. We also show the E. coli drug efflux system is conditionally essential for growth, and that the platform can be used to investigate efflux pump inhibitor specificities. We believe EKO-36 and the efflux platform will have widespread application for the study of drug efflux.