A Fluorescent Microplate Assay Quantifies Bacterial Efflux and Demonstrates Two Distinct Compound Binding Sites in AcrB

Abstract

A direct assay of efflux by Escherichia coli AcrAB-TolC and related multidrug pumps would have great value in discovery of new Gram-negative antibiotics. The current understanding of how efflux is affected by the chemical structure and physical properties of molecules is extremely limited, derived from antibacterial data for compounds that inhibit growth of wild-type E. coli. We adapted a previously described fluorescent efflux assay to a 96-well microplate format that measured the ability of test compounds to compete for efflux with Nile Red (an environment-sensitive fluor), independent of antibacterial activity. We show that Nile Red and the lipid-sensitive probe DiBAC 4 -(3) [bis-(1,3-dibutylbarbituric acid)-trimethine oxonol] can quantify efflux competition in E. coli. We extend the previous findings that the tetracyclines compete with Nile Red and show that DiBAC 4 -(3) competes with macrolides. The extent of the competition shows a modest correlation with the effect of the acrB deletion on MICs within the compound sets for both dyes. Crystallographic studies identified at least two substrate binding sites in AcrB, the proximal and distal pockets. High-molecular-mass substrates bound the proximal pocket, while low-mass substrates occupied the distal pocket. As DiBAC 4 -(3) competes with macrolides but not with Nile Red, we propose that DiBAC 4 -(3) binds the proximal pocket and Nile Red likely binds the distal site. In conclusion, competition with fluorescent probes can be used to study the efflux process for diverse chemical structures and may provide information as to the site of binding and, in some cases, enable rank-ordering a series of related compounds by efflux.T ripartite multidrug efflux pumps are widely represented across Gram-negative bacteria. The two best studied examples are AcrAB-TolC of Escherichia coli and MexAB-OprM of Pseudomonas aeruginosa (1, 2). The normal physiological function for the E. coli AcrB pump is to protect bacterial cells from bile salts inside the human host (3). However, it appears that bacterial efflux pumps and their components may also serve other diverse functions (4-7). AcrB is a proton-driven antiporter that derives its energy from the proton motive force that exists across the bacterial cytoplasmic membrane (8, 9). AcrB is organized as a homotrimer and interacts with two other components, the outer membrane partner TolC and the small periplasmic adapter lipoprotein AcrA (10, 11). The broad substrate preference of AcrB includes bile salts and detergents as well as diverse structurally unrelated antibiotics and chemicals (12-14), making it a vital player in determining intrinsic antibiotic resistance (15).AcrAB-TolC is the best-characterized bacterial efflux pump with regard to structure (16), with crystal structures solved for the individual components (17-22). The most recent high-resolution crystals show each monomer in a different configuration, lending support to a peristaltic mechanism for pump function (19). The three monomer forms, called Access (A), Binding...

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