Elucidating the interaction of antibiotics with multi-drug efflux-pumps

Secondary transporters of the RND family, such as AcrB, are principal contributors to non-specific multidrug resistance (MDR). They do this by actively pumping antibiotics out of the periplasm of Gram-negative bacteria. Despite the progress in structural understanding of the RND-pump function, only a limited number of drug-bound complexes have been resolved. Finding out the binding mode of several important classes of antibiotics including fluoroquinolones and beta-lactams would inform the development of novel therapeutics. We have identified that the major cause for the lack of drug-transporter complex structures is that RND-transporters use detergents as their primary substrates.

We will target the problem by employing an alternative solubilisation-strategy of the transporter-antibiotic complexes, styrene-maleic acid co-polymer (SMA) which we recently successfully demonstrated in single-particle studies of AcrB. We will use this technology to solve structures of the wild-type and G288D mutant of Salmonella AcrB, creating a shift in the antibiotic selectivity of the pump.

These crystal structures will provide crucial information about the mode of fluoroquinolone binding and mechanisms of MDR, while the success of SMA-based crystallisation will allow effective antibiotic-soaking that will usher a new era of research into antibiotics and pump inhibitors.