The emergence and spread of antimicrobial resistant bacteria is one of today’s most significant public health problems as they are recalcitrant to effective treatment. A key mechanism used by microorganisms to achieve simultaneous resistance to multiple compounds is through pumping out antimicrobials before they reach their intracellular target. Through various efflux pump associated mechanisms, enhanced bacterial survival provides a window within which further adaptive resistance mechanisms can be acquired. Therefore, it is of fundamental importance to understand exactly how these multidrug efflux proteins recognise and export such a diverse array of compounds, to enable the rational design of drugs that are not recognised by these systems or compounds that can act as efflux pump inhibitors (EPIs) that obstruct their action and potentiate the activity of currently ineffectual antimicrobials.
One efflux protein, QacA, can be found in staphylococci where it confers resistance to multiple compounds commonly used as antiseptics and disinfectants. Recently a cryo-EM structure of a mutant variant of QacA was made available allowing the overall structure and configuration of the 14 transmembrane segments that this protein is comprised of to be deciphered. Here, we have extended the story to include the identification of features of the protein required for the binding and transport of antimicrobial compounds. This has been achieved by applying an integrated approach. Construction and analysis of a large set of site-directed mutants identified residues potentially comprising the drug binding site(s). These data were integrated with a combination of docking studies, long-timescale molecular dynamics simulations, and resistance and checkerboard assays with combinations of QacA substrates and potential EPIs. This has allowed the identification of the first set of compounds that potentiate the action of the QacA drug efflux pump and provided the basis for further EPI development.