Molecular mechanism of multidrug efflux pumps and their role in bacterial resistance to antibiotics

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The project will develop a novel approach in tackling bacterial resistance to antibiotics. This project targets a molecular system that is the basis of one of the major mechanisms of multidrug resistance, proton-driven multidrug efflux in bacteria. Until now, much effort has been dedicated to combating efflux to increase therapeutic antibiotic concentrations. We adopt a radically new approach, in which we aim to understand, uncouple and facilitate efflux to dissipate its transmembrane proton drive. Transmembrane proton gradient provides energy drive for membrane functions and is the hallmark of living cells. Enhancing proton dissipation through specific channels severely impairs vital cellular functions, generates heat and is lethal to cells. To achieve this, we target a molecular system that bacteria deploy to overcome antibiotic attacks. One natural defence mechanism in E. coli involves a trans-cell envelope protein complex, AcrAB/TolC, which functions as a molecular pump to remove xenotoxic compounds and protects bacteria from hydrophobic drugs and antibiotics. These pumps are driven by membrane proton gradients and are notorious for their role in multidrug resistance. We will investigate the mechanism and regulation of the AcrAB/TolC pump using hydrophobic antibiotics erythromycin and tetracycline, as well as fluorescent hydrophobic compounds to monitor pump function. We will use advanced molecular biology in combination with high performance computing, cutting edge magnetic resonance, electron and superresolution microscopy tools to map the precise details of proton flow through the ArcB pump and its coupling to substrate efflux. We aim to identify bottlenecks in substrate efflux that are modulated by proton flow and seek modulators that constitutively open the proton channel. We will screen our compound libraries for such drugs with the aim of dissipating bacterial proton motive force into heat that "cooks" the pathogen.Experimental plan:We aim to map the detailed path of proton flow through the inner membrane component, AcrB, of the trans-periplasmic molecular complex AcrAB/TolC and its structural coupling to substrate efflux. To do this, we will use a combination of modelling, mutagenesis and efflux assays, and structure characterisation.Model of AcrB membrane trimer showing Tyr residues and key elements of the pump, as well as the gating loop (pink, blue) 1. We will use computational tools to carry out in silico mutagenesis of key residues in AcrB to understand proton flow and its coupling to efflux2. We combine point mutagenesis with efflux and PMF dissipation assays to validate functionally the proposed proton and substrate flow paths. 3. We will assess conformational consequences of these mutations on the overall structure of the protein using cryoEM and will validate protonation sites by NMR. 4. Combining this knowledge, we will model the conformational coupling/uncoupling of substrate and proton flow and will screen our libraries for compounds that widen the proton channel and allosterically enhance efflux independent proton flow.Using the AcrAB/TolC as a proton flow enhancer is very appealing, as bacteria express the pump in response to antibiotic challenge, which augments the effect. In addition, occluding substrate flow through TolC can retain antibiotics in the periplasm and enhance the antibiotic efficacy.