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

该项目将开发一种新的方法来解决细菌对抗生素的耐药性。这个项目的目标是一个分子系统，它是细菌多药耐药的主要机制之一--质子驱动的多药外排的基础。到目前为止，人们一直致力于对抗外排以增加治疗性抗生素浓度。我们采用了一种全新的方法，我们的目标是了解、解偶联和促进外流来驱散其跨膜质子驱动。跨膜质子梯度为膜功能提供能量驱动，是活细胞的标志。通过特定通道增加质子的耗散会严重损害细胞的重要功能，产生热量，对细胞是致命的。为了实现这一点，我们瞄准了细菌为克服抗生素攻击而部署的分子系统。大肠杆菌的一种自然防御机制涉及跨细胞包膜蛋白复合体AcrAB/TolC，它的功能是作为一个分子泵来清除外源有毒化合物，并保护细菌免受疏水药物和抗生素的伤害。这些泵由膜质子梯度驱动，因其在多药耐药中的作用而臭名昭著。我们将使用疏水抗生素红霉素和四环素以及荧光疏水化合物来研究AcrAB/TolC泵的机制和调节，以监测泵的功能。我们将使用先进的分子生物学结合高性能计算、尖端磁共振、电子和超分辨率显微镜工具来绘制通过ArcB泵的质子流动的精确细节及其与基材外流的耦合。我们的目标是找出底物外流中受质子流调制的瓶颈，并寻找能够结构性地打开质子通道的调节剂。我们将为这类药物筛选我们的化合物文库，目的是将细菌质子驱动力转化为热量，从而“烹饪”病原体。实验计划：我们的目标是绘制质子通过跨质分子复合体AcrAB/TolC的内膜成分AcrB的详细路径，以及它与底物外排的结构耦合。为此，我们将使用建模、诱变和外排分析以及结构特征相结合的方法。AcrB膜三聚体模型显示Tyr残基和泵的关键元件，以及门控回路(粉色、蓝色)1。我们将使用计算工具在计算机中对AcrB中的关键残基进行诱变，以了解质子流动及其与fflux2的耦合。我们将点突变与外流和PMF耗散分析相结合，在功能上验证了所提出的质子和衬底流动路径。3.我们将使用CryoEM评估这些突变对蛋白质整体结构的构象影响，并将通过核磁共振验证质子化位点。4.结合这些知识，我们将对底物和质子流的构象耦合/去偶联进行建模，并将筛选我们的库，以寻找拓宽质子通道并变构增强外排非依赖性质子流的化合物。使用AcrAB/TolC作为质子流增强剂非常有吸引力，因为细菌表达泵以响应抗生素的挑战，这将增强作用。此外，闭塞底物流经TolC可以将抗生素保留在周质中，提高抗生素的疗效。

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.