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

多药外排泵的分子机制及其在细菌耐药性中的作用

• 批准号: 2745697
• 金额: --
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2745697
• 关键词: Molecular; mechanism; multidrug; efflux; pumps

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.

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。该项目的目标是一个分子系统，这是多药耐药的主要机制之一，质子驱动的细菌多药外排的基础。到目前为止，许多努力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，我们的目标是理解，解偶联和促进外排，以消散其跨膜质子驱动。跨膜质子梯度为膜功能提供能量驱动，是活细胞的标志。通过特定通道增强质子耗散严重损害重要的细胞功能，产生热量并对细胞致命。为了实现这一目标，我们瞄准了细菌用来克服抗生素攻击的分子系统。大肠杆菌的一种天然防御机制。大肠杆菌涉及跨细胞包膜蛋白复合物AcrAB/TolC，其作为分子泵起作用以去除异种毒性化合物并保护细菌免受疏水性药物和抗生素的侵害。这些泵由膜质子梯度驱动，并且因其在多药耐药性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监测泵功能，以研究AcrAB/TolC泵的机制和调节。我们将使用先进的分子生物学与高性能计算，尖端的磁共振，电子和超分辨率显微镜工具相结合，以绘制通过ArcB泵的质子流的精确细节及其与基底流出的耦合。我们的目标是确定瓶颈的底物流出的质子流调制，并寻求调制器组成打开质子通道。我们将筛选这些药物的化合物库，目的是将细菌的质子动力消散成热量，“烹饪”pathogene.Experimental计划：我们的目标是绘制质子流通过跨周质分子复合物AcrAB/TolC的内膜组件AcrB的详细路径及其与底物外排的结构耦合。AcrB膜三聚体模型显示了Tyr残基和泵的关键元件，以及门控环（粉色，蓝色）1.我们将使用计算工具进行AcrB中关键残基的计算机诱变，以了解质子流及其与efflux 2的耦合。我们结合联合收割机点诱变与外排和PMF耗散测定功能验证所提出的质子和底物流动路径。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，并通过NMR验证质子化位点。4.结合这些知识，我们将模拟底物和质子流的构象偶联/解偶联，并将筛选我们的文库中的化合物，拓宽质子通道和变构增强外排独立的质子流。使用AcrAB/TolC作为质子流增强剂是非常有吸引力的，因为细菌表达泵响应抗生素的挑战，这增强了效果。此外，封闭通过TolC的底物流可以将抗生素保留在周质中并增强抗生素功效。