Antibiotic resistance in Gram-negative bacteria: structure and function of TolC-dependent multidrug efflux pumps

细菌性疾病在全世界都是一种不祥的威胁，造成巨大的痛苦、死亡和经济损失，但在我们的一生中，抗生素保护我们免受最坏的情况。然而现在，越来越多的耐多药细菌威胁着世界末日的到来，在这种情况下，即使是微不足道的感染也可能变得严重，往往危及生命，手术和化疗几乎不可能。英国首席医疗官萨利·戴维斯（Sally Davies）去年承认了这一问题的严重性，他表示，这一问题与恐怖主义和全球变暖相提并论，世界卫生组织（World Health organization）将这一问题描述为“对人类健康的三大威胁之一”。英国首相戴维•卡梅伦（David Cameron）委托进行的一项调查显示，如果现在不采取行动，到2050年，抗生素耐药性可能导致3亿人过早死亡。因此，对抗生素（药物）耐药性进行复杂的研究，以了解细菌如何在抗生素治疗中存活下来，并向我们展示对抗这种威胁的新方法，比以往任何时候都更加重要。在我们的实验室里，我们几十年来建立了一个循序渐进的研究计划，在这个计划中，我们结合了一系列强大的实验方法来研究致病细菌的生物学。在这里，我们寻求继续资助，以进一步分析细菌在其细胞包膜中建立的机制，以排出沙门氏菌，大肠杆菌和假单胞菌等细菌具有多重耐药能力的抗生素。这些药物外排机制都由三部分组成，横跨内外脂质膜和中间空间，即外周质，并使用充满能量的转运体将抗生素的货物结合并运送或“泵送”到一个叫做TolC的细胞出口管道，我们已经展示了TolC作为一个向细胞外部开放的通用细胞“垃圾槽”。第三个组件，环塑料适配器，帮助运输器组装和工作与TolC出口管道。这些三方多药外排泵在其特定成分和成分间相互作用的细节以及它们结合和排出细菌细胞的抗生素范围方面存在差异。我们已经成功地描述了所有泵共同的TolC出口管道和适配器组件的结构和作用，并定义了它们之间的相互作用以及最广泛的一类药物外排泵中的转运体。这最终导致了第一个完整组装的多药物外排泵的精确原子视图，并深入了解了它如何打开以允许外排，以及如何抑制外排。我们现在的目标是进一步完善我们现有的泵模型，并利用我们来之不易的专业知识，将重点转移到定义其他两类抗生素泵的成分结构和相互作用，这两类抗生素泵使用不同的转运蛋白，因此组装方式不同。我们还旨在激发新的方法来详细建立泵如何结合和转移活细菌细胞中的药物。通过这些方法，我们希望可视化所有不同的药物外排泵是如何在细菌细胞包膜内组装和运作的。通过这样做，我们将更好地了解细菌感染我们和抵抗化疗的策略，并通过识别泵中的弱点，为我们抑制和抵消它们开辟新的途径。

Bacterial diseases present an ominous threat throughout the world, causing great suffering, death, and economic cost, but for our lifetime antibiotics have protected us from the worst. Now however, the increasing prevalence of multidrug resistant bacteria threatens an apocalyptic scenario in which even trivial infections could become serious, often life threatening, and surgery and chemotherapy become nigh impossible. The severity of the problem is recognised by Chief Medical Officer Sally Davies who in the last year stated the problem is on a par with terrorism and global warming, and the World Health Organisation described the problem as "one of the three greatest threats to human health". A review commissioned by Prime Minister David Cameron suggested that without action now, antibiotic resistance could be responsible for 300 million premature deaths by 2050. It is therefore more important than ever to perform sophisticated studies on antibiotic (drug) resistance to learn how bacteria survive antibiotic treatment, and show us new ways to combat this threat.In our laboratory we have over decades built up a step-wise programme of research in which we combine a range of powerful experimental approaches to study the biology of pathogenic (disease-causing) bacteria. Here we seek a continuation in funding to further our analyses of the machineries that bacteria build in their cell envelope to expel antibiotics underpinning the ability of bacteria like Salmonella, E. coli and Pseudomonas to become multidrug resistant. These drug efflux machineries are all made up of three components and span across the inner and outer lipid membranes and intervening space, the periplasm, and use energized transporters to bind and deliver or 'pump' their cargo of antibiotics to a cell exit duct called TolC which we have shown acts as a universal cell 'trash chute' opened to the outside of the cell. The third component, the periplasic adaptor, helps the transporter assemble and work with the TolC exit duct.These tripartite multidrug efflux pumps differ in the detail of their specific components and inter-component interactions, and the range of antibiotics they bind and eject from the bacterial cell. We have succeeded in describing the structure and action of the TolC exit duct and adaptor components common to all the pumps, and defined their interactions with each other and the transporter in the most widespread class of drug efflux pump. This culminated in the first precise atomic view of a complete assembled multidrug efflux pump, and gave an insight into how it opens to allow efflux, and how this might be inhibited. We now aim to further refine our existing pump model, and exploit our hard-won expertise to shift focus to define the component structures and interactions in the two other classes of antibiotic pump that use different transporters and therefore assemble differently. We also aim to instigate new approaches to establish in detail how the pumps bind and translocate the drugs from the living bacterial cell. In these ways we hope to visualize how all the different drug efflux pumps assemble and operate within the bacterial cell envelope. In so doing we will offer a better understanding of the strategies bacteria employ to infect us and, resist chemotherapy, and by identifying weak points in the pumps open up new ways for us to inhibit and counteract them.