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

革兰氏阴性菌的抗生素耐药性：TolC 依赖性多药外排泵的结构和功能

基本信息

批准号：
MR/N000994/1
负责人：
Vassilis Koronakis
金额：
$ 206.16万
依托单位：
University of Cambridge
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2015
资助国家：
英国
起止时间：
2015 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=MR%2FN000994%2F1
关键词：
Antibiotic resistance Gram negative bacteria

项目摘要

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.

细菌性疾病在全世界构成了不祥的威胁，造成了巨大的痛苦、死亡和经济损失，但在我们的一生中，抗生素保护了我们免受最坏的影响。然而，现在，多重耐药细菌的日益流行威胁着世界末日的情景，即使是微不足道的感染也可能变得严重，往往危及生命，手术和化疗几乎是不可能的。首席医疗官萨莉·戴维斯（Sally Davies）承认了这个问题的严重性，她在去年表示，这个问题与恐怖主义和全球变暖相提并论，世界卫生组织将这个问题描述为“人类健康的三大威胁之一”。首相大卫卡梅隆委托进行的一项审查表明，如果现在不采取行动，抗生素耐药性可能会在2050年前导致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生存，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重要。在我们的实验室，我们已经建立了一个逐步的研究计划，其中我们联合收割机结合一系列强大的实验方法来研究致病（致病）细菌的生物学。在这里，我们寻求继续提供资金，以进一步分析细菌在其细胞包膜中构建的机制，以排出抗生素，从而支持沙门氏菌、大肠杆菌等细菌的能力。大肠杆菌和假单胞菌成为多重耐药。这些药物外排机制都是由三个组成部分和跨越内外脂质膜和干预空间，周质，并使用通电的转运蛋白绑定和交付或“泵”他们的货物抗生素到细胞出口管称为TolC，我们已经表明作为一个通用的细胞“垃圾槽”打开到细胞外。第三个组件，周质适配器，帮助转运蛋白组装并与TolC出口管一起工作。这些三重多药外排泵在其特定组件和组件间相互作用的细节上有所不同，以及它们结合并从细菌细胞中排出的抗生素的范围。我们已经成功地描述了所有泵共有的TolC出口导管和适配器组件的结构和作用，并定义了它们彼此之间的相互作用以及最广泛的药物外排泵中的转运蛋白。这最终导致了第一个完整组装的多药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中的组件结构和相互作用上，这两类抗生素泵使用不同的转运蛋白，因此组装方式不同。我们的目标还在于激发新的方法来详细确定泵如何结合和转运活细菌细胞中的药物。通过这些方式，我们希望可视化所有不同的药物外排泵如何在细菌细胞包膜内组装和操作。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开辟抑制和抵消它们的新方法。