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Mapping the interactions within multidrug efflux pump assemblies.

绘制多药物外排泵组件内的相互作用。

基本信息

批准号：
BB/N002776/1
负责人：
Vassiliy Bavro
金额：
$ 55.73万
依托单位：
University of Essex
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2016
资助国家：
英国
起止时间：
2016 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FN002776%2F1
关键词：
Mapping interactions within multidrug efflux

项目摘要

Multidrug-resistant bacterial infections are one of principal challenges facing medicine today. A group of bacteria known as the Gram-negative are particularly resistant to the action of antibiotics, as they have evolved a secondary membrane around their cells, preventing easy entry of the antibiotics. No new antibiotics targeting this group have been developed for over 40 years and the need to find and exploit novel bacterial weakness points is of great importance for both human and veterinary medicine. One of the central mechanisms underlying their multidrug resistance, is the action of the of the so-called multidrug-efflux pumps. These assemble from 3 components, spanning the double membrane and pumping out antibiotics, lowering their effective concentration in the cell thus rendering them ineffective. While pumps are very diverse, they share a common outer membrane protein (OMP). Deactivation or removal of OMPs dramatically increases bacterial sensitivity to antibiotics, suggesting that targeting the OMP may be an effective therapeutic approach. Yet none of the currently available drugs target the pump assembly process. Partially this is due to lack of information on the interactions between pump components to design targeted inhibitors. This project specifically aims to close the gap in our understanding of pump intercomponent interactions with the view of disrupting their assembly.The study of full pump assemblies has been hindered by their complexity and transient association of their elements, preventing effective usage of standard structural approaches such as X-ray crystallography.Here, we will overcome these bottlenecks by innovative usage of multiple approaches. The first is the application of the novel technique of X-ray Radiolytic Footprinting (XRF), allowing to study transient and heterogeneous proteins in solution. XRF is a mapping technique, which is based on oxidation of the surface-exposed parts of the protein by usage of highly reactive hydroxyl radicals. These radicals are created by splitting water molecules in solution by usage of high-energy X-rays. The pattern of oxidation is detected by the molecular weight differences of fragmented proteins using a technique called mass-spectrometry. Comparison of the modification of a given protein on its own with the modification obtained in the presence of its binding partner reveals zones of protection, or footprints, corresponding to the interaction surfaces between the proteins. By using this approach we will map the tripartite pump complex and will use the information to specifically target the binding interfaces by mutagenesis to disrupt the association of the OMP with the rest of the pump. We will characterise the effect of these mutations on antibiotic resistance of the cells to identify crucial residues.Furthermore, we will use modified MFP proteins with truncated and scrambled hairpins to dissect their role in the assembly of the pump, allowing to distinguish between the two currently contradictory models of assembly. Recently, we have shown that the hairpin-domain of the MFP binds the OMP with higher affinity than the corresponding full-length protein. Furthermore it binds in an energy-independent fashion. However, unlike the full-length protein the binding of the hairpin does not produce a functional pump. Here, we will exploit these findings by further engineering stabilising interactions of the hairpin with its target OMP, and will test its capability to outcompete native MFPs and inhibit the function of the pump in vivo. This project will further our fundamental understanding of the pump assembly and settle the long-standing debate on the mode of MFP-OMP interaction. Demonstrating competitive inhibition of the MFP-OMP binding interface as viable strategy will pave the way to future design of a completely novel class of drugs targeting the assembly process providing a powerful tool in the fight against drug-resistance.

多重耐药细菌感染是当今医学面临的主要挑战之一。一组被称为革兰氏阴性的细菌对抗生素的作用特别有抵抗力，因为它们在细胞周围进化出了二级膜，防止抗生素轻易进入。40多年来没有开发出针对这一群体的新抗生素，寻找和开发新的细菌弱点对人类和兽医学都非常重要。其多药耐药的核心机制之一是所谓的多药外排泵的作用。它们由3种成分组成，跨越双膜并泵出抗生素，降低它们在细胞中的有效浓度，从而使它们无效。虽然泵非常多样化，但它们共享一个共同的外膜蛋白（OMP）。OMP的失活或去除显著增加了细菌对抗生素的敏感性，这表明靶向OMP可能是一种有效的治疗方法。然而，目前可用的药物中没有一种针对泵组装过程。部分原因是缺乏关于泵组件之间相互作用的信息，无法设计靶向抑制剂。本项目旨在缩小我们对泵组件间相互作用的理解与破坏其组装的差距。完整的泵组件的研究一直受到其复杂性和其元素的瞬时关联的阻碍，阻碍了标准结构方法如X射线晶体学的有效使用。在这里，我们将通过多种方法的创新使用来克服这些瓶颈。首先是应用X射线辐射分解足迹（XRF）的新技术，可以研究溶液中的瞬时和异质蛋白质。XRF是一种映射技术，其基于通过使用高反应性羟基自由基氧化蛋白质的表面暴露部分。这些自由基是通过使用高能X射线分裂溶液中的水分子而产生的。氧化的模式是通过使用一种称为质谱法的技术检测碎片蛋白质的分子量差异来检测的。将给定蛋白质自身的修饰与在其结合配偶体存在下获得的修饰进行比较，揭示了对应于蛋白质之间的相互作用表面的保护区或足迹。通过使用这种方法，我们将绘制三重泵复合物的图谱，并使用这些信息通过突变来专门靶向结合界面，以破坏OMP与泵其余部分的结合。我们将研究这些突变对细胞抗生素耐药性的影响，以确定关键的残基。此外，我们将使用具有截短和乱序发夹的修饰MFP蛋白来剖析它们在泵组装中的作用，从而区分目前两种相互矛盾的组装模型。最近，我们已经表明，发夹结构域的MFP结合OMP具有更高的亲和力比相应的全长蛋白质。此外，它以能量独立的方式结合。然而，与全长蛋白质不同，发夹的结合不产生功能性泵。在这里，我们将利用这些发现，通过进一步工程稳定的相互作用的发夹与其目标OMP，并将测试其能力，以胜过天然MFP和抑制泵在体内的功能。该项目将进一步加深我们对泵组件的基本理解，并解决长期存在的关于MFP-OMP相互作用模式的争论。证明MFP-OMP结合界面的竞争性抑制作为可行的策略将为未来设计靶向组装过程的全新药物类别铺平道路，从而提供对抗耐药性的强大工具。