The structure and function of the beta-barrel assembly machinery: an Achilles heel of Gram-negative pathogens

抗生素自上个世纪中期被发现以来，已经彻底改变了医疗保健，抗生素和其他抗微生物药物已经成为现代医学不可或缺的一部分，我们依赖它来解决细菌感染。然而，抗生素的使用在医疗保健和农业中都有了巨大的增长。不幸的是，这导致了抗生素耐药性（AMR）的大幅上升，导致感染并被抗生素靶向的细菌对我们赖以杀死它们的药物产生了抗药性。AMR的出现对社会构成了紧迫的威胁，并对人类健康构成了巨大且日益严重的问题。除了细菌感染引起的各种疾病外，医院获得性感染对我们的健康构成越来越大的威胁。在英格兰，每年约有30万名患者受到医院获得性感染的影响，NHS每年的费用超过10亿英镑。随着AMR的传播，导致这些感染的细菌现在对我们抗生素库中的几乎所有药物都具有耐药性。因此，迫切需要发现新的抗生素，我们的拨款申请正是关注这个主题。一类主要的细菌，即所谓的革兰氏阴性细菌，含有引起人类疾病的病原体，包括霍乱、鼠疫和淋病。革兰氏阴性菌也是许多医院获得性感染的原因，特别是肺炎和尿路感染。这些包括不动杆菌属、埃希氏菌属、嗜血杆菌属、军团菌属和假单胞菌属中的细菌。我们迫切需要开发新的抗生素，通过打击细菌库中的新目标来预防细菌感染：确定并瞄准细菌的“致命弱点”，使它们对现有的抗菌剂敏感或直接杀死它们。其中一个潜在的目标是一种称为β-桶装配机器的蛋白质复合物，或BAM，它位于两层保护膜的外部，这两层保护膜有助于保护革兰氏阴性细菌免受环境（以及许多抗生素）的影响。我们知道BAM对于细菌的存活和毒力是必不可少的。它的作用是将细菌细胞中产生的新蛋白质插入外膜。然而，BAM如何实现这一点尚不清楚，尽管去年使用晶体学或低温电子显微镜解决了复合物的几个3D结构。在拟议的工作中，我们的目标是使用最新的技术，包括冷冻电子显微镜和单分子FRET实验，以确定OMPs如何折叠并插入到拥挤的细菌外膜BAM。我们的目标是确定BAM在将蛋白质插入膜中时的3D结构。连同在试管和生物体本身的功能研究，我们的研究计划将揭示BAM如何发挥作用的分子细节;它如何识别其OMP底物;它如何在没有外部能源的情况下将OMP折叠成拥挤的OM;以及它如何在细菌OM中组织。除了使我们能够开发新的方法来询问这个迷人的分子机器的功能外，从长远来看，我们的目标是利用所获得的信息为开发新的途径铺平道路，以对抗革兰氏阴性病原体引起的感染。

Antibiotics have revolutionized healthcare since their discovery in the middle of the last century, and antibiotics and other antimicrobial drugs have become an integral part of modern medicine, which we rely on to tackle bacterial infections. The use of antibiotics, however, has increased enormously, both in healthcare and in agriculture. Unfortunately, this has led to huge rise in antimicrobial resistance (AMR), where the bacteria that cause infections and are targeted by antibiotics become resistant to the drugs that we rely on to kill them. The emergence of AMR poses an urgent threat to society and a massive and growing problem for human health. In addition to the vast array of diseases caused by bacterial infection, hospital-acquired infections are an ever-increasing threat to our health. Approximately 300,000 patients a year are affected by hospital-acquired infections in England, costing the NHS in excess of one billion pounds a year. As AMR spreads, the bacteria that cause these infections are now becoming resistant to almost all of the drugs in our antibiotic arsenal. There is thus an urgent need to discover new antibiotics, and it is on this topic that our grant application is focused.A major class of bacteria, the so-called Gram-negative bacteria, contains pathogens causing diseases in humans that include cholera, plague and gonorrhoea. Gram-negative bacteria are also responsible for many hospital-acquired infections, especially pneumonia and urinary tract infections. These include bacteria in the genera Acinetobacter, Escherichia, Haemophilus, Legionella, and Pseudomonas. We urgently need to develop new antibiotics able to prevent bacterial infection by hitting new targets in the bacterial armory: identifying and targeting the bacterial 'Achilles heel' to either render them susceptible to existing anti-bacterial agents or to kill them directly.One such potential target is a protein complex called the beta-barrel assembly machinery, or BAM, which sits in the outer of two protective membranes that helps to shield Gram-negative bacteria from their environment (and from many antibiotics). We know that BAM is essential for bacterial survival and virulence. Its role is to insert new proteins that are made in the bacterial cell into this outer membrane. How BAM achieves this, however, is not known despite several 3D structures of the complex being solved using crystallography or cryo-electron microscopy in the last year. In the proposed work, we aim to use the very latest techniques, including cryo-electron microscopy, and single molecule FRET experiments to determine how OMPs are folded and inserted into the crowded bacterial outer membrane by BAM. Our aim is to determine the 3D structure of BAM caught in the act of inserting a protein into a membrane. Together with functional studies in test tubes and in the organism itself, our programme of research will reveal the molecular details of how BAM functions; how it recognizes its OMP substrates; how it folds OMPs into the crowded OM in the absence of an external energy source; and how it is organized in the bacterial OM. As well as enabling us to develop new methods to interrogate the function of this fascinating molecular machine, in the long term we aim to use the information gained to pave the way towards developing new routes to combating infections caused by Gram-negative pathogens.