Breaking the Barrier: Mapping protein interactions in the bacterial outer membrane as targets for new antimicrobials

纵观人类历史，细菌一直是疾病和死亡的巨大负担，随着疫苗和抗生素的发现，这种负担只会减少。我们现在面临着抗菌素耐药性上升的浪潮，并正在经历一场缓慢发展的医院获得性耐药细菌感染大流行。除了更好的预防、控制和监测外，迫切需要确定新的目标，我们可以针对这些目标开发新的抗生素药物。特别令人担忧的是革兰氏阴性细菌。在被美国疾病控制中心确定为紧急威胁的5种微生物中，有3种是革兰氏阴性细菌，尽管正在试验的新抗生素少得令人担忧，但目标革兰氏阴性细菌就更少了。膜及其相关蛋白质构成了目前跨越多个疾病领域的大多数药物靶点，很大程度上是因为膜是细胞内和细胞间许多区隔和交流的基础。革兰氏阴性细菌有一个独特的额外的保护性外膜(OM)，保护细菌免受环境的影响。OM是毒素和抗生素的主要屏障，对细菌的生长、毒力、致病机理和生物膜的形成(这对确定许多感染是重要的)至关重要。所有生物膜都有两个两亲性脂分子(通常是磷脂)的小叶，它们形成一个双层，每个小叶中的脂类是不同的(不对称)。细菌OM可能是生物学中膜不对称的最显著的例子，内叶以磷脂为主(与正常膜一样)，外叶以脂多糖分子(细菌膜所特有的)为主。完整的外膜蛋白(OMP)都具有桶状结构，因此进化到在与其他膜上的蛋白质不同的环境中折叠和发挥功能：由于内毒素聚集在一起，它们经历了一个非常坚硬的膜。此外，它们在膜中不太移动，它们的构象和相互作用由与其他蛋白质和脂多糖的相互作用决定，这些蛋白质和脂多糖在其他膜中缺失，但对细菌的生长和生存至关重要。因此，OM是一个迷人的环境，可以为抗菌干预提供丰富的新靶点。在这项MRC计划拨款中，我们将整合对细菌OM的功能和结构研究，以及在蛋白质结构(和蛋白质相互作用)预测方面的最新创新，以及我们设计能够结合目标蛋白质的新蛋白质的能力。在试管和整个细菌细胞中工作，我们将学习OMP如何自然折叠并嵌入外膜，它们之间以及当它们在外膜中时如何与LPS分子相互作用，以及这些相互作用如何影响蛋白质的工作方式和细菌的生长。最终，我们希望利用这些发现来阐明杀死细菌的新方法，或者至少削弱它们的防御，以便其他药物可以杀死它们。项目拨款是至关重要的，因为它将使我们能够建立一支才华横溢的团队，能够共同努力，以更快的速度和更大的规模做出发现，这是通过单独的较小规模的项目拨款无法实现的，它还将使我们能够将英国置于这一重要研究领域的前沿。

Across human history, bacteria have been responsible for a huge burden of disease and mortality that only lessened with the discovery of vaccination and antibiotics. We now face a rising tide of antimicrobial resistance, and are experiencing a slow-moving pandemic of hospital-acquired infections by drug-resistant bacteria. Alongside better prevention, control, and surveillance, there is an urgent need to identify new targets against which we can develop new antibiotic drugs. Of particular concern are the Gram-negative group of bacteria. Of the five microorganisms identified as urgent threats by the US Centres for Disease Control, three are Gram-negative bacteria, and while there are worryingly few new antibiotics in trials, even fewer target Gram-negative bacteria. Membranes, and the proteins associated with them, constitute the majority of current drug targets across multiple disease areas, largely because membranes are the basis for much compartmentalisation and communication in and between cells. Gram-negative bacteria have a unique, additional, protective outer membrane (OM) that shields the bacterium from its environment. The OM is a major barrier to toxins and antibiotics, and is critical for bacterial growth, virulence, pathogenesis, and the formation of biofilms (which are important for establishing many infections). All biological membranes have two leaflets of amphipathic lipid molecules (typically phospholipids) that form a bilayer, and the lipids in each leaflet are different (asymmetric). The bacterial OM is perhaps the most striking example of membrane asymmetry in biology, with an inner leaflet dominated by phospholipids (as in normal membranes), and an outer leaflet dominated by lipopolysaccharide molecules (which are unique to the bacterial membrane). Integral outer membrane proteins (OMPs), which all have a barrel-shaped structure, have thus evolved to fold and function in a different environment to proteins in other membranes: they experience a very rigid membrane because the lipopolysaccharide clumps together. Furthermore, they don't move around very much in the membrane, and their conformations and interactions are dictated by interactions with other proteins and lipopolysaccharide that are missing in other membranes, but essential for bacterial growth and survival. The OM is thus a fascinating environment that could provide a rich source of new targets for antibacterial interventions. In this MRC programme grant, we will integrate functional and structural studies on the bacterial OM, with the latest innovations in protein structure (and protein interaction) prediction, and in our ability to design new proteins that can bind target proteins. Working in the test tube and with whole bacterial cells, we will learn how OMPs naturally fold up and become embedded within the outer membrane, how they interact with each other and with LPS molecules when they're in that membrane, and how these interactions affect the ways proteins work and how bacteria grow. Ultimately, we want to use these discoveries to illuminate new ways of killing bacteria, or at least weakening their defences so that other drugs can kill them. A programme grant is essential because it will allow us to build a talented team that can work together to make discoveries at a pace and scale that would be impossible via individual, smaller project grants, and it will allow us to place the UK at the forefront of this vital area of research.