Molecular basis of outer membrane stabilisation by the energised Tol-Pal system in Gram-negative bacteria

细菌对地球上大多数生物的健康和幸福都很重要。人类的例子包括它们在我们的肠道微生物群中对食物消化的重要性。细菌也可能是致病性的，这意味着它们可以感染组织和器官，如果不及时治疗，可能会致命。现代医学很大程度上依赖于通过使用抗生素对肺炎和败血症等细菌感染进行有效治疗。细菌中抗生素耐药性的增加正日益使我们的许多一线抗生素失效。目前的建议侧重于外膜，这是导致革兰氏阴性细菌（如大肠杆菌、铜绿假单胞菌和肺炎克雷伯菌）耐药的主要因素之一。外膜是一个高度不可渗透的屏障，它排除了许多类型的抗生素，这些抗生素对缺乏外膜的革兰氏阳性细菌是有效的。在革兰氏阴性菌的分裂过程中，新复制的子细胞必须将其单独的外膜固定在下面的细胞壁上。这个过程需要能量的输入。因为外膜是一个“无能量”的环境，必要的能量是由细菌内膜上的电化学梯度提供的。一种复杂的、多蛋白质的蛋白质纳米机器被称为toll - pal，它的任务是传递能量。toll - pal利用这种能量来源，以一种未知的机制稳定外膜。我们最近发现了toll - pal是如何实现这一复杂功能的，在这个过程中，我们发现了一种我们称之为“动员和捕获”的生物机制。目前的提案试图利用这些进步。我们将研究五种toll - pal组分的所有离散相互作用以及它们如何在细菌的膜中移动，以了解如何利用细胞的能量将外膜固定在细胞壁上。作为这项工作的一部分，我们还将利用我们开发的一项新技术，通过该技术，可以将革兰氏阴性细菌中的带电复合物捕获在其激活状态下，以便随后使用被称为粘菌素的抗菌蛋白进行结构解剖。通过这项工作，我们将获得迄今为止最详细的关于革兰氏阴性细菌外膜是如何稳定的，以及像toll - pal这样的纳米机器是如何实现这一目标的，这些知识可以在未来与细菌感染的斗争中得到利用。

Bacteria are important for the health and well-being of most living organisms on earth. Examples in humans include their importance in our gut microbiomes for the digestion of food. Bacteria can also be pathogenic, meaning they can infect tissues and organs which, if left untreated, can be lethal. Much of modern medicine relies on the effective treatment of bacterial infections, such as pneumonia and sepsis, through the use of antibiotics. The rise of antibiotic resistance in bacteria is increasingly rendering many of our frontline antibiotics ineffective.The current proposal focuses on the outer membrane, one of the main factors contributing to antibiotic resistance in Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. The outer membrane is a highly impermeable barrier that excludes many types of antibiotics that are otherwise effective against Gram-positive bacteria that lack an outer membrane. During the process of division in Gram-negative bacteria newly replicated daughter cells must have their individual outer membranes pinned to the underlying cell wall. This process requires the input of energy. Because the outer membrane is an 'energy-less' environment the requisite energy is provided by the electrochemical gradient across the inner membrane of the bacterium. A complex, multiprotein protein nanomachine known as Tol-Pal is tasked with this transfer of energy. Tol-Pal taps into this energy source to stabilise the outer membrane by an unknown mechanism.We discovered recently how Tol-Pal achieves this complex function, in the process identifying a biological mechanism we call 'mobilisation-and-capture'. The current proposal seeks to capitalise on these advances. We will investigate all the discrete interactions of the five Tol-Pal components and how they move in the membranes of the bacterium in order to understand how the cell's energy is exploited to pin the outer membrane to the cell wall. As part of this work, we will also exploit a new technology we have developed whereby energised complexes in Gram-negative bacteria can be trapped in their activated states for subsequent structural dissection using antibacterial proteins known as colicins.Through this work we will obtain the most detailed view yet of how the outer membrane is stabilised in Gram-negative bacteria and how nanomachines like Tol-Pal work to achieve this, knowledge that can be exploited in the future fight against bacterial infections.