Targeting SurA Dynamics: An Achilles Heel in Bacterial Outer Membrane Biogenesis

2017年2月，世界卫生组织发布了有史以来第一份耐药“优先病原体”名单--对人类健康构成最大威胁的12个细菌家族的目录。在这些细菌中，有9种是革兰氏阴性菌。革兰氏阴性细菌的外膜(OM)保护它们免受抗生素等潜在破坏性分子的伤害。OM充满了外膜蛋白(OMP)，创建了一种对细菌生存至关重要的通透性屏障，并赋予细菌对多种类型抗生素的耐药性。因此，打破这一障碍是产生新抗菌剂的一条诱人的途径。OMPS的生物合成始于细胞质，结束于它们的折叠和插入OM，在那里它们形成圆柱形的桶状结构。分子伴侣苏拉(Sura)护送这些新制造的未折叠的OMP通过周质到达OM。扰乱苏拉蛋白的伴侣功能会导致细菌活力丧失，并影响抗生素耐药性，这表明苏拉蛋白是控制革兰氏阴性病原菌的一个有吸引力的靶点。目前有效操纵SurA活性的挑战之一是，我们对这种大型、多结构域的伴侣机器如何工作以确保OMP安全通过周质的分子基础了解非常有限。虽然苏拉的晶体结构已知，但大量证据表明，苏拉的功能主要依赖于苏拉中的动态运动，涉及其三个结构域的相互依赖的重排及其与未折叠的OMP的瞬时相互作用。在这项提议中，我们将利用溶液核磁共振(NMR)方面的最新、令人兴奋的发展，以及其他生物物理和计算技术以及试管和活细菌中的功能分析，这将使我们能够以原子分辨率‘观察’动态苏拉伴侣机制的运行。利用这些实验，我们将能够理解Sura中的构象重排如何使其具有伴侣功能。此外，我们还将阐明SurA与其OMP底物之间的瞬时和动态相互作用如何实现有效的底物识别和防止OMP聚集。最后，我们将探索如何在体内控制Sura的功能和OMP的生物合成，为开发新的抗菌剂铺平道路，这些抗菌剂会削弱病原菌如何构建OM，削弱它们对抗生素的防御。

In February 2017 the World Health Organisation published its first ever list of antibiotic-resistant "priority pathogens" - a catalogue of 12 families of bacteria that pose the greatest threat to human health. Of these, 9 are Gram-negative bacteria. The outer membrane (OM) of Gram-negative bacteria protects them from potentially damaging molecules such as antibiotics. The OM is packed with outer membrane proteins (OMPs), creating a permeability barrier that is critical for bacterial survival and endows bacterial resistance to many types of antibiotics. Breaking this barrier is thus an attractive route towards the generation of new antibacterial agents. The biosynthesis of OMPs starts in the cytoplasm and ends with their folding and insertion into the OM, where they form cylindrical, barrel-like structures. The molecular chaperone SurA escorts these newly made unfolded OMPs through the periplasm to the OM. Perturbing SurA chaperone function leads to a loss of bacterial viability and affects antibiotic resistance, demonstrating that SurA is an attractive target to control Gram-negative pathogens. One of the current challenges of effective manipulation of SurA activity is our very limited understanding of the molecular basis of how this large, multidomain chaperone machine works to ensure the safe journey of OMPs through the periplasm. While the crystal structure of SurA is known, a wealth of evidence suggests that SurA function predominantly relies on dynamic motions in SurA involving interdependent rearrangements of its three domains and their transient interactions with unfolded OMPs. In this proposal we will exploit the very latest, exciting, developments in solution nuclear magnetic resonance (NMR), along with other biophysical and computational techniques and functional assays in test tubes and in living bacteria, that will enable us to 'watch' the dynamic SurA chaperone machinery in action with atomic resolution. Using these experiments we will be able to understand how conformational rearrangements in SurA enable its chaperone function. In addition, we will elucidate how the transient and dynamic interactions between SurA and its OMP substrates enable efficient substrate recognition and prevent OMP aggregation. Finally, we will explore how SurA function and OMP biosynthesis can be controlled in vivo, paving the way towards new antibacterial agents that impair how pathogenic bacteria build their OM, weakening their defences against antibiotics.