Delivery and clearance of outer membrane proteins to the bacterial outer membrane

外膜蛋白向细菌外膜的递送和清除

• 批准号: BB/X015653/1
• 负责人: Neil Ranson
• 金额: $ 87.57万
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX015653%2F1
• 关键词: Delivery; clearance; outer; membrane; proteins

Gram-negative bacteria are a type of micro-organism that are absolutely pervasive in Nature, and we lead lives that are intimately entangled with their biology. such bacteria and have enormous implications for our everyday lives, because they live in our guts, infect our bodies, and are in our environment. They therefore impact human health and wellbeing, are important for biotechnology, sustainable agricultural economies and much more. Gram-negative bacteria differ from other bacteria in that they possess an outer membrane (or OM) that is rich in proteins and complex lipopolysaccharides (LPS). This OM plays a vital role in protecting the bacteria from their environment, for example it is a formidable barrier to toxins, including those such antibiotics that we intentionally use to try to poison them. The OM is critical for their normal growth, and for their pathogenesis in plant, animal and human disease. However, the creation of the OM is uniquely challenging because each component from which it is made, whether that be a protein or lipid molecule, is made inside the bacterial cell, and exported across the inner membrane to a space between the inner and outer membranes called the periplasm. Those components then have to before be incorporated into the OM. The periplasm is devoid of ATP, the molecule that is typically used to power such processes across Nature and so the choreography of the different steps in OM biogenesis must be powered and controlled in a different way to typical protein machineries inside the cell. Bacteria have thus evolved elaborate machineries to build, maintain and adapt their OMs, dependent on growth conditions. A single, essential protein complex, the beta-barrel assembly machinery (or BAM complex), is required to fold and insert outer membrane proteins (OMPs) into the OM. Although substantial recent progress has revealed the structure of BAM and given initial insights into the mechanisms by which OMPs may be inserted into the OM, two critical areas of BAM function remain poorly characterised, namely how are OMPs delivered to BAM and what happens if OMP folding stalls? The rates of OMP synthesis, folding and degradation are finely balanced, and their dysregulation can be bactericidal1. Understanding how these processes operate is thus crucial to understanding OM biogenesis and may reveal an Achilles heel by which bacterial growth can be halted. The focus of this application is to provide new insight into these important questions. We will use an integrated structural molecular biology approach to determine the 3D structure of complexes between BAM and the periplasmic molecular chaperone SurA, which delivers OMPs to BAM, and complexes that include substrate OMPs. We will then use mass spectrometry methods to look at the dynamics and interactions made in such complexes, and functional assays to understand how OMP delivery works. We will also look at what happens when OMP folding goes wrong, and how BAM cooperates with proteins that degrade misfolded OMPs, using the same toolkit of techniques.

革兰氏阴性细菌是自然界中绝对普遍存在的一种微生物，我们的生活与它们的生物学密切相关。这些细菌对我们的日常生活有着巨大的影响，因为它们生活在我们的肠道中，感染我们的身体，并且存在于我们的环境中。因此，它们影响人类健康和福祉，对生物技术、可持续农业经济等非常重要。革兰氏阴性细菌与其他细菌的不同之处在于，它们拥有富含蛋白质和复杂脂多糖 (LPS) 的外膜 (OM)。这种有机物质在保护细菌免受环境侵害方面发挥着至关重要的作用，例如，它是毒素的强大屏障，包括我们故意用来毒害细菌的抗生素。 OM 对于它们的正常生长以及植物、动物和人类疾病的发病机制至关重要。然而，OM 的创建具有独特的挑战性，因为制造 OM 的每种成分，无论是蛋白质还是脂质分子，都是在细菌细胞内制造的，并穿过内膜输出到内膜和外膜之间的空间。称为周质。然后，这些组件必须先合并到 OM 中。周质缺乏 ATP，这种分子通常用于为自然界中的此类过程提供动力，因此 OM 生物发生中不同步骤的编排必须以与细胞内典型蛋白质机器不同的方式提供动力和控制。因此，细菌进化出了复杂的机器来构建、维持和适应它们的有机体，这取决于生长条件。需要一个单一的必需蛋白质复合物，β-桶组装机器（或 BAM 复合物）来折叠外膜蛋白 (OMP) 并将其插入 OM 中。尽管最近取得的实质性进展揭示了 BAM 的结构，并初步了解了 OMP 插入 OM 的机制，但 BAM 功能的两个关键领域仍不清楚，即 OMP 如何传递到 BAM 以及如果 OMP 折叠会发生什么摊位？ OMP 合成、折叠和降解的速率非常平衡，它们的失调可能具有杀菌作用。因此，了解这些过程如何运作对于了解 OM 的生物发生至关重要，并可能揭示阻止细菌生长的致命弱点。该应用程序的重点是为这些重要问题提供新的见解。我们将使用集成的结构分子生物学方法来确定 BAM 和周质分子伴侣 SurA（将 OMP 传递至 BAM）之间的复合物以及包含底物 OMP 的复合物的 3D 结构。然后，我们将使用质谱方法来观察此类复合物中的动力学和相互作用，并使用功能分析来了解 OMP 传递的工作原理。我们还将研究当 OMP 折叠出错时会发生什么，以及 BAM 如何使用相同的技术工具包与降解错误折叠的 OMP 的蛋白质配合。