Structure, Dynamics and Activity of the Bacterial Secretosome

所有的细胞都被膜所包围，膜由称为磷脂的双层脂肪分子组成。细胞膜就像一层分子的“皮肤”，把细胞的内部保持在里面，并把不同的生化反应分开。这一障碍需要以受控的方式穿越，以允许营养物质的输入，废物的去除，以及与外界的沟通。这是通过存在于各种膜中的各种蛋白质来实现的。我们对膜蛋白赋予的各种生物功能有了很大的了解，如运输、呼吸、光合作用。然而，我们对膜是如何形成的，以及蛋白质在生物合成过程中的跨膜转运和进入膜的转运知之甚少，我们的建议旨在全面了解蛋白质是如何输出的--蛋白质分泌。分泌（简称“Sec”）机制对生命至关重要，对每个生物体中的每个细胞都是如此。该项目涉及细菌细胞中的这一过程。细菌分泌蛋白质用于广泛的膜和细胞外活性，包括：细胞粘附、致病性、抗生素降解，还包括保护性细胞壁或包膜的生物发生。被称为革兰氏阴性菌的主要种类具有由具有肽聚糖（PG）层的周质组成的细胞壁，所述周质被外膜包围。细胞壁的生物发生依赖于通过Sec机制从细胞内部分泌蛋白质。周质中的蛋白质可以很容易地折叠并保留在那里，而那些注定要到达细胞表面的蛋白质则被运送到另一个称为BAM复合物的运输机器，以便掺入外膜。从内膜到外膜通过包膜的旅程知之甚少。我们关心的问题是：蛋白质如何快速有效地通过这个非常拥挤和具有挑战性的环境？我们已经发现，内（Sec）和外膜（BAM）的蛋白质转运机制以及与质量控制有关的各种辅助因子相互作用，形成一个跨越细菌包膜的组装体，我们称之为细菌分泌体。一个连续的管道通过信封的存在将有深远的影响，我们的理解外膜生物发生，包括上述问题的答案。该项目将利用互补的专业知识，在生物化学和新的突破性技术的高分辨率成像（电子显微镜）和准确的质量测量（质谱）。这种强大的组合将使我们能够检查组装的活性，结构和动力学，以了解它是如何工作的蛋白质是如何通过内膜传递到周质和外膜的。这些知识很重要，因为它将启发我们对细菌生理学的基本方面的理解，以及整个生物学的膜间运输。此外，我们对细菌分泌体的理解的新进展将有助于制定策略，以折衷包膜生物发生及其再生能力，这对生存至关重要。这将为我们对抗抗菌素耐药性（AMR）提供急需的弹药。

All cells are surrounded by membranes, made up from a double layer of fatty molecules called phospholipids. Cell membranes act as a molecular "skin", keeping the cell's insides in, and separating different biochemical reactions. This barrier needs to be traversed in a controlled manner to allow the import of nutrients, and removal of waste products, and for communication with the outside world. This is achieved by a wide range of proteins that reside in the various membranes. We understand a great deal about the diverse biological functions that membrane proteins bestow, such as transport, respiration, photosynthesis. However, we know very little about how membranes are formed, or about the necessary transport of proteins across or into membranes during their biogenesis.Our proposal aims to gain a complete understanding about how proteins are exported-protein secretion. The secretory ('Sec' for short) machinery is essential for life-for every cell in every organism. The project concerns this process, in bacterial cells. Bacteria secrete proteins for a wide range of membrane and extracellular activities including for: cell adherence, pathogenicity, the degradation of antibiotics, including also the biogenesis of the protective cell wall or envelope. A major class of bacteria known as Gram-negatives, possess a cell wall composed of a periplasm with a peptidoglycan (PG) layer, surrounded by an outer-membrane. The biogenesis of the cell wall is dependent on protein secretion from the cell interior through the Sec machinery. Proteins of the periplasm can readily fold and remain there, while those destined for the cell surface are delivered to another transport machine called the BAM complex, for incorporation into the outer-membrane. The journey from the inner- to the outer-membrane through the envelope, is poorly understood. We are concerned with the question: how do proteins make their way rapidly and efficiently through this very crowded and challenging environment? We have discovered that the protein transport machineries of the inner- (Sec) and outer-membranes (BAM) as well as various accessory factors concerned with quality control interact to form an assembly that spans the bacterial envelope, which we have called the bacterial secretosome. The existence of a contiguous conduit through the envelope will have far reaching implications for our understanding of outer-membrane biogenesis, including an answer to the question posed above.The project will harness complementary expertise in biochemistry and new breakthrough technologies for high resolution imaging (electron microscopy) and accurate mass measurements (mass spectrometry). This powerful combination will allow us to examine the activity, structure and dynamics of the assembly in order to understand how it works-how proteins are delivered through the inner-membrane to the periplasm and the outer-membrane. This knowledge is important because it will enlighten our understanding of a fundamental aspect of bacterial physiology, and inter-membrane transport throughout biology. Moreover, new advances in our understanding of the bacterial secretosome will help develop strategies to compromise envelope biogenesis and its regenerative capabilities-essential for survival. This would generate much needed ammunition in our fight against antimicrobial resistance (AMR).