Hijacking the Sec machinery in bacterial warfare

所有的细胞都被膜包围，膜由一层叫做磷脂的脂肪分子组成。细胞膜扮演着分子“皮肤”的角色，将细胞内部隔离开来，分离不同的生化反应。需要以有控制的方式打破这一障碍，以允许营养物质、废物的运输以及与外部世界的沟通；这是通过广泛的膜插入蛋白质实现的。我们对膜蛋白所具有的运输、呼吸、光合作用等多种生物学功能有了很深的了解。然而，我们对膜是如何形成的，或者新的蛋白质是如何穿过或进入膜的了解要少得多。我们的实验室旨在更多地了解蛋白质是如何进入和离开细胞的。蛋白质，如激素和抗体，通常由细胞通过分泌(简称SEC)机制输出，这是生命所必需的。简单的细菌细胞还分泌蛋白质用于许多目的：形成保护性细胞壁，用于生存和抗生素耐药性(AMR)；黏附在表面；以及引发疾病。这项提议涉及的发现是，细菌还会产生蛋白质，这些蛋白质可以绕过细菌的细胞膜和细胞壁进入其他细菌细胞。这种活动在细菌竞争期间特别重要，有助于确定哪些细菌在肠道等细菌群落中生存，以及它们如何对外部因素做出反应，例如致病细菌的到来。细菌为占上风而部署的武器之一是接触依赖生长抑制物(CDI)毒素。这个项目与最近发现的CDI毒素劫持SEC系统以输入竞争对手细胞有关。这项工作将建立在目前一项分析蛋白质如何出口的研究基础上。大多数临床相关细菌被两层膜包围，每一层膜都有自己的输出机制。我们最近发现，这些机制--内膜的SEC和外膜的BAM--直接相互作用。这两个相同的复合体也被CDI毒素劫持，所以我们的假设是，这种相互作用对进口和出口都很重要。该项目的目标是通过分析其结构和测量反向蛋白转运(IMPORT)的活性，了解该组件是如何被用于毒素进口的。为此，该项目将利用生物化学方面的互补专业知识和高分辨率电子冷冻显微镜成像方面的新突破性技术。这些科学方法将阐明CDI毒素是如何劫持分泌机制进入细胞质的。该项目的结果将对提供对一个跨越生物学广度的基本过程--蛋白质运输--的新理解具有重要意义。此外，我们获得的信息还可以进一步利用。首先，如果我们能够复制和适应CDI毒素部署的机制，这将允许将定制蛋白输送到细菌中--这一壮举目前很难实现。例如，这可能有助于将有毒蛋白质作为一种策略来杀死特定的细菌，以开发下一代抗生素。此外，更良性的应用可能是进口蛋白质，这些蛋白质旨在赋予新的合成活动，用于学术研究和商业化的技术创新。

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. The barrier needs to be breached in a controlled manner to allow transport of nutrients, waste products and for communication with the outside world; this is achieved by a wide range of membrane-inserted proteins. We understand a great deal about the diverse biological functions that membrane proteins bestow, such as transport, respiration, photosynthesis. However, we know much less about how membranes are formed, or about the how new proteins are transported across or into membranes.Our lab aims to understand more about how proteins are able to get in and out of the cell. Proteins, such as hormones and antibodies, are normally exported by cells via the secretory ('Sec' for short) machinery, which is essential for life. Simple bacterial cells also secrete proteins for many purposes: to form a protective cell wall, for survival and antibiotic resistance (AMR); to stick to surfaces; and to cause disease. This proposal concerns the discovery that bacteria also produce proteins that can enter into other bacterial cells, bypassing their membranes and cell wall. This activity is particularly important during bacterial competition, and helps determine which bacteria survive in bacterial communities such as the gut, and how they respond to external factors, e.g. the arrival of disease-causing bacteria. One of the weapons that bacteria deploy to gain the upper hand are Contact-Dependent growth Inhibitor (CDI) toxins. This project relates to a recent discovery that CDI toxins hijack the Sec system for import into rival cells.The work will build on a current study analysing how proteins are exported. Most clinically relevant bacteria are surrounded by two membranes, each of which has their own export machinery. We have recently discovered that these machineries -Sec in the inner membrane and BAM in the outer membrane- interact directly with one another. These same two complexes are also hijacked by CDI toxins, so our hypothesis is that this interaction is important both for import as well as export.The objectives of the project are to understand how this assembly is co-opted for toxin import through an analysis of its architecture and measurement of reversed protein transport (import) activity. To do so the project will harness complementary expertise in biochemistry and new breakthrough technologies in imaging by high-resolution electron cryo-microscopy. These scientific methods will illuminate how the CDI toxin hijacks the secretion machinery for its passage into the cytoplasm.The results of the project will be important in terms of delivering new understanding of a fundamental process -protein trafficking- that spans the breadth of biology. Moreover, the information we gain could be further exploited. First of all, if we were able to copy and adapt the mechanism deployed by CDI toxins this would allow the delivery of bespoke proteins into bacteria -a feat that is currently very difficult to achieve. This could be useful, for example, for the delivery of toxic proteins as a strategy to kill specific bacteria for the development of next generation antibiotics. Additionally, a more benign application could be for the import of proteins designed to bestow new synthetic activities for technical innovation useful in academic research and for commercialisation.