Deciphering the molecular principles of bacterial metabolosome biogenesis

沙门氏菌等致病菌在哺乳动物肠道中大量繁殖，可导致人类严重的健康问题，包括食物中毒、大规模肠道炎症和心血管疾病。据估计，2017年全球有535，000例沙门氏菌胃肠道感染病例，2018年欧盟有91，857例。沙门氏菌细胞产生一种特殊的纳米级细胞器，称为细菌微区室。这些细胞器提供了一套独特的代谢优势，使沙门氏菌成为宿主肠道恶劣环境中的主要物种。细胞器使用由数千种蛋白质组成的外壳来隔离用于1，2-丙二醇利用（Pdu）的多种酶。这种独特的结构允许Pdu细胞器保护细菌细胞免受有毒代谢物的侵害，并增强细胞的代谢。虽然Pdu细胞器对细菌病原体代谢的重要性是赞赏，很少有人知道细菌细胞如何产生，然后调节这些细胞器，赋予适应性细胞代谢，在复杂的肠道环境中生存，我们最近报道了确切的蛋白质化学计量的Pdu细胞器，并建立了一个新的结构模型的细胞器。我们还开发了用荧光标记物标记Pdu蛋白和耗尽靶蛋白的系统，这样我们就可以使用显微镜跟踪特定的构建蛋白，并研究它们在细菌细胞中的功能。使用开发的系统，我们已经发现，货物酶和壳蛋白在沙门氏菌中独立自组装。我们还表明，Pdu细胞器的位置和运动被限制在细菌细胞内。站在这些令人兴奋的科学和技术突破以及具有互补专业知识的成熟研究团队的基础上，我们现在的目标是深入探索Pdu细胞器是如何合成的，以及Pdu细胞器的组织如何在沙门氏菌细胞内协调。我们将首先确定单个建筑蛋白质经历的多步组装以形成更高级的组装，识别构成酶和壳组装的蛋白质，并阐明酶和壳组装如何结合在一起以形成完整的细胞器。在我们计划的第二部分，我们将研究驱动货物组装的小连接蛋白的结构和功能，以具有“液体状”动态相，并确定相分离机制对于介导蛋白质相互作用和功能蛋白质细胞器的形成至关重要。最后，我们将使用最先进的荧光显微镜来探测Pdu细胞器是如何产生，定位和调节以执行细菌细胞中的重要功能的。这个雄心勃勃的多学科研究项目具有基础和应用意义。Pdu MCP代表了揭示蛋白质自组装原理和生物学中多蛋白质复合物产生的理想模型系统。我们将学习数千种蛋白质如何组装在一起以在细菌细胞内构建功能实体的基本物理和化学，并确定细胞如何精确有效地控制代谢细胞器的形成和功能。我们预计，我们的研究结果将提供更深入的了解天然细菌细胞器和蛋白质组装的生物合成和维护。这项研究可能会为生物“工厂”的工程设计策略提供信息，以增强不同生物技术应用中的细胞代谢和能量生产。此外，我们发现介导Pdu细胞器组装所需的基本蛋白质-蛋白质相互作用可能代表新的治疗靶点，以破坏Pdu细胞器的产生，从而消除沙门氏菌在人类肠道中茁壮成长的能力。

Pathogenic bacteria, such as Salmonella, thrive in mammalian intestines and can cause severe health issues in human, including food poisoning, massive gut inflammation and cardiovascular disease. There were estimated 535,000 cases of Salmonella gastrointestinal infections worldwide in 2017, and 91,857 cases in the EU in 2018. Salmonella cells produce a specialised nano-scale organelle, known as the bacterial microcompartment. These organelles provide a suite of unique metabolic advantages that allow Salmonella to become the predominant species in the hostile environment of the host gut. The organelle uses a shell that is made of thousands of proteins to sequester multiple enzymes used for 1,2-propanediol utilisation (Pdu). This unique structure allows the Pdu organelles to protect bacterial cells from toxic metabolites and to enhance the cell's metabolism. Although the importance of Pdu organelles for the metabolism of bacterial pathogens is appreciated, little is known about how bacterial cells generate and then modulate these organelles to confer adaptive cellular metabolism to survive in the sophisticated gut environment.We have recently reported the exact protein stoichiometry of Pdu organelles and have established a new structural model of the organelle. We have also developed systems for tagging Pdu proteins with fluorescent markers and depleting target proteins, so that we can track specific building proteins using microscopes and study their functions in bacterial cells. Using the developed systems, we have discovered that the cargo enzymes and shell proteins self-assemble independently in Salmonella. We have also shown that the locations and movement of Pdu organelles are confined within the bacterial cell. Standing on these exciting scientific and technical breakthroughs and an established research team with complementary expertise, we now aim to do an in-depth exploration of how Pdu organelles are synthesized and how the organisation of Pdu organelles is coordinated within the Salmonella cell. We will first determine the multi-step assembly that individual building proteins undergo to form higher-ordered assemblies, identify the proteins that make up the enzyme and shell assemblies, and elucidate how enzyme and shell assemblies associate together to form an intact organelle. In the second section of our programme, we will characterise the structures and functions of small linker proteins that drive the assembly of cargos to have a "liquid-like" dynamic phase and ascertain that the phase separation mechanism is vital for mediating the protein interactions and formation of a functional protein organelle. Finally, we will use state-of-the-art fluorescence microscopy to probe how the Pdu organelles are generated, located, and modulated to perform such important functions in bacterial cells.This ambitious and multidisciplinary research project has both fundamental and applied significance. Pdu MCPs represents an ideal model system for uncovering the principles of protein self-assembly and the generation of multi-protein complexes in biology. We will learn the basic physics and chemistry of how thousands of proteins assemble together to build a functional entity within a bacterial cell, and determine how the cell precisely and efficiently controls the formation and function of metabolic organelles. We anticipate that our findings will provide a deeper understanding of the biosynthesis and maintenance of natural bacterial organelles and protein assemblies. The research may inform strategies for the engineering of biological "factories" for the enhancement of cell metabolism and energy production in diverse biotechnological applications. Moreover, the essential protein-protein interactions that we find are required to mediate the assembly of Pdu organelles could represent novel therapeutic targets to disrupt the production of Pdu organelles and thus ablate the ability of Salmonella to thrive in the human gut.