Activation of Peptidoglycan Synthesis by Outer Membrane Proteins.

细菌细胞壁肽聚糖在大多数细菌的包膜中形成一个连续的层，即所谓的“小囊”，它对维持细胞完整性和细胞形状至关重要。像大肠杆菌这样的革兰氏阴性细菌有一个非常薄的主要是单层的小囊，夹在两层细胞膜之间，内层细胞膜，细胞质膜和外膜。细菌细胞的生长和分裂需要控制肽聚糖层的扩大，这涉及50多种已知的酶和蛋白质，但其确切机制在很大程度上仍然未知。我们自己和其他实验室的数据支持一种模型，在这种模型中，小囊被由肽聚糖合成酶和水解酶组成的多酶复合物放大，这些酶复合物在细胞内部由细菌细胞骨架的成分控制。这个模型现在已经根据我们最近的令人兴奋的结果进行了修订：我们的工作表明，肽聚糖的合成也由小囊外的新型脂蛋白LpoA和LpoB控制，它们固定在外膜上，与主要的肽聚糖合成酶相互作用并激活。这些外膜激活剂的发现极大地改变了我们对肽聚糖生长的看法，并根据我们的假设，提出了细菌在生长过程中调节其肽聚糖层表面密度的机制。关于lpo介导的肽聚糖合酶的激活还有许多未解的问题，这些问题将在本研究计划中得到解决。在这个项目中，我们的目标是阐明新的肽聚糖合成激活剂的生理作用，它们在外膜定位的重要性以及它们对肽聚糖合成酶和肽聚糖合成复合物活性的影响。我们将进一步研究肽聚糖合酶中激活剂与其对接结构域之间的相互作用，并确定lpo对接结构域复合物的共晶结构。这一部分将提供对lpo介导的肽聚糖合成酶激活机制的见解。最后，我们将探讨外膜肽聚糖合酶激活剂也与肽聚糖水解酶相互作用并影响其活性的可能性，这是一个有吸引力的假设，我们有初步的数据。该项目将涉及各种分子生物学、生物化学和结构生物学技术。预期的结果将大大扩展我们对模型细菌大肠杆菌肽聚糖合成的分子机制的知识，大肠杆菌是一种重要的病原体，根据健康保护局（HPA）的说法，它是英国每年约2万例菌血症的最常见原因。我们的预期结果将与其他革兰氏阴性细菌相关，包括病原体，如流感嗜血杆菌，已知具有必需的lpoA基因，沙门氏菌，克雷伯氏菌，肠杆菌，沙雷氏菌和柠檬酸杆菌。肽聚糖组装的生物合成途径是我们最重要的抗菌剂，β -内酰胺（如青霉素）和糖肽的目标。由于肽聚糖对细菌是必需的和特异性的，并且不存在于人体中，因此它代表了抗菌治疗的理想靶点。我们的研究可能会产生知识，可用于开发治疗抗生素耐药细菌急需的新型抗生素，抗生素耐药细菌的传播日益被视为对公共卫生的威胁。

The bacterial cell wall peptidoglycan forms a continuous layer, a so-called 'sacculus', in the envelope of most bacteria and it is essential to maintain cell integrity and cell shape. Gram-negative bacteria like Escherichia coli have a very thin and mainly single-layered sacculus, which is sandwiched between the two cell membranes, the inner, cytoplasmic membrane and the outer membrane. Growth and division of a bacterial cell requires the controlled enlargement of the peptidoglycan layer, which involves more than 50 known enzymes and proteins but the precise mechanisms have remained largely unknown. Data from our own and other laboratories favour a model in which the sacculus is enlarged by multi-enzyme complexes made of peptidoglycan synthases and hydrolases, which are controlled from inside the cell by components of the bacterial cytoskeleton. This model has now been revised based on our recent, exciting results: our work has shown that peptidoglycan synthesis is also controlled from outside the sacculus by novel lipoproteins, LpoA and LpoB, which are anchored to the outer membrane and which interact with, and activate, the major peptidoglycan synthases. The discovery of these outer membrane activators has dramatically changed our view on peptidoglycan growth and, according to our hypothesis, suggests a mechanism by which bacteria regulate the surface density of their peptidoglycan layer during growth. There are many unanswered questions on the Lpo-mediated activation of peptidoglycan synthases which will be addressed in this research proposal. Within the proposed project we aim to clarify the physiological role of the new peptidoglycan synthesis activators, the importance of their localization to the outer membrane and their effect on the activities of peptidoglycan synthases and of peptidoglycan synthesis complexes. We will further study the interactions between the activators and their docking domains in the peptidoglycan synthases, and we aim to determine the co-crystal structures of Lpo-docking domain complexes. This part will provide insights into the mechanisms of Lpo-mediated activation of peptidoglycan synthases. Finally, we will explore the possibility that outer membrane peptidoglycan synthase activators also interact with and affect the activities of peptidoglycan hydrolases, an appealing hypothesis for which we have preliminary data. The project will involve a variety of molecular biology, biochemistry and structural biology techniques. The expected results will substantially expand our knowledge on the molecular mechanisms of peptidoglycan synthesis in the model bacterium Escherichia coli, which is an important pathogen and, according to the Health Protection Agency (HPA), the most common cause of bacteraemia in the UK with ca. 20 000 cases per year. Our expected results will be relevant to other Gram-negative bacteria including pathogens like Haemophilus influenzae, which is known to have an essential lpoA gene, Salmonella, Klebsiella, Enterobacter, Serratia and Citrobacter. The biosynthetic pathway of peptidoglycan assembly is the target of our most important antimicrobials, the beta-lactams (like penicillin) and glycopeptides. Because peptidoglycan is essential and specific for bacteria, and is not present in humans, it represents an ideal target for antimicrobial therapy. Our research may generate knowledge that could be used to develop novel antibiotics that are urgently needed for the treatment of antibiotic-resistant bacteria the spread of which is increasingly seen as a threat to public health.