Membrane steps in bacterial cell wall synthesis

细菌被高度复杂的细胞被膜包围，其中包含必需的肽聚糖层或球囊，一种由聚糖链组成的网状分子，由短肽连接，围绕细胞膜。球囊提供机械强度以抵抗几个大气压的细胞膨压（渗透压），保护细胞免于破裂并保持其细胞形状。细菌细胞的生长和分裂需要肽聚糖层的受控扩大，这涉及60多种已知的酶和蛋白质，但它们如何共同作用以生长细胞壁的精确机制在很大程度上仍然未知。肽聚糖的合成始于细胞质中可溶性结构单元的形成。这些都配备了载体脂质C55-P（磷酸十一异戊烯醇），导致最终的前体脂质II，它是跨细胞膜运输和聚合的球囊生长。在聚合反应期间，载体脂质被释放，并且它被再循环用于进一步的前体运输。这些膜的步骤知之甚少：C55-P所需的转运蛋白是未知的，脂质II转运蛋白（翻转酶）和一些脂质II聚合酶的身份目前在该领域的激烈竞争，不同的整合膜蛋白已被建议执行这些功能。该提案旨在通过使用生物化学和细胞技术的组合确定所有相关蛋白质的活性和相互作用来破译模型细菌大肠杆菌中的膜步骤。我们的目标是在蛋白脂质体中单独或组合重建所有膜步骤以进行功能分析，我们将支持生化实验与突变表型和体内相互作用的细胞研究。我们已经为这个项目获得了大量的初步数据。我们已经可以从大肠杆菌中纯化出所有“困难的”整合膜蛋白。大肠杆菌以足够的量将它们重组在蛋白脂质体中，我们表明所有酶都是有活性的。预期的结果将大大扩展我们对模式细菌大肠杆菌中肽聚糖合成分子机制的了解，大肠杆菌是一种重要的病原体，并且根据健康保护局（HPA）的说法，它是英国菌血症最常见的原因。每年2万例。世卫组织于2017年2月发布的新抗生素研发优先病原体清单包括10种革兰氏阴性菌。我们的预期结果将与其他革兰氏阴性病原体如嗜血杆菌、沙门氏菌、克雷伯氏菌、肠杆菌、沙雷氏菌和柠檬酸杆菌以及革兰氏阳性细菌相关。肽聚糖组装的生物合成途径是我们最重要的抗菌剂β-内酰胺类（如青霉素）和糖肽类（如万古霉素）的靶点。由于肽聚糖对细菌是必需的和特异性的，并且不存在于人类中，因此它代表了抗菌治疗的理想靶标。我们的研究可能会产生可用于开发新抗生素的知识，这些抗生素迫切需要用于治疗耐药性细菌，其传播越来越被视为对公共健康的威胁。

Bacteria are surrounded by a highly complex cell envelope which contains the essential peptidoglycan layer or sacculus, a net-like molecule made of glycan chains connected by short peptides which surrounds the cell membrane. The sacculus provides mechanical strength to resist the cell's turgor (osmotic pressure) of several atmospheres, protecting the cell from bursting and maintaining its cell shape. Growth and division of a bacterial cell requires the controlled enlargement of the peptidoglycan layer, which involves more than 60 known enzymes and proteins but the precise mechanisms of how they work together to grow the cell wall have remained largely unknown. Peptidoglycan synthesis starts in the cytoplasm with the formation of soluble building blocks. These are equipped with the carrier lipid C55-P (undecaprenol phosphate) leading to the final precursor lipid II, which is transported across the cell membrane and polymerized for sacculus growth. During the polymerisation reaction, the carrier lipid is released, and it gets recycled for further rounds of precursor transport. These membrane steps are poorly understood: the required transporter for C55-P is unknown and the identity of the lipid II transporter (flippase) and some of the lipid II polymerases are currently hotly contested in the field; different integral membrane proteins have been suggested to perform these functions. The proposal aims to decipher the membrane steps in the model bacterium Escherichia coli by determining the activities and interactions of all proteins involved using a combination of biochemical and cellular techniques. We aim to reconstitute all membrane steps alone or in combination in proteoliposomes for functional analysis, and we will back biochemical experiments up with cellular studies on mutant phenotypes and in vivo interactions. We have acquired a large amount of preliminary data for this project. We can already purify all the 'difficult' integral membrane proteins from E. coli in sufficient quantity and reconstitute them in proteoliposomes, and we show that all the enzymes are active.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. The WHO priority pathogens list for Research&Development of new antibiotics published in February 2017 includes 10 Gram-negative species. Our expected results will be relevant to other Gram-negative pathogens like Haemophilus, Salmonella, Klebsiella, Enterobacter, Serratia and Citrobacter, and to Gram-positive bacteria.The biosynthetic pathway of peptidoglycan assembly is the target of our most important antimicrobials, the beta-lactams (e.g. penicillin) and glycopeptides (e.g. vancomycin). 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.