Activation of Peptidoglycan Synthesis by Outer Membrane Proteins.

外膜蛋白激活肽聚糖合成。

• 批准号: BB/I020012/1
• 负责人: Waldemar Vollmer
• 金额: $ 60.92万
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI020012%2F1
• 关键词: Activation; Peptidoglycan; Synthesis; Outer; Membrane

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.

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“囊”，它对维持细胞完整性和细胞形状至关重要。革兰氏阴性菌如大肠杆菌具有非常薄且主要为单层的球囊，其夹在两个细胞膜（内膜、细胞质膜和外膜）之间。细菌细胞的生长和分裂需要肽聚糖层的受控扩大，这涉及50多种已知的酶和蛋白质，但精确的机制在很大程度上仍然未知。来自我们自己和其他实验室的数据支持这样一种模型，在该模型中，球囊被由肽聚糖酶和水解酶组成的多酶复合物扩大，肽聚糖酶和水解酶由细菌细胞骨架的组分从细胞内部控制。基于我们最近令人兴奋的结果，该模型现已得到修订：我们的工作表明，肽聚糖的合成也由新型脂蛋白LpoA和LpoB从球囊外部控制，这些脂蛋白锚定在外膜上，与主要的肽聚糖酶相互作用并激活。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，根据我们的假设，提出了一种细菌在生长过程中调节其肽聚糖层表面密度的机制。有许多未回答的问题，脂蛋白介导的肽聚糖酶的激活，这将在本研究计划中解决。在拟议的项目中，我们的目标是澄清新的肽聚糖合成激活剂的生理作用，其定位到外膜的重要性和它们对肽聚糖酶和肽聚糖合成复合物的活性的影响。我们将进一步研究激活剂和它们在肽聚糖酶中的对接结构域之间的相互作用，并且我们的目标是确定Lpo-对接结构域复合物的共晶结构。这一部分将提供深入了解脂蛋白介导的肽聚糖酶激活的机制。最后，我们将探讨外膜肽聚糖合成酶激活剂也相互作用，并影响肽聚糖水解酶的活动，一个有吸引力的假设，我们有初步的数据的可能性。该项目将涉及多种分子生物学、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中肽聚糖合成的分子机制的了解，大肠杆菌是一种重要的病原体，根据健康保护局（HPA）的说法，它是英国细菌血症的最常见原因。每年20 000例。我们的预期结果将与其他革兰氏阴性菌相关，包括已知具有必需lpoA基因的流感嗜血杆菌、沙门氏菌、克雷伯氏菌、肠杆菌、沙雷氏菌和柠檬酸杆菌等病原体。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）和糖肽的靶标。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。

项目成果

Elongated structure of the outer-membrane activator of peptidoglycan synthesis LpoA: implications for PBP1A stimulation.

• DOI: 10.1016/j.str.2014.04.017
• 发表时间: 2014-07-08
• 期刊: STRUCTURE
• 影响因子: 5.7
• 作者: Jean, Nicolas L.;Bougault, Catherine M.;Lodge, Adam;Derouaux, Adeline;Callens, Gilles;Egan, Alexander J. F.;Ayala, Isabel;Lewis, Richard J.;Vollmer, Waldemar;Simorre, Jean-Pierre
• 通讯作者: Simorre, Jean-Pierre

Solution NMR assignment of LpoB, an outer-membrane anchored Penicillin-Binding Protein activator from Escherichia coli.

• DOI: 10.1007/s12104-014-9557-z
• 发表时间: 2015-04
• 期刊: BIOMOLECULAR NMR ASSIGNMENTS
• 影响因子: 0.9
• 作者: Jean, Nicolas L.;Bougault, Catherine M.;Egan, Alexander J. F.;Vollmer, Waldemar;Simorre, Jean-Pierre
• 通讯作者: Simorre, Jean-Pierre

Discovery of chlamydial peptidoglycan reveals bacteria with murein sacculi but without FtsZ.

• DOI: 10.1038/ncomms3856
• 发表时间: 2013
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Pilhofer, Martin;Aistleitner, Karin;Biboy, Jacob;Gray, Joe;Kuru, Erkin;Hall, Edward;Brun, Yves V.;VanNieuwenhze, Michael S.;Vollmer, Waldemar;Horn, Matthias;Jensen, Grant J.
• 通讯作者: Jensen, Grant J.

Outer-membrane lipoprotein LpoB spans the periplasm to stimulate the peptidoglycan synthase PBP1B

• DOI: 10.1073/pnas.1400376111
• 发表时间: 2014-06-03
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Egan, Alexander J. F.;Jean, Nicolas L.;Simorre, Jean-Pierre
• 通讯作者: Simorre, Jean-Pierre

In vitro peptidoglycan synthesis assay with lipid II substrate.

• DOI: 10.1007/978-1-62703-245-2_17
• 发表时间: 2013-01-01
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Biboy, Jacob;Bui, Nhat Khai;Vollmer, Waldemar
• 通讯作者: Vollmer, Waldemar