Dynamics of bacterial peptidoglycan synthesis

细菌肽聚糖合成动力学

• 批准号: 9197654
• 负责人: YVES V BRUN
• 金额: $ 85.19万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-05 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9197654
• 关键词: Address; Affect; Alpha Cell; Amino Acids; Anabolism; Animal Model; Anti-Bacterial Agents; Antibiotics; Architecture; Bacillus subtilis; Back; Bacteria; Bacterial Model; Biochemistry; Biological Models; Cell Shape; Cell Size; Cell Wall; Cell division; Cell physiology; Cells; Cellular biology; Chemicals; Collection; Coupled; Cytokinesis; Data; Development; Dipeptides; Discipline; Enzymes; Escherichia coli; Future; Genes; Genetic; Goals; Gram-Negative Bacteria; Gram-Positive Bacteria; Growth; Hydrolysis; Image Analysis; Individual; Intervention; Laboratories; Lead; Liquid substance; Mechanics; Methods; Microfluidic Microchips; Microfluidics; Microscopy; Modeling; Modification; Morphogenesis; Morphology; N-Acetylmuramoyl-L-alanine Amidase; Organic Synthesis; Organism; Outcome; Peptidoglycan; Peripheral; Polymers; Polysaccharides; Process; Property; Proteins; Public Health; Publishing; Research; Research Personnel; Resolution; Role; Series; Shapes; Side; Site; Solvents; Streptococcus pneumoniae; Structure; Surface; Technology; Testing; Therapeutic Agents; Thick; Thinness; Time; Work; bacterial resistance; base; cell envelope; cell type; comparative; design; expectation; feeding; fluorophore; forward genetics; gene synthesis; improved; insight; macromolecule; method development; nanochannel; novel; pressure; public health relevance; retinal rods; screening; spatiotemporal; tool

DESCRIPTION (provided by applicant): The peptidoglycan (PG) cell wall has long been an attractive target for antibiotic intervention since the late stages of its synthesis take place on he solvent accessible surface of bacterial cells. This essential macromolecule defines bacterial size and shape and provides cells with mechanical strength to resist cell envelope breakdown. Additionally, recent research has demonstrated the importance of the spatiotemporal coordination of PG biosynthesis for bacterial growth, revealing a vulnerability that can be exploited for the development of new antibiotics. The development of methods to enable spatiotemporal tracking of PG synthesis in live bacterial cells is critical to advancing the understanding of the mechanisms of PG synthesis dynamics. In the absence of such methods, identification of new antibiotic targets or identification of novel antibiotic agents will remain elusive. This project has three specific aims that are focused on a long-term goal of elucidating the mechanisms of PG synthesis dynamics. The first Specific Aim seeks to design and develop a series of D-amino acid- and dipeptide-based fluorogenic probes, with optimized photophysical properties, that when coupled with integrated nanochannel and microfluidic devices, and automated image analysis tools, will propel the study of PG dynamics to an unprecedented level of spatiotemporal resolution. The subsequent specific aims will utilize these tools and approaches to analyze the mechanisms of PG dynamics in bacterial model systems with differing cell shapes and cell envelope architectures. In Specific Aim 2, the probes and methods developed under specific aim 1 will be employed to test two major and long-standing hypotheses regarding the spatiotemporal coordination of the elongation and division PG synthesis machineries as well as the coordination between PG hydrolysis and synthesis in the principal model for ovoid-shaped cells, Streptococcus pneumoniae. In Specific Aim 3, PG spatiotemporal dynamics will be examined at an unprecedented resolution for the major model species for rod-shaped Gram negative bacteria with a thin layer of PG, E. coli, and for rod-shaped Gram-positive bacteria with a thick layer of PG in the model organism, B. subtilis. Furthermore, Aim 3 will leverage a high-throughput microscopy screening platform, the availability of a comprehensive strain collection in which each gene has been separately deleted, and the powerful genetics of both species, to systematically and randomly screen for genes involved in PG synthesis dynamics. The comparative analysis of the three model systems will identify the core principles of PG dynamics and how they can be modified to yield different outcomes in dynamics, cell shape and cell envelope architecture. Aims 2 and 3 will feed back into Aim 1 and lead to the design of improved probes and nanochannel configurations. The highly integrated approach, coupled with the individual expertise of the investigators, will provide an unprecedented understanding of PG synthesis and dynamics that can be used to uncover new antibacterial targets, an important step toward addressing the critical need for the discovery of new antibiotics.

描述（由申请人提供）：肽聚糖（PG）细胞壁长期以来一直是抗生素干预的一个有吸引力的目标，因为其合成的后期阶段发生在细菌细胞的溶剂可及的表面上。这种重要的大分子决定了细菌的大小和形状，并为细胞提供机械强度以抵抗细胞包膜破裂。此外，最近的研究证明了 PG 生物合成的时空协调对于细菌生长的重要性，揭示了可用于开发新抗生素的脆弱性。开发能够对活细菌细胞中 PG 合成进行时空追踪的方法对于增进对 PG 合成动力学机制的理解至关重要。如果没有这样的方法，新抗生素靶点的鉴定或新型抗生素药物的鉴定将仍然难以实现。该项目有三个具体目标，重点是阐明 PG 合成动力学机制的长期目标。第一个具体目标旨在设计和开发一系列基于 D-氨基酸和二肽的荧光探针，具有优化的光物理特性，当与集成纳米通道和微流体设备以及自动图像分析工具结合使用时，将推动以下研究： PG 动态达到前所未有的时空分辨率水平。后续的具体目标将利用这些工具和方法来分析具有不同细胞形状和细胞包膜结构的细菌模型系统中的PG动力学机制。在具体目标2中，在具体目标1下开发的探针和方法将用于测试关于伸长和分裂PG合成机制的时空协调以及PG水解和合成之间的协调的两个主要且长期存在的假设。卵形细胞肺炎链球菌的主要模型。在具体目标 3 中，将以前所未有的分辨率检查主要模型物种的 PG 时空动力学，包括具有薄层 PG 的杆状革兰氏阴性菌、大肠杆菌和具有厚层的杆状革兰氏阳性菌。模型生物枯草芽孢杆菌中的 PG 层。此外，Aim 3将利用高通量显微镜筛选平台、每个基因都被单独删除的综合菌株库的可用性以及两个物种强大的遗传学，系统地、随机地筛选参与PG合成动力学的基因。三个模型系统的比较分析将确定 PG 动力学的核心原理，以及如何修改它们以在动力学、细胞形状和细胞包膜结构方面产生不同的结果。目标 2 和 3 将反馈到目标 1 中，并导致改进探针和纳米通道配置的设计。这种高度集成的方法，加上研究人员的个人专业知识，将提供对 PG 合成和动力学的前所未有的了解，可用于发现新的抗菌靶点，这是满足发现新抗生素的关键需求的重要一步。