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有吸引力的靶标，因为其合成的晚期发生在细菌细胞的溶剂易访问表面上。这种必不可少的大分子定义了细菌的大小和形状，并为细胞提供了机械强度以抵抗细胞包膜分解。此外，最近的研究表明，PG生物合成对细菌生长的时空协调的重要性，揭示了可以利用的脆弱性，用于开发新的抗生素。实现活细胞细胞中PG合成的时空跟踪方法的发展对于促进对PG合成动力学机制的理解至关重要。在没有这种方法的情况下，鉴定新的抗生素靶标或新型抗生素剂的鉴定将仍然难以捉摸。该项目的三个特定目标集中在阐明PG合成动力学机制的长期目标上。 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.随后的特定目的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架构的细菌模型系统中PG动力学的机制。在特定目标2中，将采用特定目标1开发的探针和方法，以测试有关伸长和PG合成机器的时空协调的两个主要和长期的假设，以及PG水解和合成中PG水解与卵巢形状模型的合成之间的协调。在特定的目标3中，PG时空动力学将以前所未有的分辨率进行研究，用于用于杆状革兰氏革兰氏阴性细菌的主要模型物种，其薄层PG，大肠杆菌，杆状革兰氏阳性细菌在模型生物体中具有厚的PG层PG。此外，AIM 3将利用高通量显微镜筛选平台，每个基因已分别删除的综合菌株收集的可用性以及两种物种的强大遗传学，以系统地和随机筛选参与PG合成动力学的基因。对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中产生不同的结果。 AIM 2和3将反馈到AIM 1中，并导致改进的探针和纳米通道配置的设计。高度综合的方法，再加上研究人员的个人专业知识，将提供对PG合成和动态的前所未有的理解，可用于发现新的抗菌靶标，这是解决新抗生素的关键需求的重要一步。