Dynamics of bacterial peptidoglycan synthesis

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，因为其合成的后期阶段发生在细菌细胞的溶剂可及表面上。这种必需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被膜破裂。此外，最近的研究已经证明了PG生物合成的时空协调对于细菌生长的重要性，揭示了可以用于开发新抗生素的脆弱性。开发能够时空跟踪活细菌细胞中PG合成的方法对于推进对PG合成动力学机制的理解至关重要。在缺乏此类方法的情况下，新抗生素靶标的鉴定或新抗生素剂的鉴定将仍然是难以捉摸的。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。第一个特定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，具有优化的生物物理特性，当与集成的纳米通道和微流体设备以及自动图像分析工具相结合时，将推动PG动力学的研究达到前所未有的时空分辨率水平。随后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PG动力学的机制。在具体目标2中，根据具体目标1开发的探针和方法将用于测试两个主要和长期存在的假设，即关于延伸和分裂PG合成机制的时空协调以及卵形细胞主要模型肺炎链球菌中PG水解和合成之间的协调。在具体目标3中，将以前所未有的分辨率检查具有薄层PG的杆状革兰氏阴性细菌的主要模式种属的PG时空动态，E。大肠杆菌，而对于在模式生物中具有厚层PG的杆状革兰氏阳性菌，B。枯草杆菌。此外，Aim 3将利用高通量显微镜筛选平台，每个基因已被单独删除的综合菌株收集的可用性，以及两个物种的强大遗传学，以系统和随机地筛选参与PG合成动力学的基因。三个模型系统的比较分析将确定PG动力学的核心原则，以及如何修改它们以产生不同的动力学结果，细胞形状和细胞包膜结构。目标2和3将反馈到目标1，并导致改进的探针和纳米通道配置的设计。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理解，可用于发现新的抗菌靶标，这是解决发现新抗生素的关键需求的重要一步。

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.