Dynamics of bacterial peptidoglycan synthesis

描述(申请人提供)：多肽聚糖(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，因为它的合成后期发生在细菌细胞的溶剂可及表面。这种重要的大分子决定了细菌的大小和形状，并为细胞提供了抵抗细胞膜破裂的机械强度。此外，最近的研究证明了PG生物合成的时空协调对细菌生长的重要性，揭示了一个可用于开发新抗生素的漏洞。能够在活细菌细胞中时空跟踪PG合成的方法的发展对于促进对PG合成动力学机制的理解是至关重要的。在缺乏这种方法的情况下，识别新的抗生素靶标或识别新的抗生素制剂将仍然难以捉摸。这个项目有三个特定的目标，集中在一个长期目标，即阐明PG合成动力学的机制。第一个具体目标是设计和开发一系列基于D-氨基酸和二肽的荧光探针，具有优化的光物理性质，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，将把PG动力学的研究推向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膜结构的细菌模型系统中PG动力学的机制。在特定目标2中，在特定目标1下开发的探针和方法将被用来检验两个主要的和长期存在的假说，即在卵形细胞的主要模型-肺炎链球菌中，关于PG合成机制的伸长和分裂的时空协调以及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研究模式生物枯草杆菌(B.subtilis)中具有薄层PG的杆状革兰氏阴性菌和具有厚层PG的杆状革兰氏阳性菌的主要模式物种的PG时空动力学。此外，AIM 3将利用高通量显微镜筛选平台、可获得的全面菌株集合(其中每个基因已分别删除)以及两个物种的强大遗传学，系统和随机地筛选参与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，以及如何对它们进行修改，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。AIMS 2和AIMS 3将反馈到AIMS 1，并导致改进的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，这是朝着解决发现新抗生素的迫切需求迈出的重要一步。

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.