Spatial organization of mycobacterial cell wall biosynthesis

分枝杆菌细胞壁生物合成的空间组织

• 批准号: 9884733
• 负责人: Yasu S Morita
• 金额: $ 23.09万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-05 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9884733
• 关键词: Address; Amino Acids; Anabolism; Animal Model; Antibiotics; Area; Bacillus subtilis; Bacterial Infections; Binding; Binding Proteins; Biochemical; Biochemistry; Biotinylation; Cell Fractionation; Cell Wall; Cell membrane; Cells; Complex; Daptomycin; Data; Detection; Dissociation; Drug Targeting; Drug resistance; Enzymes; Evaluation; Fluorescence Microscopy; Foundations; Genetic; Goals; Growth; In Vitro; Intracellular Membranes; Knowledge; Label; Laboratories; Ligase; Link; Lipids; Membrane; Metabolic; Microscopic; Microscopy; Mission; Modeling; Mycobacterium smegmatis; Mycobacterium tuberculosis; Peptidoglycan; Physiology; Play; Polymerase; Polymers; Process; Production; Proteins; Proteomics; Public Health; Publishing; Research; Resolution; Rod; Role; Route; Testing; Text; Therapeutic; Tuberculosis; United States National Institutes of Health; Work; antimicrobial; cell envelope; cell growth; clinically significant; extracellular; fight against; innovation; insight; interest; muramyl-NAc-(pentapeptide)pyrophosphoryl-undecaprenol; mycobacterial; pathogen; periplasm; polymerization; resistant strain; synthetic enzyme

PROJECT SUMMARY/ABSTRACT There is a fundamental gap in understanding how peptidoglycan (PG) is biosynthesized in a spatially coordinated fashion to support the polar growth of Mycobacterium tuberculosis (Mtb). This gap represents an important problem because the elongation of the cell envelope, an essential process of bacterial growth, is incomprehensive without understanding the precise mechanism of spatially coordinated PG precursor biosynthesis, transport and cell wall integration. The intracellular membrane domain (IMD) is a discrete area of the plasma membrane (PM) particularly enriched in the subpolar region of actively growing mycobacterial cells. The long-term goal is to understand the role of PM partitioning in mycobacterial physiology and to identify vulnerabilities in this process. As the next step to achieve this goal, the overall objective of this proposal is to gain the fundamental insights into the spatial compartmentalization of PG assembly, so that the IMD can be evaluated as a target for inhibiting the assembly of the PG layer. The central hypothesis is that the IMD is a region of the PM where polyprenol-linked PG precursors are synthesized. The rationale is that characterizing the PM partitioning of the PG biosynthesis will lay the foundation for understanding the role of the IMD in the cell wall elongation, thereby beginning to understand how the robust pathogen Mtb produces and maintains its highly complex cell wall. Guided by published studies and preliminary data from the applicant’s laboratories, this hypothesis will be tested by pursuing two specific aims: 1) Determine the subcellular localization of proteins that synthesize, transport and polymerize PG precursors; 2) Determine the localized production of polyprenol-linked PG precursors and PG polymer. Under the first aim, the working hypothesis, that PG biosynthetic enzymes are spatially and biochemically segregated in the PM, will be addressed by quantitative and super resolution microscopy and by subcellular fractionation. Under the second aim, the working hypothesis, that precursors and polymerized PG are in distinct PM regions, will be determined by bioorthogonal metabolic labeling of PG for microscopic detection and in vitro biotinylation of PG precursors for biochemical detection. The project is innovative because it combines synergistic expertise of two laboratories to dissect the role of compartmentalized PM in mycobacterial PG synthesis, a substantive departure from the status quo in both concept and execution. The proposed research is significant because it reveals whether PM partitioning organizes PG synthesis, an established drug target of high clinical significance. The key data obtained from this study will form the basis to further investigate the role of the IMD in cell wall elongation of Mtb, enhancing our evaluation of IMD disruption as a new route for inhibiting PG synthesis and mycobacterial cell growth. !

项目摘要/摘要 在理解肽聚糖(PG)是如何在空间上被生物合成的过程中，存在着一个根本的空白 协调支持结核分枝杆菌(结核分枝杆菌)极地生长。这一差距代表着一个 这是一个重要的问题，因为细胞包膜的伸长是细菌生长的一个基本过程 对空间配位PG前驱体的精确机制缺乏全面的了解 生物合成、运输和细胞壁整合。细胞内膜结构域(IMD)是一个离散的区域 质膜(PM)尤其富含在生长活跃的分枝杆菌细胞的亚极区。 长期目标是了解PM分配在分枝杆菌生理学中的作用，并确定 这一过程中的漏洞。作为实现这一目标的下一步，这项提议的总体目标是 对PG组件的空间分区有基本的了解，以便IMD可以 被评估为抑制PG层的组装的目标。中心假设是IMD是一种 PM的区域，在那里合成聚戊烯醇联的PG前体。其基本原理是， PG生物合成的PM划分将为理解IMD在生物合成中的作用奠定基础 细胞壁伸长，从而开始了解强大的病原体Mtb是如何产生和维持其 高度复杂的细胞壁。以已发表的研究和申请人实验室的初步数据为指导， 这一假说将通过追求两个具体目标来检验：1)确定细胞内的亚细胞定位 合成、运输和聚合PG前体的蛋白质；2)确定定位的 聚戊烯醇交联型PG前驱体和PG聚合物的生产。在第一个目标下，工作 假设PG生物合成酶在PM中在空间和生化上是分离的，将是 通过定量和超分辨率显微镜以及亚细胞分离来解决。在第二个下面 目的，将确定工作假设，即前体和聚合PG位于不同的PM区域 生物正交代谢法用于PG前体的显微检测和体外生物素化 用于生化检测。该项目具有创新性，因为它结合了两个项目的协同专业知识 实验室剖析分隔PM在分枝杆菌PG合成中的作用，这是一种实质性的 在理念和执行上都背离现状。这项拟议的研究意义重大，因为它 揭示PM分割是否组织PG合成，PG合成是一个具有很高临床意义的既定药物靶点。 这项研究获得的关键数据将成为进一步研究IMD在细胞壁中的作用的基础 Mtb的延长，增强了我们对IMD中断作为抑制PG合成和 分枝杆菌细胞生长。 好了！