Spatial organization of mycobacterial cell wall biosynthesis

项目总结/摘要 在理解肽聚糖（PG）如何在空间上生物合成方面存在根本性的差距。 协调的方式来支持结核分枝杆菌（Mtb）的极性生长。这一差距代表了 这是一个重要的问题，因为细胞被膜的伸长是细菌生长的一个基本过程， 不了解空间配位的PG前体的精确机制， 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）特别是在活跃生长的分枝杆菌细胞的亚极区富集。 长期目标是了解PM分配在分枝杆菌生理学中的作用， 这个过程中的漏洞。作为实现这一目标的下一步，本提案的总体目标是 获得对PG组装的空间划分的基本见解，以便IMD可以 作为抑制PG层的组装的目标进行评价。核心假设是IMD是一个 合成聚戊烯醇连接的PG前体的PM区域。理由是， PG生物合成的PM分配将为理解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如何产生和维持其 高度复杂的细胞壁。根据已发表的研究和申请人实验室的初步数据， 这一假设将通过追求两个具体目标来检验：1）确定 合成、运输和转运PG前体的蛋白质; 2）确定定位的 生产聚戊烯醇连接的PG前体和PG聚合物。在第一个目标下， 假设PG生物合成酶在PM中空间和生物化学分离， 通过定量和超分辨率显微镜和亚细胞分级来解决。根据第二个 目的，工作假设，即前体和聚合PG是在不同的PM区域，将被确定 通过PG的生物正交代谢标记用于显微镜检测和PG前体的体外生物素化 用于生化检测。该项目是创新的，因为它结合了两个 实验室解剖分区PM在分枝杆菌PG合成的作用，一个实质性的 在理念和执行上脱离现状。这项研究意义重大，因为它 揭示了PM分区是否组织PG合成，这是一个具有高度临床意义的既定药物靶点。 本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 延长Mtb，增强了我们对IMD破坏作为抑制PG合成的新途径的评价， 分枝杆菌细胞生长。 !

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. !