肽聚糖生物合成在高流动性微膜区与隔膜区之间动态区域化进行的研究

As the major component of cell wall and one of the most valuable antibiotics target, peptidoglycan plays important protective roles in both gram-negative and gram-positive bacteria. Little is known about how the many reactions in its biosynthesis is temporally and spatially organized to ensure highly efficient synthesis of new cell wall during the cell division process. Based on circumstantial supporting evidence, we hypothesize that synthesis of the key precursor lipid II is confined and compartmented in regions of increased fluidity (RIF), which are newly found membrane microdomains in bacteria. We further hypothesize that these precursor-carrying domains are randomly formed and distributed in early cell growth stage and then delivered to the lateral elongasomes and the central peptidoglycan synthetic sites in the septum in the division process. Here we propose to provide direct evidence for compartmentation of this biosynthesis and explore its role in cell physiology and antibiotic mechanism of drugs, using Bacillus subtilis as a model. Specifically, we will express each and every enzyme in the biosynthesis as a fusion protein with monomeric superfolder green fluorescent protein and co-localize with the fluorescent marker of the fluidic membrane microdomains using fluorescence microscopy. In addition, we will label antibiotics with fluorescent dyes and allow them to bind membrane-associated precursors or intermediates for their co-localization with the RIF marker, in order to demonstrate that the biosynthetic precursors are also compartmented like the enzymes. Moreover, we will use time-lapsed total internal reflection fluorescent microscopes to monitor how the fluorescently labeled proteins or precursors are delivered to the lateral and septal peptidoglycan synthetic sites in the division process. By co-localization with a fluorescence-labeled actin protein and septal protein involved in generation of the RIF and divisome, respectively, the regulation mechanism of this dynamic change will be explored. Furthermore, peptidoglycan-targeting antibiotics will be used to observe whether they affect the formation of membrane microdomains and the subcellular localization of the biosynthetic enzymes and precursors as an additional mechanism to augment their bactericidal potency. Through these proposed studies, a new mode of temporal and spatial organization of proteins and precursors will likely be established for efficient synthesis of peptidoglycan, both as an important physiological process and as a valid antibiotic target.

肽聚糖在各种细菌起重要的保护作用，是细胞壁的最主要组成成分，也是最重要的抗菌药物靶标之一。我们对它的生物合成中众多的合成反应在时间与空间上如何组织，协调仍然知之甚少。综合文献中的新旧发现和我们近期的一些研究结果，我们提出合成脂质II前体的所有酶和膜上的所有中间体形成一个合成体，集中定位于近期发现的高流动性微膜区（高流区）。该合成体分布于细胞膜的各个角落，在细胞分裂前期就开始合成累积脂质II，然后随细胞分裂的进行由分裂体引导沿类肌蛋白细胞骨架向位于细胞膜边侧的延长合成体和隔膜区的糖肽链合成体源源不断输送用于高效合成新细胞壁。我们将以枯草芽胞杆菌为模型为此提供证据。这项研究将建立起肽聚糖生物合成在时间与空间上的新组织模式，并把这种组织模式确认为有效的抗生素靶标，为进一步理解这种生物合成模式在亚细胞水平上的调控及发展新的抗生素药物打下坚实基础。

肽聚糖是微生物细胞壁的主要组成部分，也是最重要的药物靶标之一。它的生物合成牵涉众多的合成酶和细胞骨架蛋白，但我们对这些生物大分子如何在时间与空间上如何组织，协调以完成繁复的合成步骤所知有限。本项目以枯草芽胞杆菌为模型，通过对肽聚糖前体相关的合成酶及其它蛋白进行荧光标记并与高流动性微膜区共定位，研究它们与延长合成体及隔膜区的分裂体的关系。研究结果表明肽聚糖前体合成相关的所有酶及其合成中间体在高流动性微膜区与延长合成体的合成酶与骨架蛋白形成多蛋白复合体，定位于细胞的侧膜。同时， 这些肽聚糖前体合成相关的所有酶及其合成中间体也与分裂体的合成酶与架构蛋白相互作用，共同定位于隔膜的高流动性微膜区。进一步，我们以磷脂合成中的磷酸酰化转移酶为例，发现膜周边蛋白通过两条对称的α-螺旋多肽定位于高流动性微膜区，定位机理十分独特，是以疏水作用最小化为基础。当点突变使得该转移酶无法正确定位时，细菌的生长出现了明显的缺陷，首次显示了高流动性微膜区具有重要的生物功能。 最后，我们对破坏高流动性微膜区的达托霉素的膜吸收过程进行研究，取得重要进展并提出其膜表面吸收及膜插入的详细分子机制。 这些研究成果建立起了肽聚糖生物合成在时间与空间上的新组织模式，并证明了组织这种协同模式的高流动性微膜区是有效的抗生素靶标，为进一步理解这种生物合成模式在亚细胞水平上的调控及发展新的抗生素药物打下坚实基础。

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