Mechanism of Cellulose Synthesis and Transport Across Biological Membranes

纤维素合成和跨生物膜运输的机制

基本信息

批准号：
10061615
负责人：
Jochen Zimmer
金额：
$ 54.73万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-05 至 2022-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10061615
关键词：
3-Dimensional Address Anabolism Architecture Bacteria Binding Biochemical Biological Biological Assay Biological Models Biophysics Biopolymers C-terminal Cell membrane Cells Cellulose Chemicals Chromosomal translocation Complex Couples Crystallization Data Deposition Electron Microscopy Enzymes Fiber Glucans Glucose Glycosides Green Algae Health Hospitals Human Hydrolase In Vitro Individual Infection Kinetics Length Lipids Measurement Membrane Microbial Biofilms Modeling Molecular Conformation Nucleic Acids Oligosaccharides Planet Earth Polymers Polysaccharides Polystyrenes Process Property Reaction Regulation Research Resolution Risk Roentgen Rays Side Slide Structure Surface System Testing Transmembrane Transport Urochordata Vascular Plant Vesicle Work X-Ray Crystallography antimicrobial aptamer bacterial community biophysical techniques cell envelope cellulose synthase experimental study genetic analysis genetic linkage analysis in vivo insight microbial community nanodisk novel therapeutics optical traps periplasm prevent proteoliposomes reconstitution single molecule small molecule

项目摘要

Cellulose is the most abundant biopolymer on earth. It is a linear polymer of glucose molecules primarily formed by vascular plants but also by green algae, bacteria, and even tunicates. Bacterial cellulose is frequently found in biofilms, which are sessile bacterial communities encased in a 3-dimensional matrix of polysaccharides, proteinaceous fibers, and nucleic acids. Biofilm bacteria are less susceptible to anti-microbial treatments and are responsible for about 80% of hospital-derived infections, thereby posing a significant risk to human health. Developing novel therapeutics to treat or prevent biofilm infections requires a detailed mechanistic understanding of how the biofilm constituents, in particular polysaccharides, are synthesized and deposited outside the cell. The proposed research seeks to provide this information. Bacterial cellulose biosynthesis is an ideal model system to study the mechanism and regulation of exo- polysaccharide secretion. Gram-negatives produce and secrete cellulose via a multi-subunit complex consisting of the inner membrane BcsA and BcsB subunits, the periplasmic BcsZ hydrolase, as well as the outer membrane subunit BcsC. Our previous work provided detailed mechanistic insights into how the inner membrane-integrated BcsA-B complex elongates the cellulose chain and translocates the polymer across the plasma membrane. While current data explain how cellulose is extended, we currently have no information on how cellulose biosynthesis initiates. This question will be addressed biochemically in Aim 1a by reconstituting the initiation reaction in vitro from cell-free expressed 'uninitiated' cellulose synthase. BcsA processively elongates cellulose and pushes the polymer into a transmembrane channel formed by its own membrane-spanning region. Structural snapshots of different cellulose synthase states during cellulose synthesis and membrane translocation provide insights into conformational changes during this process. Yet a precise analysis of energetic requirements for and processivity rates of cellulose translocation is currently missing. We will address these questions on a single molecule level using an optically trapped and catalytically active BcsA-B complex in Aim 1b. Past the inner membrane and in Gram-negatives, cellulose must cross the periplasm and the outer membrane before reaching the biofilm matrix. This section of the translocation path is most likely formed by a direct interaction of periplasmic and outer membrane components with the BcsA-B complex at the inner membrane. In Aim 2 we seek to reconstitute outer membrane transport of cellulose from nanodisc and proteoliposome- reconstituted components for detailed kinetic, biochemical, and interaction studies. This information will support our efforts to determine the structure of an inner and outer membrane-spanning cellulose synthase complex as outlined in Aim 3. We will use X-ray crystallography and/or electron microscopy to determine the structure of individual periplasmic and outer membrane components as well as their complexes with BcsA-B.

纤维素是地球上最丰富的生物聚合物，它主要是葡萄糖分子的线性聚合物。由维管植物形成，也由绿藻、细菌甚至被囊动物形成。常见于生物膜中，生物膜是包裹在 3 维矩阵中的固着细菌群落多糖、蛋白质纤维和核酸对抗菌剂不太敏感。大约 80% 的医院源性感染是由其造成的，从而对开发治疗或预防生物膜感染的新疗法需要详细的研究。了解生物膜成分（特别是多糖）如何合成和合成的机制拟议的研究旨在提供这些信息。细菌纤维素生物合成是研究外切酶机制和调控的理想模型系统革兰氏阴性菌通过多亚基复合物产生和分泌纤维素。由内膜 BcsA 和 BcsB 亚基、周质 BcsZ 水解酶以及我们之前的工作提供了关于内膜亚基 BcsC 的详细机械见解。膜整合的 BcsA-B 复合物延长纤维素链并将聚合物跨过虽然目前的数据解释了纤维素是如何延伸的，但我们目前还没有相关信息。纤维素生物合成是如何开始的。这个问题将在目标 1a 中通过重构来解决无细胞表达的“未启动”纤维素合酶的体外起始反应。 BcsA 不断延长纤维素并将聚合物推入由其形成的跨膜通道纤维素合成过程中不同纤维素合酶状态的结构快照。合成和膜易位提供了对该过程中构象变化的洞察。目前正在对纤维素易位的能量需求和加工率进行精确分析我们将使用光学捕获和催化在单分子水平上解决这些问题。目标 1b 中的活性 BcsA-B 复合物。穿过内膜并且在革兰氏阴性菌中，纤维素必须穿过周质和外膜在到达生物膜基质之前，这部分易位路径很可能是由直接形成的。周质和外膜成分与内膜 BcsA-B 复合物的相互作用。在目标 2 中，我们寻求从纳米圆盘和蛋白脂质体中重建纤维素的外膜运输- 这些信息将用于详细的动力学、生化和相互作用研究的重组成分。支持我们确定跨膜内膜和外膜纤维素合酶结构的努力复合物如目标 3 中所述。我们将使用 X 射线晶体学和/或电子显微镜来确定单个周质和外膜成分及其与 BcsA-B 的复合物的结构。