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：
9016558
负责人：
Jochen Zimmer
金额：
$ 29.79万
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 2017-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9016558
关键词：
Address Alginates Anabolism Antibiotics Architecture Bacteria Biochemical Biological Biological Assay Biological Models Biological Process Biopolymers C-terminal Cells Cellulose Chronic Complex Coupled Crystallography Data Dental Plaque Detergents Diphosphates Endocarditis Enzymes Escherichia coli Experimental Designs Glucans Glucosamine Goals Gram-Negative Bacteria Growth Health Heart Human Immune system In Vitro Infection Inflammation Integral Membrane Protein Ion Channel Kinetics Laboratories Length Link Lipid Bilayers Lipids Membrane Microbial Biofilms Molecular Biology Monitor Mutagenesis Nature Nucleic Acids Nucleotides Periplasmic Proteins Phase Polymers Polysaccharides Predisposition Process Protein translocation Proteins Pseudomonas aeruginosa Reaction Risk Role Site Staphylococcus aureus Streptococcus mutans Streptococcus viridans Structure Surface System Techniques Tissues Transferase Vesicle base biological systems cell envelope cellulose synthase crosslink cystic fibrosis patients experience extracellular in vitro Assay in vitro activity in vivo insight interest membrane synthesis mutant novel pathogenic bacteria polypeptide prevent proteoliposomes reconstitution research study structural biology three dimensional structure tool tooth surface

项目摘要

DESCRIPTION (provided by applicant): Bacterial biofilms are best described as multi-cellular, usually sessile, bacterial aggregates stabilized by extracellular polysaccharides and are implicated in a number of pathogenic conditions. For example, chronic biofilm infections of Pseudomonas aeruginosa are commonly found in cystic fibrosis patients. Endocarditis, the inflammation of the heart chamber and valve, can be caused by biofilms of Staphylococcus aureus and Streptococcus viridans, and dental plaques are biofilms of Streptococcus mutans and sanguinis on the surface of the teeth. Biofilm bacteria pose a particular risk to human health because of their low susceptibility to common antibiotics and the host immune system. The polysaccharides typically found in bacterial biofilms include ¿-1,6 linked N-acetyl-glucosamine, alginate, and cellulose. By using the bacterial cellulose synthase machinery as a model system, we propose to determine how extracellular polysaccharides are synthesized and transported across the bacterial cell envelope. This process is particularly interesting because extracellular polysaccharides are synthesized inside the cell from nucleotide diphosphate-activated precursors and can grow to several microns in length, yet they are efficiently secreted to reach the site of their biological function. We combine the tools of molecular and structural biology to first, identify the essential components required for cellulose synthesis and membrane translocation, second, to reconstitute cellulose biosynthesis in vitro from purified components, and third, to determine the 3-dimensional structure of the catalytically active subunit of the cellulose synthase complex. We developed a novel in vitro asay for celulose synthesis, demonstrating that the iner membrane components of the cellulose synthase machinery (BcsA and BcsB) are required for celulose synthesis and translocation. While BcsA is the catalytically active subunit, BcsB is an auxiliary subunit that most likely associates with BcsA; however, its precise role during cellulose synthesis is unclear. Therefore, based on our in vitro assay, we propose to define the minimal core of the BcsB subunit required for cellulose synthesis (Aim 1). To ultimately prove that the BcsA and BcsB components are sufficient for cellulose synthesis and translocation, we have to reconstitute the reactions in vitro from purified components. Thus, our second aim is to purify the BcsA and BcsB subunits and to reconstitute cellulose synthesis and membrane translocation in proteoliposomes. To gain mechanistic insights into the process of cellulose biosynthesis, biochemical data obtained from aims 1 and 2 must be integrated with structural information on the key enzymes. Therefore, the third aim of this proposal is to solve the 3-dimensional structure of the cellulose synthase subunit BcsA by x- ray crystallography. Overall, we undertake a multi-disciplinary approach to reveal how one of nature's most abundant polymers is synthesized and translocated across biological membranes.

描述（由申请人提供）：细菌生物膜最好被描述为由胞外多糖稳定的多细胞、通常无柄的细菌聚集体，并且与许多致病性疾病有关，例如，铜绿假单胞菌的慢性生物膜感染常见于囊肿。纤维化患者的心内膜炎（心室和瓣膜的炎症）可能是由葡萄球菌生物膜引起的。金黄色葡萄球菌和草绿色链球菌以及牙菌斑是牙齿表面变形链球菌和血链球菌的生物膜，因为它们对常见抗生素和宿主免疫系统中常见的多糖的敏感性较低，因此对人类健康构成特殊风险。细菌生物膜包括 ¿ -1,6 连接的 N-乙酰氨基葡萄糖、藻酸盐和纤维素通过使用细菌纤维素合酶机制作为模型系统，我们建议确定细胞外多糖是如何合成并穿过细菌细胞包膜的。因为细胞外多糖是在细胞内由核苷酸二磷酸激活的前体合成的，并且可以生长到几微米长度，但它们可以有效地分泌到达其生物学功能的位点。我们结合分子和结构生物学的工具，首先，确定纤维素合成和膜转位所需的基本成分，其次，从纯化的成分中在体外重建纤维素生物合成，第三，确定催化活性的三维结构我们开发了一种用于纤维素合成的新型体外测定法，证明了纤维素合酶机制的内膜成分（BcsA 和 BcsB）。虽然 BcsA 是催化活性亚基，但 BcsB 是最有可能与 BcsA 相关的辅助亚基；然而，其在纤维素合成过程中的确切作用尚不清楚，因此，根据我们的体外测定，我们建议。定义纤维素合成所需的最小 BcsB 亚基核心（目标 1）最终证明 BcsA 和 BcsB 成分是足够的。对于纤维素合成和易位，我们必须在体外从纯化的组分中重建反应，因此，我们的第二个目标是纯化 BcsA 和 BcsB 亚基并重建蛋白脂质体中的纤维素合成和膜易位，以获得对过程的机制见解。对于纤维素生物合成，从目标 1 和 2 获得的生化数据必须与关键酶的结构信息相结合。因此，该提案的第三个目标是通过 X 射线晶体学解析纤维素合酶亚基 BcsA 的 3 维结构总体而言，我们采用多学科方法来揭示自然界最丰富的聚合物之一是如何合成并跨生物膜转运的。