Mechanism of cellulose synthesis and transport across biological membranes

描述（由申请人提供）：细菌生物膜最好描述为由胞外多糖稳定的多细胞的、通常是固着的细菌聚集体，并且与许多致病性病症有关。例如，铜绿假单胞菌的慢性生物膜感染通常在囊性纤维化患者中发现。心内膜炎，心脏腔室和瓣膜的炎症，可以由金黄色葡萄球菌和草绿色链球菌的生物膜引起，牙菌斑是牙齿表面上的变形链球菌和血链球菌的生物膜。生物膜细菌对人类健康构成特别的风险，因为它们对常见抗生素和宿主免疫系统的敏感性较低。通常在细菌生物膜中发现的多糖包括ω-1，6连接的N-乙酰基-葡糖胺、藻酸盐和纤维素。通过使用细菌纤维素合成酶机制作为一个模型系统，我们建议，以确定胞外多糖是如何合成和运输穿过细菌细胞包膜。这个过程特别有趣，因为胞外多糖是在细胞内从核苷酸二磷酸活化的前体合成的，并且可以生长到几微米长，但它们被有效地分泌到其生物功能的位点。我们结合联合收割机的分子和结构生物学的工具，第一，确定纤维素合成和膜转位所需的基本组件，第二，重组纤维素合成酶在体外纯化的成分，第三，以确定纤维素合成酶复合物的催化活性亚基的三维结构。我们开发了一种新的体外纤维素合成方法，证明纤维素合成酶的内膜成分（BcsA和BcsB）是纤维素合成和转运所必需的。虽然BcsA是催化活性亚基，但BcsB是最有可能与BcsA结合的辅助亚基;然而，其在纤维素合成过程中的确切作用尚不清楚。因此，基于我们的体外试验，我们建议定义纤维素合成所需的BcsB亚基的最小核心（目的1）。为了最终证明BcsA和BcsB组分足以用于纤维素合成和转运，我们必须从纯化的组分在体外重构反应。因此，我们的第二个目标是纯化BcsA和BcsB亚基，并在脂蛋白体中重建纤维素合成和膜转运。为了获得纤维素生物合成过程的机理见解，从目标1和2获得的生物化学数据必须与关键酶的结构信息相结合。因此，本建议的第三个目的是通过X射线晶体学解析纤维素合成酶亚基BcsA的三维结构。总的来说，我们采取多学科的方法来揭示自然界中最丰富的聚合物之一是如何合成和跨生物膜转运的。

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.