Mechanism of cellulose synthesis and transport across biological membranes

描述（由申请人提供）：细菌生物膜最好被描述为多细胞的，通常是无根的，由细胞外多糖稳定的细菌聚集体，并与许多致病条件有关。例如，铜绿假单胞菌的慢性生物膜感染常见于囊性纤维化患者。心内膜炎，即心腔和瓣膜的炎症，可由金黄色葡萄球菌和翠绿链球菌的生物膜引起，而牙斑是牙齿表面的变形链球菌和血源性链球菌的生物膜。生物膜细菌对人类健康的风险特别大，因为它们对普通抗生素和宿主免疫系统的敏感性很低。通常在细菌生物膜中发现的多糖包括-1,6连接的n -乙酰氨基葡萄糖，海藻酸盐和纤维素。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在细菌细胞包膜中运输的。这个过程特别有趣，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，可以长到几微米长，但它们有效地分泌到它们的生物功能位点。我们结合分子生物学和结构生物学的工具，首先，确定纤维素合成和膜易位所需的基本成分，其次，从纯化的成分在体外重建纤维素生物合成，第三，确定纤维素合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纤维素合成体外实验，证明了纤维素合酶机制的内膜组分（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是辅助亚基，最有可能与BcsA结合；然而，它在纤维素合成中的确切作用尚不清楚。因此，基于我们的体外实验，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组分对纤维素的合成和转运是足够的，我们必须用纯化的组分在体外重建反应。因此，我们的第二个目标是纯化BcsA和BcsB亚基，并重建蛋白质脂质体中的纤维素合成和膜易位。为了深入了解纤维素生物合成过程的机理，从目标1和目标2中获得的生化数据必须与关键酶的结构信息相结合。因此，本课题的第三个目的是利用x射线晶体学技术解决纤维素合酶亚基BcsA的三维结构。总的来说，我们采用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.