Mechanism of cellulose synthesis and transport across biological membranes

描述(由申请人提供)：细菌生物膜最好的描述是由细胞外多糖稳定的多细胞的，通常为固着的细菌聚集体，并与许多致病条件有关。例如，囊性纤维化患者中常发现慢性生物膜感染的铜绿假单胞菌。心内膜炎，即心腔和瓣膜的炎症，可由金黄色葡萄球菌和绿色链球菌的生物膜引起，而牙菌斑是牙齿表面的变形链球菌和血球菌的生物膜。由于生物膜细菌对常见抗生素和宿主免疫系统的敏感性较低，因此对人类健康构成了特别的威胁。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成酶机制作为一个模型系统，我们建议确定胞外多糖是如何合成的，并通过细菌细胞被膜运输。这个过程特别有趣，因为胞外多糖是由二磷酸核苷酸激活的前体在细胞内合成的，可以生长到几个微米长，但它们被有效地分泌到它们的生物功能部位。我们结合了分子生物学和结构生物学的工具，首先确定了纤维素合成和膜转运所需的必要成分，其次，从纯化的成分中重建了纤维素的体外生物合成，第三，确定了纤维素合成酶复合体催化活性亚基的三维结构。我们建立了一种新的纤维素合成体外分析方法，证明了纤维素合成酶机械(BCSA和BCSB)的内膜成分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是一种辅助亚基，很可能与BCSA有关；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(目标1)。为了最终证明BCSA和BCSB组分足以进行纤维素的合成和转运，我们必须从纯化的组分中重建体外反应。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转位。为了从机制上深入了解纤维素的生物合成过程，从AIMS 1和AIMS 2获得的生化数据必须与关键酶的结构信息相结合。因此，本方案的第三个目标是用X射线结晶学方法解决纤维素合成酶亚基BCSA的三维结构。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和跨生物膜转移的。

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.