Structural investigation of the multi-membrane spanning bacterial cellulose synthase (Bcs) supramolecular complex

摘要 细菌已经进化出几种机制来在恶劣的环境因素(如抗菌剂)中生存 由相互竞争的有机体或在临床环境中产生的药物、宿主防御和其他外部力量。在……里面 对这些环境因素的反应仍然知之甚少，群体感应浮游细菌开始 产生一种三维凝胶状细胞外基质，由分泌的多糖、脂类、蛋白质、 以及被称为生物膜的核酸。生物膜的形成使固着的大菌落有可能存活下来 通过保护核心居民免受脱水或抗菌剂的扩散，通常情况下 对浮游细菌有效。后者在临床环境中已成为一个极其重要的因素 据估计，60%-70%的医院感染是由附着在医疗设备上的生物膜细菌引起的 插入设备。机会细菌包括铜绿假单胞菌、致病性大肠杆菌和 许多葡萄球菌代表最常见的微生物，可以在患者身上发现 囊性纤维化、尿路感染和许多医院感染。附着力是生物膜的关键。 形成，通常开始于多糖的分泌。纤维素，b-(1®4)D-葡萄糖的线性聚合物 单位，是最丰富的陆地生物聚合物之一，也是生物膜的常见成分，可能是由于 它的力量和对退化的顽固性。纤维素合成酶操纵子很复杂，不同的 细菌种类。在致病性大肠杆菌的情况下，纤维素的产生至少使用三种蛋白质，一种 99 kDa内膜合成酶(BCSA)负责合成和跨膜内转运。 膜，单程80 kDa周质蛋白(BCSB)与BCSA专有相互作用，以及125 KDA外膜孔蛋白(BCSC)具有较大的周质结构域。它很可能是周质结构域 BCSB和BCSC都与纤维素聚合物相互作用，但这还没有 演示了。本研究的目的是研究细菌中复合体的形成。 大肠杆菌BCSA-B-C的环氧糖分泌系统纤维素合成酶，并测定其3- 周质/外膜孔蛋白BCSC的空间结构。最先进的结构 分析将通过室内低温电子显微镜(CRYO-EM)和/或使用同步加速器进行 Argonne National Lab或NSLS-II先进光子源提供的X射线衍射辐射 布鲁克海文国家实验室。这些目标的完成将带来前所未有的未知信息 细菌分泌系统，并给出了多糖或相关毒力的分子描述 要素被输出到环境中。此外，结构信息可能揭示具有吸引力的目标 设计急需的新疗法来改善生物被膜的形成。

Abstract Bacteria have evolved several mechanisms to survive harsh environmental factors such as antimicrobial agents produced by competing organisms or in the clinical setting, host defenses, and other external forces. In a still poorly understood response to these environmental factors, quorum sensing planktonic bacteria begin to produce a 3-dimension gel-like extracellular matrix formed by secretion of polysaccharides, lipids, proteins, and nucleic acids known as a biofilm. Formation of the biofilm allows sessile macro-colonies to survive likely by shielding the core inhabitants from dehydration, or diffusion of antimicrobial agents that would normally be effective to a planktonic bacterium. The latter has become an extremely important in the clinical setting with an estimated 60-70% of nosocomial infections are caused by biofilm producing bacteria attached to medical insertion devices. Opportunistic bacteria including Pseudomonas aeruginosa, pathogenic Escherichia coli, and many Staphylococcal species represent the most common organisms, and can be found in patients suffering cystic fibrosis, urinary tract infections and many nosocomial infections. Adhesion is a crucial for biofilm formation, and typically begins with polysaccharide secretion. Cellulose, a linear polymer of b(1®4) D-glucose units, is one of the most abundant terrestrial biopolymers, and a frequent constituent of biofilms likely due to its strength and recalcitrance to degradation. Cellulose synthase operons are complex and vary greatly among bacterial species. In the case of pathogenic E. coli, cellulose is produced using a minimum of three proteins, a 99 kDa inner-membrane synthase (BcsA) responsible for both synthesis and translocation across the inner- membrane, a single pass 80 kDa periplasmic protein (BcsB) with obligate interactions with BcsA, and a 125 kDa outer-membrane porin (BcsC) with a large periplasmic domain. It is likely that the periplasmic domains of BcsB and BcsC both interact with the cellulose polymer and each other, however this has yet to be demonstrated. The goals of this research are to investigate complex formation in the bacterial epoxosaccharide secretion system cellulose synthase from E. coli BcsA-B-C, and to determine the 3- dimensional structure of the periplasmic/outermembrane porin protein BcsC. State-of-the-art structural analysis will be performed by in house cryogenic electron microscopy (cryo-EM) and/or use of synchrotron radiation for X-ray diffraction available from the Advance Photon Source at Argonne National Lab or NSLS-II at Brookhaven Nation Lab. Completion of these goals will result in unprecedented information of uncharted bacterial secretion systems, and give a molecular description of how polysaccharides or related virulence factors are exported into the environment. Furthermore, structural information may reveal attractive targets for designing much needed new therapeutics to ameliorate biofilm formation.