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

多膜跨细菌纤维素合酶（Bcs）超分子复合物的结构研究

• 批准号: 9468670
• 负责人: Justin Finley Acheson
• 金额: $ 5.71万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-12-01 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9468670
• 关键词: 3-Dimensional; Adhesions; Affinity; Bacteria; Binding; Biological; Biopolymers; Cell Wall; Cell membrane; Cells; Cellulose; Clinical; Complex; Crowding; Crystallization; Cystic Fibrosis; Cytosol; DNA; Dehydration; Devices; Diffusion; Electron Microscopy; Electrons; Engineering; Environment; Environmental Risk Factor; Escherichia coli; Extracellular Matrix; Fiber; Gel; Genes; Glucose; Goals; Gram-Negative Bacteria; Hospitals; Host Defense; In Vitro; Individual; Infection; Institution; Investigation; Length; Lipids; Mediating; Medical; Membrane; Microbial Biofilms; Molecular; Negative Staining; Nosocomial Infections; Nucleic Acids; Operon; Organism; Patients; Periplasmic Proteins; Photons; Planet Earth; Polymers; Polysaccharides; Production; Proteins; Pseudomonas aeruginosa; Research; Resolution; Roentgen Rays; Source; Streptavidin; Structure; System; Therapeutic; Urinary tract infection; VDAC1 gene; Virulence Factors; Work; X-Ray Crystallography; antimicrobial; antimicrobial drug; aptamer; cellulose synthase; combat; cryogenics; design; experimental study; in vivo; insight; microbial community; novel therapeutics; pathogen; pathogenic Escherichia coli; pathogenic bacteria; periplasm; protein complex; quorum sensing; response; scaffold; synchrotron radiation; therapeutic development; three dimensional structure

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.

摘要 细菌已经进化出几种机制来在恶劣的环境因素中生存，例如抗微生物剂。 由竞争性生物体或临床环境、宿主防御和其他外力产生的因子。在 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 产生由多糖、脂质、蛋白质 和被称为生物膜的核酸。生物膜的形成使得固着的大菌落能够存活， 通过保护核心居民免于脱水，或通常会 对嗜热细菌有效。后者在临床环境中已变得极其重要， 据估计，60-70%的医院感染是由附着在医疗器械上的产生生物膜的细菌引起的。 插入器械。致病性细菌，包括铜绿假单胞菌、致病性大肠杆菌和 许多葡萄球菌物种代表了最常见的生物体，并且可以在患有 囊性纤维化、尿路感染和许多医院感染。粘附性是生物膜形成的关键 形成，并且通常开始于多糖分泌。纤维素，B-（1® β 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 它的强度和抗降解能力。纤维素合酶操纵子是复杂的，并且在不同的细胞中变化很大。 细菌种类在致病性E.在大肠杆菌中，纤维素是由至少三种蛋白质产生的， 99 kDa内膜合酶（BcsA）负责合成和跨内膜转运， 膜，与BcsA具有专性相互作用的单通道80 kDa周质蛋白（BcsB），以及125 kDa外膜孔蛋白（BcsC），具有大的周质结构域。很可能是细胞的周质区 BcsB和BcsC都与纤维素聚合物相互作用，但这还有待于进一步研究。 演示。本研究的目的是研究细菌中复合物的形成 环氧糖分泌系统纤维素合成酶。coli BcsA-B-C，并测定3- 周质/外膜孔蛋白BcsC的三维结构。最先进的结构 将通过内部低温电子显微镜（cryo-EM）和/或使用同步加速器进行分析 用于X射线衍射的辐射，可从阿贡国家实验室的高级光子源或NSLS-II获得， 布鲁克海文国家实验室。这些目标的完成将导致前所未有的信息未知 细菌分泌系统，并给出了多糖或相关毒力如何分子描述 这些因素被输出到环境中。此外，结构信息可以揭示有吸引力的目标， 设计急需的新疗法以改善生物膜形成。