Probing the architecture, assembly, and function of amyloid-polysaccharide entanglements in bacterial biofilms

探究细菌生物膜中淀粉样蛋白-多糖缠结的结构、组装和功能

• 批准号: 10605820
• 负责人: Schuyler A. Chambers
• 金额: $ 6.91万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605820
• 关键词: Acceleration; Address; Adhesives; Ammonium; Amyloid; Amyloid Proteins; Amyloid fibers; Anti-Bacterial Agents; Anti-Infective Agents; Antibiotics; Architecture; Arginine; Bacteria; Bacterial Polysaccharides; Binding; Biochemical; Biological Assay; Biophysics; Biopolymers; Cell surface; Cells; Cellulose; Chemicals; Communities; Creativeness; Detection; Development; Diffusion; Disease; Disinfectants; Escherichia coli; Evaluation; Event; Exhibits; Extracellular Matrix; Extracellular Protein; Fellowship; Fluorescence Microscopy; Foundations; Future; Human Microbiome; Infection; Institution; Investigation; Isotope Labeling; Link; Magnetic Resonance; Mechanics; Mentorship; Microbial Biofilms; Modification; Molecular; NMR Spectroscopy; Nature; Nuclear Magnetic Resonance; Organism; Pathogenesis; Pathology; Polymers; Polysaccharides; Production; Research; Research Project Grants; Role; Salmonella; Salmonella enterica; Sampling; Structure; Symbiosis; Testing; Time; Tissues; Training; Universities; Urinary tract; Vancomycin; Visualization; Western Blotting; Work; bacterial community; cell community; clinically relevant; cohesion; commensal bacteria; design; efficacy evaluation; exhaust; experimental study; extracellular; immunoregulation; in vivo; member; microbial; microbiome; multidisciplinary; mutant; novel; pathogen; pathogenic bacteria; phosphoethanolamine; pressure; recruit; solid state nuclear magnetic resonance

Project Summary Bacteria are most commonly found in nature in multicellular communities termed biofilms. Biofilms are formed when bacteria synthesize, secrete, and enmesh themselves with diverse biopolymers. Beneficial bacteria in the microbiome assemble biofilms, while biofilms are unfortunately also linked to difficult-to-treat infections that exhibit increased tolerance to antibacterials and can exhaust treatment options. However, there are no blueprints for how bacteria build these tissue-like architectures and uncovering these details can accelerate discovery of new anti-infectives. E. coli, in particular, are normal residents in the healthy microbiome, but emerge as pathogens when they egress and colonize the urinary tract. E. coli, Salmonella species and other Gram-negative organisms harness specific amyloid and polysaccharide machinery to elaborate mechanically robust extracellular matrix architectures resembling baskets and blankets that surround cells and drive the formation of tissue-like biofilms. Due to the complexity of biopolymer composites, there are significant challenges associated with studying their structure and function, yet the ubiquity of these biopolymers makes them of high importance for study. This research plan is directed to test molecular hypotheses for how bacteria employ curli and phosphoethanolamine cellulose, a newly discovered chemically modified form of cellulose, to enmesh themselves in extracellular matrix (ECM). The research plan will test hypotheses regarding functional roles that we propose are ascribed to the zwitterionic phosphoethanolamine modification. Aim 1 is directed to evaluate the temporal and spatial developments of matrix assembly beyond the bacterial cell surface using fluorescence microscopy and creative functional biochemical assays. Aim 2 will implement a strategically designed solid-state nuclear magnetic resonance (NMR) approach to detect molecular contacts between polysaccharides and protein amyloids that are responsible for matrix cohesion. The functional benefit of ECM biopolymers will be determined in Aim 3, where clinically relevant antibiotics and a novel vancomycin-conjugate will be evaluated for efficacy against pEtN cellulose and curli containing biofilms. This work promises to formulate a molecular foundation for future avenues of inquiry at the host-pathogen interface, involving possible immunomodulatory roles of bacterial polysaccharides and amyloids, and possible biopolymer contributions to microbiome symbiosis and amyloid- associated disease pathologies. The fellowship candidate will receive significant training in solid-state NMR spectroscopy to study molecular interactions within E. coli biofilms and biochemical approaches to investigate bacterial communities. The considerable support and mentorship structure provided through this fellowship, the research sponsor (Prof. Lynette Cegelski) and institution (Stanford University) will facilitate the professional development of the fellowship applicant and the rigorous scientific investigation of the proposed research.

项目概要 细菌最常见于自然界的多细胞群落中，称为生物膜。生物膜形成 当细菌合成、分泌并与不同的生物聚合物结合时。体内的有益细菌 微生物组组装生物膜，而不幸的是，生物膜也与难以治疗的感染有关 对抗菌药物表现出更高的耐受性，并且可能耗尽治疗选择。然而并没有蓝图 了解细菌如何构建这些类似组织的结构，揭示这些细节可以加速发现 新的抗感染药。尤其是大肠杆菌，是健康微生物组中的正常居民，但以 当病原体排出并定植于尿道时。大肠杆菌、沙门氏菌和其他革兰氏阴性菌 生物体利用特定的淀粉样蛋白和多糖机制来制造机械坚固的 细胞外基质结构类似于篮子和毯子，围绕细胞并驱动细胞的形成 组织样生物膜。由于生物聚合物复合材料的复杂性，相关的重大挑战 随着研究它们的结构和功能，这些生物聚合物的普遍存在使得它们具有高度重要性 用于学习。该研究计划旨在测试细菌如何利用卷曲和 磷酸乙醇胺纤维素，一种新发现的化学改性形式的纤维素，可将 它们本身位于细胞外基质（ECM）中。该研究计划将测试有关功能角色的假设， 我们认为归因于两性离子磷酸乙醇胺修饰。目标 1 旨在评估 使用荧光研究细菌细胞表面之外的基质组装的时间和空间发展 显微镜和创造性的功能生化测定。目标 2 将实施战略设计的固态 核磁共振 (NMR) 方法检测多糖和蛋白质之间的分子接触 负责基质凝聚的淀粉样蛋白。 ECM 生物聚合物的功能优势将被确定 目标 3，将评估临床相关抗生素和新型万古霉素结合物的疗效 针对含有 pEtN 纤维素和 curli 的生物膜。这项工作有望为以下方面奠定分子基础： 宿主-病原体界面的未来研究途径，涉及细菌可能的免疫调节作用 多糖和淀粉样蛋白，以及生物聚合物对微生物共生和淀粉样蛋白的可能贡献 相关疾病病理。奖学金候选人将接受固态核磁共振方面的重要培训 利用光谱学研究大肠杆菌生物膜内的分子相互作用，并利用生化方法进行研究 细菌群落。通过该奖学金提供的大量支持和指导结构， 研究赞助者（Lynette Cegelski 教授）和机构（斯坦福大学）将促进专业人士 奖学金申请人的发展以及对拟议研究的严格科学研究。