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

项目摘要 细菌在自然界中最常见的是被称为生物膜的多细胞群落。生物被膜形成 当细菌合成、分泌并与不同的生物聚合物结合时。环境中的有益细菌 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，这些感染 表现出对抗菌药物的耐受性增加，并可以选择排气治疗方案。然而，没有蓝图 细菌如何构建这些组织状结构并发现这些细节可以加速发现 新的抗感染药物。尤其是大肠杆菌，是健康微生物群中的正常居民，但以 当病原体排出并定居在尿路时。大肠埃希菌、沙门氏菌和其他革兰氏阴性菌 生物利用特定的淀粉样蛋白和多糖机制来阐述机械健壮 细胞外基质结构类似篮子和毯子，包围细胞并驱动 组织状生物膜。由于生物聚合物复合材料的复杂性，存在着相关的重大挑战。 通过研究它们的结构和功能，这些生物聚合物的无处不在使它们变得非常重要 为了学习。这项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，一种新发现的化学修饰形式的纤维素 它们自身在细胞外基质(ECM)中。该研究计划将测试有关功能角色的假设 我们认为这归因于两性离子磷乙醇胺的修饰。目标1旨在评估 利用荧光技术研究细菌细胞表面外基质组装的时空变化 显微镜和创造性的功能生化分析。AIM 2将实施战略性设计的固态 核磁共振检测多糖与蛋白质分子间相互作用的方法 与基质凝聚力有关的淀粉样蛋白。ECM生物聚合物的功能效益将被确定 在目标3中，将评估临床相关的抗生素和新型万古霉素结合物的疗效 对抗含有生物膜的PETN纤维素和卷曲。这项工作有望形成一种分子基础 未来研究宿主-病原体界面的途径，涉及细菌可能的免疫调节作用 多糖和淀粉，以及可能的生物聚合物对微生物组共生和淀粉样蛋白的贡献- 相关的疾病病理学。奖学金候选人将接受固态核磁共振方面的重要培训 光谱学研究大肠杆菌生物膜内的分子相互作用和生化方法研究 细菌群落。通过这一奖学金提供的相当大的支持和指导结构， 研究赞助商(Lynette Cegelski教授)和机构(斯坦福大学)将促进专业人员 发展研究金申请者和对拟议研究进行严格的科学调查。

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.