Structure, Function, and Disruption of Microbial Amyloid Assembly and Biofilm For

描述（由申请人提供） 翻译后摘要：结构，功能和破坏微生物淀粉样蛋白组装和生物膜形成生物膜细菌肆虐在临床上。我们目前的抗生素库由细胞杀伤的单一主题的变化组成，即对细菌活力至关重要的靶向过程，但生物膜对常规抗生素和宿主防御的敏感性降低，并且已经成为严重和持续性感染性疾病的毒力标志，包括囊性纤维化，尿路感染，心内膜炎和导管感染。改进的生物膜模型对于理解功能和推动新抗感染药物的设计至关重要。在这个新的创新者奖申请中，我提出了一个非常规的研究计划，旨在实现和提供突破性的发现，需要将生物膜描述符从模糊的术语，如“胶水”和“粘液”转化为基于化学组成和分子结构的定量描述。我通过将新的全细胞固态NMR策略与化学生物学，显微镜和生物化学技术相结合来实现这一目标。从“系统的角度来看，”完整的生物膜的碳和氮NMR光谱将允许所有部分的总和分析，而选择性再耦合测量，连同稳态和瞬态生物合成标记，将分配独特的分子签名，以允许生物膜作为时间和有机体的函数。细菌淀粉样蛋白在不同门的生物膜中普遍存在，并有助于生物膜的形成和尿路致病性E。杆菌我们将从原子水平详细剖析淀粉样蛋白生物发生的分子基础。然后，我们将确定细菌如何使用这些结构作为构建生物膜结构的基石。我们还将参与鉴定干扰组装过程的化合物，并将检查破坏的性质，以提高我们对E.大肠维斯化学遗传学。在更广泛的背景下，该项目在未来五年的结果将建立一个实验基础，定量表征异质性，不溶性，非结晶组装，有助于人类感染性疾病。 公共卫生相关性：这个时代可能会被记住，因为在这个时代，传染病在全球范围内急剧复苏，这是由于治疗持续性生物膜相关传染病的挑战，抗生素耐药性的日益上升，以及药物开发管道中候选抗生素的数量减少。我们提出了一种非常规和创新的方法，这将使变革性的发现能够在分子和原子水平上理解细菌如何组装复杂的异质细胞外结构，包括功能性淀粉样蛋白纤维，以及细菌如何使用这些构建模块来构建有组织的生物膜结构。我们还将参与识别干扰组装过程的小分子，以推动新的抗淀粉样蛋白，抗生物膜和抗毒性疗法的开发。

DESCRIPTION (Provided by the applicant) Abstract: Structure, Function, and Disruption of Microbial Amyloid Assembly and Biofilm Formation Biofilm bacteria are wreaking havoc in the clinic. Our current arsenal of antibiotics is comprised of variations on the single theme of cell killing, i.e. targeting processes essential to bacterial viability, yet biofilms exhibit reduced sensitivity to conventional antibiotics and host defenses, and have emerged as virulence hallmarks of serious and persistent infectious diseases, including cystic fibrosis, urinary tract infection, endocarditis, and catheter infections. Improved biofilm models are crucial to understanding function and driving the design of new anti-infectives. In this New Innovator Award application I present an unconventional research plan designed to enable and deliver breakthrough discoveries needed to transform biofilm descriptors from vague terms like "glue" and "slime" to quantitative descriptions based on chemical composition and molecular architecture. I achieve this by integrating new whole-cell solid-state NMR strategies with chemical biology, microscopy, and biochemical techniques. From a "systems perspective," carbon and nitrogen NMR spectra of intact biofilms will permit a sum-of-all-parts analysis, while selective recoupling measurements, together with steady-state and transient biosynthetic labeling, will assign unique molecular signatures to permit biofilm profiling as a function of time and organism. Bacterial amyloids are prevalent in biofilms among diverse phyla and contribute to biofilm formation and virulence by uropathogenic E. coli. We will dissect in atomic-level detail the molecular basis of amyloid biogenesis. We will then establish how bacteria use these structures as building blocks in the construction of biofilm architectures. We will also be engaged in identifying compounds that interfere with assembly processes and will examine the nature of disruption to improve our understanding of the multi-protein amyloid machinery and biofilm manufacturing in E. coli vis-¿-vis chemical genetics. In a broader context, results of this project over the next five years will establish an experimental basis for quantitatively characterizing heterogeneous, insoluble, noncrystalline assemblies that contribute to human infectious diseases. Public Health Relevance: This era may come to be remembered as one in which infectious diseases made a dramatic worldwide resurgence, owing to the challenge of treating persistent biofilmassociated infectious diseases, the increasing rise of antibiotic resistance, and the dwindling number of candidate antibiotics in the drug development pipeline. We propose an unconventional and innovative approach that will enable transformative discoveries to understand at a molecular and atomic level how bacteria assemble complex heteropolymeric extracellular structures, including functional amyloid fibers, and how bacteria use these building blocks to construct organized biofilm architectures. We will also be engaged in identifying small molecules to interfere with assembly processes to drive the development of new anti-amyloid, anti-biofilm, and anti-virulence therapeutics.