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

描述(由申请人提供) 摘要：微生物淀粉样蛋白组装和生物膜形成的结构、功能和破坏生物膜的细菌正在临床上肆虐。我们目前的抗生素库包括单一主题的细胞杀伤的变化，即靶向过程对细菌生存至关重要，但生物膜对传统抗生素和宿主防御的敏感性降低，并已成为严重和持久传染病的毒力特征，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型抗感染药物的设计至关重要。在这个新创新者奖的申请中，我提出了一项非传统的研究计划，旨在实现和交付所需的突破性发现，将生物膜描述符从“胶”和“粘液”之类的模糊术语转变为基于化学成分和分子结构的定量描述。我通过将新的全细胞固态核磁共振技术与化学生物学、显微镜和生化技术相结合来实现这一点。从“系统的角度来看”，完整生物膜的碳和氮核磁共振谱将允许所有部分的总和分析，而选择性的重新耦合测量，以及稳态和瞬时生物合成标记，将赋予独特的分子特征，以允许作为时间和有机体的函数的生物膜简档。细菌淀粉样蛋白广泛存在于不同门类的生物膜中，并与致尿路病原性大肠杆菌的生物膜形成和毒力有关。我们将在原子水平上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积木。我们还将致力于识别干扰组装过程的化合物，并研究干扰的性质，以提高我们对大肠杆菌中多蛋白淀粉样蛋白机制和生物膜制造的理解。在更广泛的背景下，该项目在未来五年的结果将为定量描述导致人类传染病的异质、不可溶、非晶态组装奠定实验基础。 公共卫生相关性：这个时代可能会被人们铭记为传染病在全球范围内戏剧性复苏的时代，这是因为治疗与生物被膜相关的持续性传染病的挑战，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数量的减少。我们提出了一种非常规和创新的方法，这将使革命性的发现能够在分子和原子水平上了解细菌如何组装复杂的异多聚体细胞外结构，包括功能淀粉样纤维，以及细菌如何利用这些构建块来构建有组织的生物膜结构。我们还将致力于识别干扰组装过程的小分子，以推动新的抗淀粉样蛋白、抗生物膜和抗毒力疗法的开发。

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.