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Controlling Bacterial Amyloid Formation and the Influence of Curli Subunits on Pathogenic Alpha-synuclein Aggregation

控制细菌淀粉样蛋白的形成以及 Curli 亚基对致病性 α-突触核蛋白聚集的影响

基本信息

批准号：
10586077
负责人：
Matthew Richard Chapman
金额：
$ 30.43万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-01 至 2025-03-31
项目状态：
未结题

项目摘要

Escherichia coli (E.coli) and other enteric bacteria are a major cause of human diseases. Biofilm formation contributes greatly to bacterial persistence and antimicrobial resistance in the host. Many Enterobacteriaceae, such as E. coli, produce functional amyloid fibers called curli as a major proteinaceous component of their extracellular matrix. It is now clear that functional amyloids are widespread, with examples found throughout cellular life. The curli system in E. coli provides a rich and high throughput genetic and biochemical toolbox for the study of amyloid formation. The work has contributed to a curli assembly model where the main fiber component CsgA and the minor subunit CsgB are secreted through the outer membrane-located CsgG. CsgB attaches to the surface of the cell and templates the folding of CsgA into an amyloid fiber. CsgA subunits that inappropriately polymerize in the periplasm are inhibited from amyloid accumulation via CsgC. The exquisitely- controlled curli biogenesis system ensures that E. coli is not exposed to the potentially cytotoxic outcomes of amyloid formation. Uncontrolled or inappropriate amyloid formation results in several neurodegenerative diseases, including Parkinson’s. The hallmark of Parkinson’s disease is the amyloid aggregation of alpha-synuclein, although it is unknown how amyloid formation is initiated. Colonization of mice with curli amyloid producing bacteria results in alpha-synuclein amyloid formation and Parkinson’s like symptoms. Furthermore, purified CsgA protein can accelerate alpha-synuclein amyloid formation in vitro. Therefore, it is imperative to learn how E. coli controls curli amyloid formation and how CsgA can accelerate alpha-synuclein amyloid formation. Interestingly, CsgC from E. coli can inhibit CsgA and alpha-synuclein amyloid formation. Knowledge gained from the following experiments will have implications for microbial pathogenesis, general protein folding, and amyloid biogenesis, thus paving the way for new therapies that rationally target these critical biological processes. In Aim 1 The mechanism and specificity of CsgC will be revealed, including how CsgC functions to inhibit CsgA and alpha- synuclein polymerization at low stoichiometric ratios. In Aim 2 The relationship between the intrinsic ability of CsgA to form amyloid and its ability to accelerate alpha-synuclein amyloid formation will be assessed. Finally, in Aim 3 CsgA and CsgC-like proteins in the sequenced genomes of the human gut microbiota will be identified and biochemically characterized. Together the successful completion of these aims will give an overall understanding of the mechanism of amyloid inhibitory activity and the interactions between bacterial amyloid formation and neurodegenerative diseases.

大肠杆菌（E.coli）和其他肠道细菌是人类疾病的主要原因。生物膜形成极大地促进宿主中的细菌持久性和抗微生物剂抗性。许多肠杆菌科，如大肠大肠杆菌，产生功能性淀粉样纤维称为卷曲作为其主要的蛋白质成分，细胞外基质现在很清楚，功能性淀粉样蛋白是广泛存在的，细胞生命在E.大肠杆菌提供了一个丰富的和高通量的遗传和生化工具箱，淀粉样蛋白形成的研究。这项工作有助于建立一个curli组装模型，其中主纤维组分CsgA和次要亚基CsgB通过位于外膜的CsgG分泌。CsgB 它附着在细胞表面，并作为模板将CsgA折叠成淀粉样纤维。CsgA亚基，通过CsgC抑制周质中不适当的淀粉样蛋白积聚。精致的- 控制curli生物发生系统保证了E.大肠杆菌不会暴露于潜在的细胞毒性结果淀粉样蛋白形成。不受控制或不适当的淀粉样蛋白形成导致几种神经退行性疾病，包括帕金森等帕金森病的标志是α-突触核蛋白的淀粉样蛋白聚集，尽管它是淀粉样蛋白是如何形成的尚不清楚。产生卷曲淀粉样蛋白的细菌在小鼠中的定殖结果在α-突触核蛋白淀粉样蛋白形成和帕金森样症状中的作用。此外，纯化的CsgA蛋白可加速体外α-突触核蛋白淀粉样蛋白形成。因此，了解E.大肠杆菌对照卷曲淀粉样蛋白的形成以及CsgA如何加速α-突触核蛋白淀粉样蛋白的形成。有趣的是，CsgC 来自大肠大肠杆菌能抑制CsgA和α-突触核蛋白淀粉样蛋白的形成。从以下方面获得的知识实验将对微生物发病机理、一般蛋白质折叠和淀粉样蛋白生物发生产生影响，从而为合理地靶向这些关键生物过程的新疗法铺平道路。在目标1 CsgC的机制和特异性将被揭示，包括CsgC如何发挥作用，抑制CsgA和α- 在低化学计量比下的突触核蛋白聚合。目标2：人的内在能力将评估CsgA形成淀粉样蛋白的能力及其加速α-突触核蛋白淀粉样蛋白形成的能力。最后，在目标3中，人类肠道微生物群的测序基因组中的CsgA和CsgC样蛋白将被鉴定和生化特征。这些目标的成功实现将为全面了解淀粉样蛋白抑制活性的机制和细菌之间的相互作用，淀粉样蛋白形成和神经变性疾病。