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

• 批准号: 9973388
• 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
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9973388
• 关键词: Adopted; Amyloid; Amyloid beta-Protein Precursor; Amyloid fibers; Antimicrobial Resistance; Bacteria; Behavioral; Binding; Biochemical; Biogenesis; Biological Assay; Biological Process; Brain; Cell surface; Chemicals; Collaborations; Disease; Ensure; Enterobacteriaceae; Escherichia coli; Extracellular Matrix; Fiber; Fluorescence Microscopy; Genes; Genetic; Genome; Gram-Negative Bacteria; Grant; Human; Human Microbiome; In Vitro; Knowledge; Lead; Learning; Life; Link; Membrane; Microbial Biofilms; Minor; Modeling; Molecular Chaperones; Mus; Nerve Degeneration; Neurodegenerative Disorders; Outcome; Parkinson Disease; Pathogenesis; Pathogenicity; Pathway interactions; Polymers; Property; Protein Subunits; Proteins; Seeds; Specificity; Structure; Symptoms; System; Therapeutic; Time; Toxic effect; Uropathogenic E. coli; Variant; Work; alpha synuclein; amyloid formation; associated symptom; biophysical properties; cytotoxic; experimental study; extracellular; gut bacteria; gut microbiota; host colonization; human disease; microbial; mutant; novel therapeutics; periplasm; polymerization; prevent; protein folding; protein misfolding; synuclein

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 极大地促进了细菌在宿主中的持久性和抗药性。许多肠杆菌科， 如大肠杆菌，会产生名为卷曲的功能性淀粉样纤维，作为其主要蛋白质成分 细胞外基质。现在很明显，功能性淀粉样蛋白广泛存在，例子遍及各处。 细胞生命。在大肠杆菌中的Curli系统提供了丰富和高通量的遗传和生化工具箱 对淀粉样蛋白形成的研究。这项工作为卷曲组装模型做出了贡献，其中主要纤维 CsgA组分和次亚基CsgB通过位于外膜的CsgG分泌。CsgB 附着在细胞表面，将CsgA折叠成淀粉样纤维。CsgA亚基 在周质中不适当的聚合通过CsgC抑制淀粉样蛋白的积累。精致的- 可控Curli生物发生系统确保大肠杆菌不会暴露于 淀粉样蛋白形成。 不受控制或不适当的淀粉样蛋白形成会导致几种神经退行性疾病，包括 帕金森氏症。帕金森病的标志是α-突触核蛋白的淀粉样聚集物，尽管它是 不知道淀粉样蛋白是如何形成的。产生卷曲淀粉样菌的小鼠的定植结果 在α-突触核蛋白淀粉样蛋白形成和帕金森样症状中。此外，纯化的CsgA蛋白可以 在体外加速α-突触核蛋白淀粉样蛋白的形成。因此，了解大肠埃希氏菌是如何控制 卷曲淀粉样蛋白形成和CsgA如何加速α-突触核蛋白淀粉样蛋白形成。有趣的是，CsgC 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 实验将对微生物的发病机制、一般的蛋白质折叠和淀粉样蛋白的生物发生产生影响。 从而为合理针对这些关键生物过程的新疗法铺平了道路。在目标1中， CsgC的机制和特异性将被揭示，包括CsgC如何发挥抑制CsgA和α-DNA的作用。 低化学计量比下的突触核蛋白聚合。在目标2中，人的内在能力与 将评估CsgA形成淀粉样蛋白及其加速α-突触核蛋白淀粉样蛋白形成的能力。最后， 在AIM 3中，人类肠道微生物区系测序基因组中的CsgA和CsgC样蛋白将被 进行了鉴定和生化表征。这些目标的成功实现将共同带来 全面了解淀粉样蛋白抑制活性的机制及细菌间的相互作用 淀粉样蛋白形成和神经退行性疾病。