Assembly of Curli Fibers by Escherichia coli

大肠杆菌组装 Curli 纤维

• 批准号: 7777796
• 负责人: Matthew Richard Chapman
• 金额: $ 35.24万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7777796
• 关键词: Amino Acid Sequence; Amyloid; Amyloid fibers; Attenuated; Bacteria; Biochemical; Biochemistry; Biogenesis; Biological; Biological Assay; Cell surface; Cells; Crystallization; Data; Employee Strikes; Enterobacteriaceae; Escherichia coli; Fiber; Genetic; Goals; Immune Cell Activation; Immunofluorescence Immunologic; Lipoproteins; Liposomes; Maps; Measures; Mediating; Membrane; Microbial Biofilms; Microscopy; Minor; Modeling; Molecular; Organelles; Pathway interactions; Peptides; Positioning Attribute; Process; Property; Protein Subunits; Proteins; Protocols documentation; Research Personnel; Resolution; Salmonella; Sequence Homology; Structure; Swelling; System; Techniques; Testing; Therapeutic; Virulence Factors; Work; amyloid formation; amyloidogenesis; base; deletion analysis; design; directed evolution; human disease; in vitro testing; in vivo; insight; polymerization; programs; protein folding; protein misfolding; research study

DESCRIPTION (provided by applicant): Escherichia coli and Salmonella spp. are a significant cause of human disease. An important virulence factor for these bacteria is curli. Curli are stable proteinacious fibers that contribute to biofilm formation, host colonization, immune activation and cell invasion. Curli also represent a compelling example of a naturally occurring amyloid fiber. Several important human diseases result from proteins that misfold into amyloid fibers. Curli assembly does not result from protein misfolding, but from a dedicated biogenesis pathway, providing a new paradigm for examining amyloidogenesis. Our long-term goal is to elucidate the molecular mechanism of curli biogenesis so that therapeutic protocols can be designed to attenuate their formation. We developed a testable model of curli assembly that suggests the major curli subunit protein CsgA is secreted from the cell as an unstructured protein that subsequently folds into a ?-sheet rich fiber on the cell surface. We hypothesize that the CsgB nucleator protein catalyzes CsgA's initial folding, but then fiber formation proceeds as a self-perpetuating process with the growing fiber tip able to serve as a template for CsgA polymerization. Secretion of CsgA and CsgB to the cell surface is dependent on the outer membrane localized CsgG protein. E. coli provides us a sophisticated genetic and biochemical system to explore the biogenesis of these unique fibers. In Aim 1 the hypothesis that CsgB presents an amyloid-like template to CsgA during nucleation will be tested. In Aim 2 the sequences in CsgA that facilitate its interaction with CsgB and drive its polymerization into an amyloid fiber will be determined. Finally, in Aim 3 we will test the hypothesis that CsgG forms a curli-specific secretion pore in the outer membrane. Collectively, the experiments described within this proposal will reveal the molecular and structural basis for CsgA secretion, nucleation and polymerization into a fiber.

描述（由申请人提供）：大肠杆菌和沙门氏菌属。是人类疾病的重要原因。这些细菌的重要毒力因子是卷。卷曲是稳定的蛋白纤维，有助于生物膜形成，宿主定植，免疫激活和细胞侵袭。 Curli还代表了自然存在的淀粉样纤维的引人注目的例子。几种重要的人类疾病是由于蛋白质折叠成淀粉样蛋白纤维的蛋白质而引起的。 Curli组装不是蛋白质错误折叠而导致的，而是由专用的生物发生途径引起的，为检查淀粉样生成提供了新的范式。我们的长期目标是阐明Curli生物发生的分子机制，以便可以设计治疗方案以减轻其形成。我们开发了一种可测试的冰磨组装模型，该模型表明，主要的Curli亚基蛋白CSGA从细胞中分泌为一种非结构化蛋白质，随后将其折叠成细胞表面上的富含？ - 呈富含纤维。我们假设CSGB成核蛋白会催化CSGA的初始折叠，但随后纤维形成作为一种自我延伸过程进行，增长的纤维尖端能够充当CSGA聚合的模板。 CSGA和CSGB向细胞表面的分泌取决于外膜局部CSGG蛋白。大肠杆菌为我们提供了一个复杂的遗传和生化系统，以探索这些独特的纤维的生物发生。在AIM 1中，将测试CSGB在成核过程中向CSGA提出类似淀粉样蛋白的模板的假设。在AIM 2中，将确定CSGA中促进其与CSGB相互作用并将其聚合驱动到淀粉样纤维的序列的序列。最后，在AIM 3中，我们将检验以下假设：CSGG在外膜中形成卷curli特定的分泌孔。总的来说，本提案中描述的实验将揭示CSGA分泌，成核和聚合成纤维的分子和结构基础。