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Curli Fiber Assembly and Function

Curli 纤维组装和功能

基本信息

批准号：
8728387
负责人：
Matthew Richard Chapman
金额：
$ 35.25万
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：
2007
资助国家：
美国
起止时间：
2007-04-01 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8728387
关键词：
Adopted Alzheimer&apos s Disease Amyloid Amyloid fibers Amyloidosis Antibiotic Therapy Bacteria Bacterial Infections Behavior Biochemical Biogenesis Biological Assay Biological Process Biology Cell surface Cells Chemicals Collaborations Communicable Diseases Communities Creutzfeldt-Jakob Syndrome Disease Enterobacteriaceae Escherichia coli Extracellular Matrix Extracellular Protein Fiber Generations Genetic Goals Grant Immune Cell Activation In Vitro Knowledge Learning Life Lipoproteins Mechanics Membrane Methods Microbial Biofilms Microscopic Minor Modeling Molecular Molecular Chaperones Molecular Probes Organism Pathogenesis Pathway interactions Polymers Property Proteins Role Signal Transduction Specificity Speed Structure Sweden System Testing Therapeutic Therapeutic Agents Toxic effect Universities Variant Vertebral column Virulence Virulence Factors Work amyloid formation base combat extracellular human disease in vitro Assay in vivo inhibitor/antagonist microbial mutant neurotoxicity novel strategies periplasm polymerization prevent protein folding protein misfolding public health relevance scaffold small molecule

项目摘要

DESCRIPTION (provided by applicant): Enteric bacteria such as Escherichia coli are a major cause of human disease. These bacteria produce curli, extracellular protein fibers that contribute to virulence. In addition to being important pathogenicity factors, with roles in host colonization, immune activation, and cell invasion, curli act as the major proteinaceous scaffold for bacterial biofilms. Curli are biophysically classified as an amyloid fiber because they adopt a cross ¿-strand fibrillar structure common to all amyloids. Amyloids have historically been associated with protein misfolding and cellular toxicity, especially neurotoxicity. Curli are not te products of protein misfolding, but instead are the result of an evolved biogenesis pathway. It is now clear that functional amyloids are widespread, with examples found in nearly all facets of cellular life. The curli system in E. coli provides a rich genetic and biochemical toolbox for the study of amyloid formation. Our long-term goal is to understand how E. coli builds an amyloid fiber, so that new therapies can be developed that rationally target this critical biological process. Knowledge gained here will have implications for both microbial pathogenesis and protein folding and misfolding. Our previous discoveries have contributed to a curli assembly model where the main fiber component CsgA and the minor subunit CsgB are secreted through the outer membrane via the lipoprotein CsgG. CsgB attaches to the surface of the cell and templates the folding of CsgA into an amyloid fiber. CsgE, an accessory protein with chaperone-like activity against CsgA, is also required for curli subunit secretion. In order to rationally develop therapeutics against virulence factors such as curli, we must better understand curli biogenesis and function. In Aim 1 we will focus on further developing and testing the curli biogenesis model. The roles of the chaperonelike accessory protein CsgE and the outer membrane lipoprotein CsgG in directing efficient CsgA transport through the periplasm will be explored. We will also investigate the mechanics of a newly discovered periplasmic chaperone activity that is dependent on the CsgC protein. In Aim 2 we will assess the ability of previously constructed CsgA and CsgB mutants to support biological function in well-developed in vivo biofilm assays. Furthermore, the specificity of amyloid seeding will be tested in polymicrobial biofilms. Finally, in Aim 3 we will develop small molecules with amyloid-altering capabilities. In collaboration with Fredrik Almqivst at Ume¿ University in Sweden, we have already identified molecules that discourage CsgA polymerization. We will further characterize these 2-pryidinone variants and screen a second generation of compounds for antiamyloid properties. The curli system has evolved as an "amyloid on purpose," and we will exploit this system in order to better understand global tenets of amyloid formation, microbial pathogenesis, and biofilm biology.

描述（由申请人提供）：肠道细菌如大肠杆菌是人类疾病的主要原因。这些细菌产生卷曲的细胞外蛋白纤维，有助于毒力。除了作为重要的致病因子，在宿主定植、免疫激活和细胞侵袭中发挥作用外，curli还作为细菌生物膜的主要蛋白质支架。卷曲纤维在生物病理学上被归类为淀粉样纤维，因为它们采用了所有淀粉样蛋白共有的交叉纤维状结构。淀粉样蛋白历来与蛋白质错误折叠和细胞毒性，特别是神经毒性有关。卷曲不是蛋白质错误折叠的产物，而是进化的生物合成途径的结果。现在很清楚，功能性淀粉样蛋白是广泛存在的，在细胞生命的几乎所有方面都有发现。在E.大肠杆菌为淀粉样蛋白形成的研究提供了丰富的遗传和生化工具箱。我们的长期目标是了解E。大肠杆菌构建淀粉样蛋白纤维，因此可以开发新的疗法，合理地针对这一关键的生物过程。这里获得的知识将对微生物发病机制和蛋白质折叠和错误折叠产生影响。我们以前的发现有助于卷曲组装模型，其中主要纤维组分CsgA和次要亚基CsgB通过脂蛋白CsgG通过外膜分泌。CsgB附着在细胞表面，并将CsgA折叠成淀粉样纤维。CsgE是一种辅助蛋白，具有针对CsgA的分子伴侣样活性，也是curli亚基分泌所需的。为了合理开发针对毒力因子如curli的治疗方法，我们必须更好地了解curli的生物起源和功能。在目标1中，我们将专注于进一步开发和测试curli生物成因模型。将探讨伴侣样辅助蛋白CsgE和外膜脂蛋白CsgG在指导有效CsgA运输通过周质的作用。我们还将研究一个新发现的周质分子伴侣的活动，这是依赖于CsgC蛋白的力学。在目标2中，我们将评估先前构建的CsgA和CsgB突变体在良好开发的体内生物膜测定中支持生物功能的能力。此外，将在多微生物生物膜中测试淀粉样蛋白接种的特异性。最后，在目标3中，我们将开发具有淀粉样蛋白改变能力的小分子。通过与瑞典Ume大学的Fredrik Almqivst合作，我们已经确定了阻碍CsgA聚合的分子。我们将进一步表征这些2-pryidinone变体，并筛选第二代化合物的抗淀粉样蛋白特性。curli系统已经进化为“有目的的淀粉样蛋白”，我们将利用这个系统，以更好地了解淀粉样蛋白形成，微生物发病机制和生物膜生物学的全球原则。