Curli Fiber Assembly and Function

描述(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胞外蛋白纤维，有助于毒力。除了是重要的致病因子，在宿主定植、免疫激活和细胞入侵中发挥作用外，Curli还是细菌生物被膜的主要蛋白质支架。卷曲在生物物理学上被归类为淀粉样纤维，因为它们采用了一种 所有淀粉样蛋白共有的交叉链纤维结构。历史上，淀粉样蛋白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，而是进化的生物发生途径的结果。现在很明显，功能性淀粉样蛋白广泛存在，在细胞生活的几乎所有方面都可以找到这种例子。大肠杆菌中的Curli系统为研究淀粉样蛋白的形成提供了丰富的遗传和生化工具箱。我们的长期目标是了解大肠杆菌是如何构建淀粉样纤维的，这样就可以开发出合理针对这一关键生物学过程的新疗法。在这里获得的知识将对微生物的发病机制和蛋白质折叠和错误折叠都有意义。我们以前的发现有助于建立卷曲组装模型，其中主要纤维成分CsgA和次要亚单位CsgB通过脂蛋白CsgG通过外膜分泌。CsgB附着在细胞表面，将CsgA的折叠模板化为淀粉样纤维。CsgE是一种辅助蛋白，具有抗CsgA的伴侣活性，也是Curli亚基分泌所必需的。为了合理开发针对Curli等毒力因子的治疗方法，我们必须更好地了解Curli的生物发生和功能。在目标1中，我们将专注于进一步开发和测试Curli生物发生模型。我们将探讨伴侣样辅助蛋白CsgE和外膜脂蛋白CsgG在引导CsgA通过周质有效转运方面的作用。我们还将研究一种新发现的依赖于CsgC蛋白的周质伴侣活性的机制。在目标2中，我们将评估以前构建的CsgA和CsgB突变体在发育良好的体内生物膜检测中支持生物学功能的能力。此外，淀粉样蛋白种子的特异性将在多菌生物膜中进行测试。最后，在目标3中，我们将开发具有改变淀粉样蛋白能力的小分子。与瑞典Ume大学的Fredrik Almchevst合作，我们已经确定了阻止CsgA聚合的分子。我们将进一步鉴定这些2-Pryidinone变体，并筛选第二代化合物的抗淀粉样蛋白特性。Curli系统已经进化为一种“故意的淀粉样蛋白”，我们将利用这一系统来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理。

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.