Protein and Chemical Modulation of Curli Amyloid Biogenesis

 描述(申请人提供)：大肠杆菌和其他肠道细菌是人类疾病的主要原因。生物被膜的形成对细菌在宿主体内的持久性和抗药性有很大的贡献。许多肠道细菌，包括大肠杆菌，都会产生名为卷曲的功能性淀粉样纤维，作为其细胞外基质的主要蛋白质成分。现在很明显，功能性淀粉样蛋白广泛存在，在整个细胞生命中都可以找到这样的例子。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和高通量的遗传和生化工具箱。我们想了解大肠杆菌如何用蛋白质抑制剂控制卷曲淀粉样蛋白的形成，以及我们如何用合理设计的化学伴侣来询问卷曲淀粉样蛋白的形成。我们假设，大肠杆菌利用周质蛋白将卷曲亚基穿梭于周质空间，以避免在周质中形成不适当的淀粉样蛋白。我们已经在大肠杆菌周质中鉴定了两个伴侣样蛋白，我们将对它们的淀粉样蛋白抑制活性的分子机制进行表征。我们还将使用合理设计的小分子来研究阻止卷曲淀粉样蛋白形成的生化阶段(S)。从以下实验中获得的知识将对微生物发病机制、一般蛋白质折叠和淀粉样生物发生产生影响，从而为合理针对这些关键生物学过程的新疗法铺平道路。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。可能在周质中不适当聚合的CsgA亚基通过CsgC抑制淀粉样蛋白的积累。为了合理开发针对淀粉样蛋白聚集和依赖淀粉样蛋白的生物膜形成的治疗方法，我们必须更好地了解卷曲的生物发生及其在生物膜发育中的作用。在目标1中，将探索伴侣样蛋白CsgE和外膜脂蛋白CsgG在引导CsgA通过周质有效转运方面的协调作用。我们还将不仅确定CsgE如何与CsgA和其他淀粉样蛋白相互作用，而且还将确定这些相互作用的后果。在目标2中，我们将描述有效的抗淀粉样蛋白因子CsgC的相互作用和特异性。我们将确定CsgC如何在低化学计量比下抑制CsgA和-突触核蛋白聚合。最后，在目标3中，我们将表征具有淀粉样蛋白抑制能力的小分子。与瑞典乌梅萨大学的Fredrik Almqvist合作，我们已经确定了阻止CsgA聚合的分子。我们将进一步表征这些模拟多肽的化合物如何抑制淀粉样蛋白聚合，评估它们的特异性，并测试它们抑制生物膜形成的能力。

 DESCRIPTION (provided by applicant): Escherichia 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 enteric bacteria, including 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. We want to learn how E. coli controls curli amyloid formation with protein inhibitors and how we can interrogate curli amyloid formation with rationally-designed chemical chaperones. We hypothesize that E. coli utilizes periplasmic proteins to shuttle the curli subunits through the periplasmic space and to avoid inappropriate amyloid formation in the periplasm. We have identified two chaperone-like proteins in the E. coli periplasm for which we will characterize the molecular mechanism of their amyloid inhibitory activity. We will also use rationally-designed small molecules to investigate the biochemical stage(s) at which curli amyloid formation can be stopped. 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. 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. CsgA subunits that might inappropriately polymerize in the periplasm are inhibited from amyloid accumulation via CsgC. In order to rationally develop therapeutics against amyloid assembly and amyloid-dependent biofilm formation, we must better understand curli biogenesis and its function in biofilm development. In Aim 1 the coordinated roles of the chaperone-like protein CsgE and the outer membrane lipoprotein CsgG in directing efficient CsgA transport through the periplasm will be explored. We will also determine not only how CsgE interacts with CsgA and other amyloidogenic proteins but also the consequences of these interactions. In Aim 2 we will characterize the interaction and specificity of the potent anti-amyloid factor CsgC. We will determine how CsgC functions to inhibit CsgA and -synuclein polymerization at low stoichiometric ratios. Finally, in Aim 3 we will characterize small molecules with amyloid-inhibiting capabilities. In collaboration with Fredrik Almqvist at Umeå University in Sweden, we have already identified molecules that discourage CsgA polymerization. We will further characterize how these peptidomimetic compounds inhibit amyloid polymerization, assess their specificity, and test for their ability to inhibit biofilm formation.