Assembly of Curli Fibers by Escherichia coli

描述（由申请人提供）：大肠杆菌和沙门氏菌。是人类疾病的重要原因。这些细菌的一个重要毒力因子是curli。 Curli 是稳定的蛋白质纤维，有助于生物膜形成、宿主定植、免疫激活和细胞侵袭。 Curli 也是天然淀粉样纤维的一个令人信服的例子。几种重要的人类疾病都是由错误折叠成淀粉样纤维的蛋白质引起的。 Curli 组装不是由蛋白质错误折叠引起的，而是由专门的生物发生途径引起的，为检查淀粉样蛋白生成提供了新的范例。我们的长期目标是阐明 Curli 生物发生的分子机制，以便设计治疗方案来减少其形成。我们开发了一种可测试的 Curli 组装模型，该模型表明主要的 Curli 亚基蛋白 CsgA 作为非结构化蛋白质从细胞中分泌，随后在细胞表面折叠成富含 β-折叠的纤维。我们假设 CsgB 成核蛋白催化 CsgA 的初始折叠，但随后纤维形成作为一个自我延续的过程进行，不断生长的纤维尖端能够作为 CsgA 聚合的模板。 CsgA 和 CsgB 向细胞表面的分泌依赖于外膜定位的 CsgG 蛋白。大肠杆菌为我们提供了复杂的遗传和生化系统来探索这些独特纤维的生物发生。在目标 1 中，将测试 CsgB 在成核过程中向 CsgA 呈递淀粉样蛋白样模板的假设。在目标 2 中，将确定 CsgA 中促进其与 CsgB 相互作用并驱动其聚合成淀粉样蛋白纤维的序列。最后，在目标 3 中，我们将检验 CsgG 在外膜中形成 curli 特异性分泌孔的假设。总的来说，该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。

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.