Assembly of Curli Fibers by Escherichia coli

描述(申请人提供)：大肠杆菌和沙门氏菌。是人类疾病的一个重要原因。对这些细菌来说，一个重要的毒力因素是卷曲。卷曲是一种稳定的蛋白质纤维，有助于生物膜的形成、宿主的定植、免疫激活和细胞入侵。卷曲也是自然产生的淀粉样纤维的一个引人注目的例子。几种重要的人类疾病是由蛋白质错误折叠成淀粉样纤维引起的。卷曲组装不是蛋白质错误折叠的结果，而是一种专门的生物发生途径，为研究淀粉样蛋白的发生提供了一种新的范式。我们的长期目标是阐明卷曲生物发生的分子机制，以便设计治疗方案来减少它们的形成。我们开发了一个可测试的卷曲组装模型，该模型表明主要的卷曲亚单位蛋白CsgA是作为一种非结构蛋白从细胞中分泌出来的，随后折叠成细胞表面的一层富纤维。我们假设CsgB核蛋白催化CsgA的初始折叠，但随后纤维形成是一个自我持续的过程，生长的纤维尖端能够作为CsgA聚合的模板。CsgA和CsgB分泌到细胞表面依赖于外膜定位的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提供淀粉样模板的假设。在目标2中，将确定CsgA中促进其与CsgB相互作用并推动其聚合为淀粉样纤维的序列。最后，在目标3中，我们将检验CsgG在外膜中形成卷曲特异性分泌孔的假设。总而言之，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。

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.