Biogenesis and Function of Bacterial Amyloid fibers

描述（申请人提供）：无。（根据应用改编）：Curli是由大肠杆菌和某些沙门氏菌产生的细胞外细胞器。查普曼博士已经证明，这些纤维在结构上和生物化学上与真核淀粉样蛋白纤维相关，真核淀粉样蛋白纤维与几种哺乳动物疾病有关，包括阿尔茨海默病、系统性淀粉样变性和海绵状脑病，如疯牛病和克雅氏病。与哺乳动物淀粉样纤维似乎是通过异常的蛋白质折叠途径形成的不同，细菌中的卷曲蛋白组装涉及一个专用的多步骤途径，需要csgBA和csgDEFG操纵子。主要卷曲亚基蛋白CsgA的聚合依赖于CsgB成核体，CsgA和CsgB向细胞表面的转运由组装因子CsgE、CsgF和CsgG介导。Specific Aim 1将确定成核和聚合反应的机制，并验证CsgB采用淀粉样结构刺激CsgA聚合的假设。纯化后的CsgB将通过圆二色性（CD）光谱、刚果红（CR）和硫黄素T （thT）结合测定来确定其结构和颜色性质。CsgB和CsgA中保守的Asn和Gln残基的重要性将通过定点诱变来确定。CsgB突变体的成核活性将在体内和使用最近开发的体外CsgA聚合试验进行评分。特异目的2将阐明卷曲生物发生的分子细节，特别强调亚单位分泌的机制。Chapman博士将测试外膜脂蛋白CsgG形成卷曲蛋白特异性孔的假设，该孔负责卷曲蛋白亚基的分泌，并且GsgG的功能依赖于CsgE。CsgG将通过EM、CD和Blue天然凝胶电泳进行纯化和表征，并通过体内抗生素敏感性试验和体外脂质体肿胀试验评估其成孔能力。查普曼博士将尝试鉴定CsgA和CsgB上介导其CsgG依赖性分泌的序列，并通过共纯化和细胞分离方法证明CsgE与CsgG的相互作用。特异性Aim 3将定义curli在刺激宿主炎症反应中的作用。纯化的CsgA（可溶性和聚合）在体外诱导人巨噬细胞炎症细胞因子的能力将被评估。不能聚合的CsgA突变体将用于验证聚合是刺激细胞因子产生所必需的假设。小鼠也将用纯化的curli或CsgA刺激IP，并测量NO的产生、细胞因子的产生、血清肌酐和结合胆红素的升高，以验证curli是一种细菌病原体相关微生物模式（PAMP）的假设，该模式直接刺激了作为感染性休克特征的调节不良的先天炎症反应。

DESCRIPTION (provided by applicant): None. (adapted from application): Curli are extracellular organelles produced by Escherichia coli and certain Salmonella species. Dr. Chapman has demonstrated that these fibers are structurally and biochemically related to eukaryotic amyloid fibers that are involved in several mammalian ailments including Alzheimer's disease, systemic amyloidosis, and spongiform encephalopathies such as mad cow disease and Creutzfeldt-Jacob disease. Unlike mammalian amyloid fibers that appear to be formed by aberrant pathways of protein folding, curli assembly in bacteria involves a dedicated multistep pathway that requires the csgBA and csgDEFG operons. Polymerization of the major curli subunit protein CsgA is dependent on the CsgB nucleator, and transport of CsgA and CsgB to the cell surface is mediated by the assembly factors CsgE, CsgF and CsgG. Specific Aim 1 will determine the mechanism of the nucleation and polymerization reactions and test the hypothesis that CsgB adopts an amyloid-like structure that stimulates CsgA polymerization. The structural and tinctoral properties of purified CsgB will be determined by circular dichroism (CD) spectroscopy and by Congo red (CR) and thioflavin T (thT) binding assays. The importance of the conserved Asn and Gln residues in CsgB and CsgA will be ascertained using site-directed mutagenesis. The nucleating activity of CsgB mutants will be scored in vivo and using a recently developed in vitro CsgA polymerization assay. Specific Aim 2 will elucidate the molecular details of curli biogenesis with special emphasis on the mechanism of subunit secretion. Dr. Chapman will test the hypothesis that the outer membrane lipoprotein CsgG forms a curli specific pore that is responsible fro the secretion of curli subunits and that GsgG function is dependant on CsgE. CsgG will be purified and characterized by EM, CD, and Blue native gel electrophoresis, and its pore-forming ability will be assessed by antibiotic sensitivity assays in vivo and liposome swelling assays in vitro. Dr. Chapman will attempt to identify sequences on CsgA and CsgB that mediate their CsgG-dependent secretion and to demonstrate interactions of CsgE with CsgG by using co-purification and cell-fractionation methods. Specific Aim 3 will define the role of curli in stimulating the host inflammatory response. The ability of purified CsgA (both soluble and polymerized) to induce inflammatory cytokines from human macrophages in vitro will be assessed. CsgA mutants that cannot polymerize will be used to test the hypothesis that polymerization is required for stimulating cytokine production. Mice will also be challenged IP with purified curli or CsgA, and NO production, cytokine production, and elevation of serum creatinine and conjugated bilirubin will be measured to test the hypothesis that curli are a bacterial pathogen-associated microbial pattern (PAMP) that directly stimulated the ill-regulated innate inflammatory response that characterizes septic shock.