Biogenesis and Function of Bacterial Amyloid fibers

描述（由申请人提供）：无。（改编自应用）：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。与哺乳动物的淀粉样纤维，似乎是由蛋白质折叠的异常途径形成，卷曲组装在细菌中涉及一个专门的多步途径，需要csgBA和csgDEFG操纵子。主要卷曲亚基蛋白CsgA的聚合依赖于CsgB成核剂，并且CsgA和CsgB向细胞表面的转运由组装因子CsgE、CsgF和CsgG介导。具体目标1将确定成核和聚合反应的机制，并测试CsgB采用刺激CsgA聚合的淀粉样结构的假设。将通过圆二色性（CD）光谱和通过刚果红（CR）和硫磺素T（thT）结合测定来确定纯化的CsgB的结构和着色性质。CsgB和CsgA中保守的Asn和Gln残基的重要性将使用定点诱变来确定。CsgB突变体的成核活性将在体内和使用最近开发的体外CsgA聚合测定进行评分。具体目标2将阐明与特别强调亚基分泌机制curli生物发生的分子细节。Chapman博士将检验以下假设：外膜脂蛋白CsgG形成一个curli特异性孔，负责分泌curli亚基，并且GsgG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溶胀试验评估其成孔能力。Chapman博士将尝试鉴定CsgA和CsgB上介导其CsgG依赖性分泌的序列，并通过使用共纯化和细胞分级分离方法证明CsgE与CsgG的相互作用。具体目标3将定义curli在刺激宿主炎症反应中的作用。将评估纯化的CsgA（可溶的和聚合的）在体外诱导来自人巨噬细胞的炎性细胞因子的能力。不能聚合的CsgA突变体将用于测试聚合是刺激细胞因子产生所需的假设。还将用纯化的curli或CsgA IP攻击小鼠，并且将测量NO产生、细胞因子产生以及血清肌酸酐和结合胆红素的升高，以检验curli是细菌病原体相关微生物模式（PAMP）的假设，其直接刺激以败血性休克为特征的调节不良的先天性炎症反应。

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.