Biogenesis and Function of Bacterial Amyloid fibers

描述(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胞外细胞器。查普曼博士已经证明，这些纤维在结构和生化上与真核细胞淀粉样蛋白纤维相关，而真核细胞淀粉样蛋白纤维与多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，细菌中的卷曲组装涉及一个专门的多步骤途径，需要csgBA和csgDEFG操纵子。主要卷曲亚基蛋白CsgA的聚合依赖于CsgB核因子，CsgA和CsgB的运输是由组装因子CsgE、CsgF和CsgG介导的。具体目标1将确定成核和聚合反应的机理，并检验CsgB采用淀粉样结构刺激CsgA聚合的假设。纯化的CsgB的结构和颜色性质将通过圆二色谱(CD)以及刚果红(CR)和硫代黄素T(THT)结合分析来确定。保守的Asn和Gln残基在CsgB和CsgA中的重要性将通过定点突变来确定。CsgB突变体的成核活性将在体内进行评分，并使用最近开发的体外CsgA聚合实验进行评分。具体目标2将阐明卷曲生物发生的分子细节，特别强调亚基分泌的机制。查普曼博士将检验这一假设，即外膜脂蛋白CsgG形成一个卷曲的特定孔，负责卷曲亚基的分泌，并且GsgG的功能依赖于CsgE。CsgG将通过EM、CD和Blue天然凝胶电泳法进行纯化和鉴定，其成孔能力将通过体内抗生素敏感性试验和体外脂质体肿胀试验进行评估。查普曼博士将尝试鉴定CsgA和CsgB上调节CsgG依赖分泌的序列，并通过共纯化和细胞分离的方法证明CsgE与CsgG的相互作用。具体目标3将确定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)在体外诱导人巨噬细胞产生炎性细胞因子的能力。不能聚合的CsgA突变体将被用来检验聚合是刺激细胞因子产生所必需的假设。还将用纯化的Curli或CsgA挑战小鼠的IP，并将测量NO产生、细胞因子产生以及血清肌酐和结合胆红素的升高，以检验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.