Control of virulence protein export in Yersinia pestis

描述(申请人提供)：鼠疫耶尔森氏菌，病原体 鼠疫，使用III型分泌系统(TTSS)来转移毒力 蛋白质，称为YOPs，进入真核细胞。YOP导出由以下因素触发 在体内与宿主细胞接触，或在体外没有钙的情况下通过生长。 鼠疫杆菌YopN、TyeA、SycN和YscB蛋白是预防YOP所必需的 在有钙离子存在的情况下，在接触真核细胞之前的分泌。 先前的研究已经证实，SycN和YscB蛋白的功能是 分泌的YopN蛋白的特异性伴侣。SycN和YscB的复合体， 而不仅仅是SycN或YscB，它直接与YopN的N-末端结构域结合。 另一方面，TyeA与YopN的C-末端结构域结合。这个项目 将分析YopN-TyeA-SycN-YscB复合体在调控中的作用 YOP分泌物。毒力蛋白分泌调控的广泛性 兴趣是因为认识到许多细菌病原体利用 这种“细胞接触”的递送系统。该项目的目标是： (I)确定YopN中SycN/YscB伴侣复合体的功能 分泌、YopN易位和Yop分泌的调节； 确定YopN-TyeA-SycN-YscB复合体阻断的机制 分泌物；以及(Iii)识别信号、传感器和信号转导 导致YopN-TyeA-SycN-YscB依赖的阻断在 YOP分泌物。该项目的目标将通过 不能阻断Yop的鼠疫杆菌特异性突变体的筛选与分析 分泌，结构性阻断YOP分泌，或特定效果 YopN-TyeA-SycN-YscB复合体中的蛋白质相互作用。 免疫沉淀实验和酵母双杂交和三杂交分析将 用于识别和表征新的蛋白质-蛋白质相互作用。 将使用生化和生物物理分析来确定 特定蛋白质或蛋白质的化学计量、构象和结合常数 蛋白质复合体。鼠疫杆菌染色体转座体插入文库将 筛选出YOP中有缺陷的特定插入突变体 分泌或在调节YOP的分泌。这些研究将提供 YopNTyeA-SycN-YscB复合体在细胞周期调控中的作用 鼠疫耶尔森氏菌的YOP分泌物。

DESCRIPTION (provided by the applicant): Yersinia pestis, the etiologic agent of plague, uses a type III secretion system (TTSS) to translocate virulence proteins, termed Yops, into eukaryotic cells. Yop export is triggered by contact with a host cell in vivo or by growth in the absence of Ca2+ in vitro. The Y. pestis YopN, TyeA, SycN, and YscB proteins are required to prevent Yop secretion in the presence of Ca2+ and prior to contact with a eukaryotic cell. Previous studies have established that the SycN and YscB proteins function as specific chaperones for the secreted YopN protein. A complex of SycN and YscB, but not SycN or YscB alone, directly binds to an N-terminal domain of YopN. TyeA, on the other hand, binds to a C-terminal domain of YopN. This project will analyze the function of the YopN-TyeA-SycN-YscB complex in the regulation of Yop secretion. The control of virulence protein secretion is of broad interest because of the realization that numerous bacterial pathogens employ such "cell contact-dependent" delivery systems. The goals of this project are: (i) to define the function of the SycN/YscB chaperone complex in YopN secretion, YopN translocation and in the regulation of Yop secretion; (ii) to identify the mechanism by which the YopN-TyeA-SycN-YscB complex blocks secretion; and (iii) to identify the signal, sensors and signal transduction pathways that lead to the removal of the YopN-TyeA-SycN-YscB-dependent block in Yop secretion. The objectives of this project will be realized through the selection and analysis of specific Y pestis mutants that fail to block Yop secretion, constitutively block Yop secretion, or effect specific protein-protein interactions within the YopN-TyeA-SycN-YscB complex. Immunoprecipitation experiments and yeast two- and three-hybrid analyses will be used to identify and characterize novel protein-protein interactions. Biochemical and biophysical analyses will be employed to determine the stoichiometry, conformation and binding constants of specific proteins or protein complexes. A Y. pestis chromosomal transposome insertion library will be screened to identify specific insertion mutants that are defective in Yop secretion or in the regulation of Yop secretion. These studies will provide insight into the role of the YopNTyeA-SycN-YscB complex in the regulation of Yop secretion in Y. pestis.