Control of virulence protein export in Yersinia pestis

鼠疫耶尔森氏菌毒力蛋白输出的控制

• 批准号: 6711577
• 负责人: Gregory V Plano
• 金额: $ 2.6万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-06-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6711577
• 关键词: Yersinia pestis; bacteria infection mechanism; bacterial proteins; biological signal transduction; host organism interaction; immunoprecipitation; intermolecular interaction; membrane permeability; protein binding; protein structure function; protein transport; secretion; secretory protein; virulence; yeast two hybrid system

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.

性状（由申请方提供）：鼠疫耶尔森菌，病原体 鼠疫使用III型分泌系统（TTSS）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不存在Ca 2+的条件下生长。 鼠疫耶尔森鼠疫菌YopN、TyeA、SycN和YscB蛋白是预防Yop 在Ca 2+存在下并在与真核细胞接触之前分泌。 以前的研究已经确定SycN和YscB蛋白的功能是 分泌的YopN蛋白的特异性伴侣。SycN和YscB的复合物， 而不是单独的SycN或YscB直接结合YopN的N-末端结构域。 另一方面，TyeA结合YopN的C末端结构域。这个项目 将分析YopN-TyeA-SycN-YscB复合物在调节 Yop分泌物。毒力蛋白分泌的调控具有广泛的 由于认识到许多细菌病原体利用 例如“细胞接触依赖性”递送系统。该项目的目标是： (i)定义SycN/YscB伴侣蛋白复合物在YopN中的功能 分泌、YopN易位和调节Yop分泌;（ii） 确定YopN-TyeA-SycN-YscB复合物阻断的机制 分泌;和（iii）鉴定信号、传感器和信号转导 导致去除YopN-TyeA-SycN-YscB依赖性阻滞的途径 Yop分泌物。该项目的目标将通过以下方式实现： 不能阻断Yop的鼠疫菌特异性突变株的筛选和分析 分泌，组成性阻断Yop分泌，或效应特异性 YopN-TyeA-SycN-YscB复合物内的蛋白质-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 用于鉴定和表征新的蛋白质-蛋白质相互作用。 将采用生物化学和生物物理分析来确定 特定蛋白质的化学计量、构象和结合常数，或 蛋白质复合物解脂耶鼠疫染色体转座体插入文库将 筛选以鉴定Yop缺陷的特定插入突变体， 分泌或调节Yop分泌。这些研究将提供 深入了解YopNTyeA-SycN-YscB复合物在调节 Y.鼠疫