CONTROL OF VIRULENCE PROTEIN EXPORT IN YERSINIA PESTIS

描述（改编自申请人的摘要）：该项目将 分析鼠疫菌LcrE蛋白的功能。LcrE是 参与调节一种独特的分泌机制， 毒力蛋白，耶尔森氏菌的Yop蛋白。Yops缺乏 可识别的信号序列，并且在没有蛋白水解的情况下分泌 处理. Yops蛋白的体外表达和分泌 钙真核细胞触发了至少一个 Yop直接从细菌表面附着进入宿主细胞。 这似乎是一个广泛感兴趣的机制，因为它似乎是 也存在于其他病原体中。本申请建议 确定LcrE靶向Yop所需的LcrE功能结构域 分泌装置，鉴定调节Yop所需的结构域 分泌，并鉴定与LcrE相互作用的蛋白质。这将是 通过LcrE的缺失分析，第二个 位点突变以鉴定与LcrE相互作用的蛋白质。蛋白 亲和层析、亲和印迹和免疫沉淀将 用于确认这些相互作用。最后，LcrE的作用 将YopE易位到真核细胞中的突变体 测定这些实验旨在绘制区域和残基 LcrE的具体功能，并揭示LcrE的功能。

DESCRIPTION (Adapted from applicant's abstract): This project will analyze the function of the LcrE protein in Yersinia pestis. LcrE is implicated in the regulation of a unique secretion mechanism for virulence proteins, the Yop proteins of Yersinia. The Yops lack identifiable signal sequences and are secreted without proteolytic processing. Expression and secretion of Yops in vitro regulated by calcium. Eucaryotic cells triggers the translocation of at least one Yop directly from the surface attached bacterium into the host cell. This appears to be a mechanism of broad interest since it appears to be present in other pathogens as well. This application proposes to identify functional domains of LcrE required to target LcrE to the Yop secretion apparatus, identify domains required to regulate Yop secretion, and identify proteins that interact with LcrE. This will be accomplished by deletion analysis of LcrE, characterization of second- site mutations to identify proteins that interact with LcrE. Protein affinity chromatography, affinity blotting and immunoprecipitation will be used to confirm these interactions. Finally, the effect of LcrE mutants on the translocation of YopE into eucaryotic cells will be determined. These experiments are designed to map regions and residues of LcrE to specific functions and reveal how LcrE functions.