CONTROL OF VIRULENCE PROTEIN EXPORT IN YERSINIA PESTIS

描述(改编自申请者的摘要)：这个项目将 分析鼠疫耶尔森氏菌LcrE蛋白的功能。LcrE是 涉及到一种独特的分泌机制的调节 毒力蛋白，耶尔森氏菌的YOP蛋白。青年队缺少 可识别的信号序列，分泌时不含蛋白水解物 正在处理。对YOPs体外表达和分泌的调节作用 钙。真核细胞触发至少一个移位 YOP直接从表面附着细菌进入宿主细胞。 这似乎是一种引起广泛兴趣的机制，因为它似乎是 在其他病原体中也存在。此应用程序建议 确定将LcrE定位到YOP所需的LcrE功能域 分泌装置，识别调节YOP所需的结构域 分泌，并确定与LcrE相互作用的蛋白质。这将是 通过LcrE的缺失分析，第二个- 位点突变以确定与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 用于确认这些交互作用。最后，LcrE的效果 YopE转位到真核细胞的突变体将是 下定决心。这些实验旨在绘制区域和残基的地图。 并揭示了LcrE是如何发挥作用的。

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.