Protein Interactions in Type III Secretion in Y. pestis

鼠疫耶尔森氏菌 III 型分泌物中的蛋白质相互作用

• 批准号: 6845693
• 负责人: Matthew L. Nilles
• 金额: $ 24.54万
• 依托单位: UNIVERSITY OF NORTH DAKOTA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6845693
• 关键词: Yersinia pestis; bacterial proteins; circular dichroism; gel filtration chromatography; matrix assisted laser desorption ionization; plasmids; protein localization; protein protein interaction; protein structure function; secretion; site directed mutagenesis

DESCRIPTION (Provided by applicant): Yersinia pestis, the causative agent of Plague, harbors a -71 KB plasmid that encodes many virulence-related proteins. Among the pCDl-encoded genes are genes specifying for the construction of a type III secretion system and the genes for the secreted effector proteins (collectively termed Yops). Type III secretion systems allow pathogenic bacteria to specifically translocate effectors into eukaryotic cells for the pathogen's benefit. The Yops exert their effects on targeted eukaryotic cells that results in a blockage of phagocytosis by immune cells. This effect allows Y. pestis to remain extracellular during the course of an infection. In Y. pestis the activity of the type III system is controlled by environmental stimuli, in vitro secretion is stimulated by the removal of calcium ions. In vivo Yops translocation is triggered by contact with eukaryotic cells. Appropriate control of type III secretion is required for the disease process as mutants defective in control are avirulent. Control of type III secretion relies on both positive and negative control circuits. LcrG and LcrV are two proteins whose interaction is required to control type III secretion in Y. pestis. LcrV is required to activate secretion while LcrG is required to inactivate secretion. Their interaction provides a linkage between positive and negative regulation in type III secretion control. The mechanism responsible for their controlling effects are largely unknown, although progress has been made. Published studies have shown that the interaction of LcrG and LcrV is required to control secretion. Additionally, a separate study demonstrated that a ratio of LcrV to LcrG, favoring LcrV, is required to activate secretion. Clearly, more work is needed to define the roles of LcrG and LcrV in the activation of secretion. Specifically, in this proposal we shall: Aim 1. Define structure-function relationships within LcrG relating to LcrV interaction and secretion-blocking. Aim 2. Screen for other proteins that may interact with LcrG. Aim 3. Refine the subcellular localization of LcrG. Aim 4. Examine mechanisms that alter LcrG levels relative to LcrV. Type III secretion systems have emerged recently as a common theme in the pathogenic mechanism of many gram-negative bacterial pathogens. Work in the yersiniae has been at the forefront of research on the characterization and function of type III mechanisms. An understanding of how these systems are controlled should lead to a deeper understanding of the intimate and dynamic interactions between pathogens and their hosts.

描述（由申请人提供）：鼠疫耶尔森氏菌（鼠疫病原体）含有一个 -71 KB 质粒，可编码许多毒力相关蛋白。 pCD1编码的基因中有指定构建III型分泌系统的基因和分泌效应蛋白的基因(统称为Yops)。 III 型分泌系统允许病原菌特异性地将效应器转移到真核细胞中，以实现病原体的利益。 Yops 对目标真核细胞发挥作用，导致免疫细胞的吞噬作用受阻。这种效应使得鼠疫耶尔森氏菌在感染过程中保持在细胞外。在鼠疫耶尔森氏菌中，III型系统的活性受环境刺激控制，通过钙离子的去除刺激体外分泌。体内 Yops 易位是通过与真核细胞接触而触发的。 疾病过程需要适当控制 III 型分泌，因为控制缺陷的突变体是无毒的。 III 型分泌的控制依赖于正控制回路和负控制回路。 LcrG 和 LcrV 是两种蛋白质，它们的相互作用是控制鼠疫耶尔森氏菌 III 型分泌所必需的。 LcrV 需要激活分泌，而 LcrG 需要失活分泌。它们的相互作用提供了 III 型分泌控制中正调节和负调节之间的联系。尽管已经取得了进展，但其控制作用的机制在很大程度上仍不清楚。已发表的研究表明，LcrG 和 LcrV 的相互作用是控制分泌所必需的。此外，一项单独的研究表明，LcrV 与 LcrG 的比例有利于 LcrV，是激活分泌所必需的。显然，需要更多的工作来确定 LcrG 和 LcrV 在分泌激活中的作用。具体来说，在本提案中，我们将： 目标 1. 定义 LcrG 内与 LcrV 相互作用和分泌阻断相关的结构-功能关系。目标 2. 筛选可能与 LcrG 相互作用的其他蛋白质。目标 3. 完善 LcrG 的亚细胞定位。目标 4. 检查相对于 LcrV 改变 LcrG 水平的机制。 III型分泌系统最近已成为许多革兰氏阴性细菌病原体致病机制的共同主题。耶尔森菌的工作一直处于 III 型机制的特征和功能研究的前沿。了解这些系统是如何控制的应该有助于更深入地了解病原体与其宿主之间的密切和动态相互作用。