Site-specific Proteolysis of the Legionella Type IV Secretion System

军团菌 IV 型分泌系统的位点特异性蛋白水解

基本信息

批准号：
9510237
负责人：
HOWARD A SHUMAN
金额：
$ 24.3万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-01-12 至 2019-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9510237
关键词：
Acanthamoeba castellanii Alleles Amoeba genus Attention Bacteria Bacteriophages Biochemical Biological Caliber Cells Cleaved cell Complex Coupling Cytosol Detergents Engineering Exposure to Face Genes Genetic Genetic Conjugation Horizontal Gene Transfer Image Individual Legionella Legionella pneumophila Legionnaires&apos Disease Location Maps Measures Membrane Proteins Methods Molecular Conformation Monitor Mutation Analysis Nucleic Acids Nucleoproteins Peptide Hydrolases Peptide Signal Sequences Phenotype Physiologic pulse Play Predisposition Property Proteins Proteolysis Research Design Resolution Role Site Structure System TEV protease Temperature Testing Time Type IV Secretion System Pathway Virulence base biochemical tools cell envelope cell type density genetic analysis in vivo insight macrophage mutant nanocomplexes nanomachine new technology novel pathogen periplasm protein complex protein protein interaction three dimensional structure tomography wireless fidelity

项目摘要

SUMMARY Type IV secretion systems (TFSS) are present in many different species of bacteria and play important roles both in horizontal gene transfer and in the virulence properties of several pathogens. When bacteria infect host cells, TFSS translocate proteins called “effectors” to the target cell. The effector proteins can exert a wide variety of cell biological and biochemical changes on the host that are beneficial for the infecting pathogen. Although considerable effort has been directed at understanding the functions of many TFSS effectors, much less attention has been directed at detailed studies of the TFSS. Detailed genetic analysis of TFSS however has not been carried out in any systematic way. Genetic analysis has the potential to provide important functional information that will not come from structural studies alone. The information provided by genetic analysis will contribute to a better understanding of TFSS in several ways. The identification of permissive and non-permissive sites in TFSS components will facilitate the eventual use of biochemical tools that can be tailored to provide information about protein-protein interactions and accessibility to different types of functional probes. Identification of mutant alleles with conditional or partial phenotypes will also provide important information about the functional roles of TFSS components. We will carry out a systematic, detailed mutational analysis of permissive and non-permissive sites in the Dot/Icm TFSS components DotL, DotH< DotG, and DotE of Legionella pneumophila. This TFSS is the major virulence determinant of L. pneumophila and is capable of both effector translocation to host cells and bacterial conjugation. We will use the Mu-based Entranceposon™ method to introduce 15 bp insertions (ent) into the dot/icm genes. We will screen the mutants for the ability to survive grazing by Acanthamoeba castellanii, a typical L. pneumophila host. Wild-type L. pneumophila grows within and kills A. castellanii but dot/icm Legionella mutants are digested and killed by the amoebae. Once permissive sites have been determined in the dot/icm genes, we will introduce recognition sites for TEV protease. This protease specifically recognizes a heptamer sequence that is otherwise absent in Legionella. We will study the effects of cleaving the Dot/Icm proteins that contain the TEV recognition site (TevS) in vivo under different conditions. We will also collaborate with a lab that can visualize the TFSS with Cryo-EM tomography. TEV-cleaved TFSS complexes will be examined by Cryo-EM tomography to identify the locations of specific components within the complex. This approach will allow us to determine if individual Dot/Icm components are required during translocation or if they are required for assembly of a functional TFSS but dispensable for its activity.

概括 IV 型分泌系统 (TFSS) 存在于许多不同种类的细菌中，并且发挥着重要作用在细菌的水平基因转移和毒力特性中发挥作用。感染宿主细胞后，TFSS 将称为“效应器”的蛋白质转移到靶细胞。宿主发生多种有利于感染的细胞生物学和生化变化尽管人们付出了相当大的努力来了解许多 TFSS 的功能。对于 TFSS 的详细遗传分析的关注较少。然而，TFSS 尚未以任何系统的方式进行，遗传分析有潜力提供这一点。重要的功能信息不仅仅来自结构研究提供的信息。遗传分析将有助于通过多种方式更好地理解 TFSS 的识别。 TFSS 组件中的许可和非许可位点将促进生化工具的最终使用可以定制以提供有关蛋白质-蛋白质相互作用和不同类型的可及性的信息具有条件或部分表型的突变等位基因的鉴定也将提供。关于TFSS组件的功能作用的重要信息我们将进行系统、详细的介绍。 Dot/Icm TFSS 组件 DotL、DotH< 中允许和非允许位点的突变分析嗜肺军团菌的 DotG 和 DotE 该 TFSS 是嗜肺军团菌的主要毒力决定因素。并且能够将效应器易位至宿主细胞和细菌接合，我们将使用基于 Mu 的。 Entranceposon™ 方法将 15 bp 插入 (ent) 引入 dot/icm 基因中我们将筛选突变体。卡斯氏棘阿米巴 (Acanthamoebacastellanii)（一种典型的嗜肺军团菌野生型宿主）在放牧中生存的能力。 pneumophila 在 A.castellanii 内生长并杀死它，但 dot/icm Legionella 突变体会被 A.castellanii 消化并杀死。一旦在 dot/icm 基因中确定了允许位点，我们将引入识别。 TEV 蛋白酶位点该蛋白酶特异性识别七聚体序列，而该序列在其他情况下不存在。我们将研究切割含有 TEV 识别位点的 Dot/Icm 蛋白的效果。（TevS）在不同条件下的体内实验我们还将与一个可以使用 TFSS 进行可视化的实验室合作。冷冻电镜断层扫描将通过冷冻电镜断层扫描检查 TEV 切割的 TFSS 复合物，以识别这种方法将使我们能够确定复合体中特定组件的位置。在易位过程中或在组装功能性 TFSS 时需要使用 Dot/Icm 组件但对其活动来说是可有可无的。