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

• 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
• 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' 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

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.

总结 IV型分泌系统（TFSS）存在于许多不同种类的细菌中， 在水平基因转移和几种病原体的毒力特性中的作用。当细菌 当TFSS感染宿主细胞时，TFSS将被称为“效应子”的蛋白质转运到靶细胞。效应蛋白可以发挥 宿主上各种各样的细胞生物学和生物化学变化，这些变化有利于感染 病原体尽管人们已经付出了相当大的努力来了解许多TFSS的功能 效应器，少得多的注意力一直针对TFSS的详细研究。详细的基因分析 然而，过渡时期社会保障制度并没有以任何系统的方式实施。基因分析有可能提供 这些重要的功能信息不仅仅来自结构研究。提供的资料 遗传分析将有助于更好地了解TFSS在几个方面。的识别 TFSS组件中的许可和非许可位点将促进生物化学工具的最终使用 可以定制以提供关于蛋白质-蛋白质相互作用的信息， 功能性探针。鉴定具有条件或部分表型的突变等位基因也将提供 有关TFSS组件功能角色的重要信息。我们将进行系统的、详细的 Dot/Icm TFSS组分DotL、DotH< 嗜肺军团菌的DotG和DotE。该TFSS是L.嗜肺 并且能够将效应子易位至宿主细胞和细菌接合。我们将使用基于Mu 将15 bp插入（ent）引入dot/icm基因的Entranceposon™方法。我们会筛选变种人 对卡氏阿米巴（Alamoebacastellanii）的放牧生存能力，一个典型的L.嗜肺宿主。野生型L 嗜肺菌生长并杀死A.但是dot/icm军团菌突变体被castellanii消化并杀死， 变形虫一旦在dot/icm基因中确定了允许位点， TEV蛋白酶的位点。该蛋白酶特异性识别在细胞中不存在的七聚体序列。 军团菌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。我们还将与一个可以可视化TFSS的实验室合作， 冷冻电磁断层扫描将通过Cryo-EM断层扫描检查TEV切割的TFSS复合物，以鉴定 复杂的特定组件的位置。这种方法将使我们能够确定， 在移位过程中或如果功能性TFSS的组装需要Dot/Icm组件， 但它的活性却很高。