The interface between L. pneumophila manipulation of host endoplasmic reticulum and innate immune subterfuge

嗜肺军团菌操纵宿主内质网与先天免疫诡计之间的界面

• 批准号: 10554261
• 负责人: Ralph R. Isberg
• 金额: $ 66.14万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-20 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10554261
• 关键词: Adaptor Signaling Protein; Aerosols; Amoeba genus; Autophagocytosis; Bacteria; Bacterial Proteins; Binding; Cell-Free System; Cells; Chemistry; Collaborations; Cytoplasm; Detection; Development; Disease; Docking; Electron Microscopy; Endoplasmic Reticulum; Engineering; Eukaryotic Initiation Factors; Event; Family; Future; Goals; Growth; Immune; Immune response; Immune signaling; Immunologic Surveillance; Infection; Label; Legionella; Legionella pneumophila; Legionnaires' Disease; Life Style; Link; Locales; Macrophage; Masks; Mediating; Membrane; Messenger RNA; Microbe; Modeling; Modification; Molecular; Morphology; Organelles; Organism; Pathway interactions; Peptide Initiation Factors; Peripheral; Play; Process; Protein Family; Protein Synthesis Inhibition; Protein translocation; Proteins; Proteomics; Reaction; Regulation; Resistance; Ribosomes; Role; Rough endoplasmic reticulum; Site; Solid; Source; Speed; Structure; System; Testing; Transcript; Transfection; Translation Initiation; Translations; Tubular formation; Ubiquitin; Ubiquitination; Vacuole; Viral; Virulence; Work; Xenopus laevis; antimicrobial; arm; aspirate; experimental study; helicase; microbial; microorganism; mutant; pathogen; pathogenic bacteria; phosphoric diester hydrolase; prevent; prion-like; response; ribosome profiling; trafficking; transcriptome sequencing; translation factor; ubiquitin isopeptidase

Many pathogens grow within host cells by building membrane-bound replication compartments essential for growth. The compartments provide important protection from host innate immune cytoplasmic surveillance systems and interfere with trafficking organisms into degradative compartments. Growth in the compartment is driven by microbial proteins which promote compartment construction, protection against innate immune surveillance and provide regulatory control over the host cell. How these different arms of the pathogen are coordinated is poorly understood. A bacterium that uses this strategy is Legionella pneumophila, which grows in a vacuole within macrophages during pneumonic disease. A group of over 300 Legionella proteins are translocated into host cells, controlling all aspects of the intracellular lifestyle, including formation of the replication compartment, blocking host cell translation, and preventing cytosolic recognition of the replication compartment. The organism hijacks host tubular endoplasmic reticulum (ER) as one of the earliest steps in replication compartment formation, an event promote by the bacterial Sde proteins. The proposed studies will test two models for Sde function. First, it will test the model that the Sde proteins controls ER tubule formation, replication compartment formation, and immune avoidance by promoting a three-step pathway. Secondly, it will test the model that manipulation of host translation initiation plays a role in coordinating replication vacuole construction and immune avoidance. The first model proposes that the Sde deubiquitinase activity liberates free ubiquitin (Ub) from the polyUb on the replication compartment. This would serve as a pool for ADPribosyltransferase and phosphodiesterase domains to promote Ub modification of the tubular ER protein Rtn4 and host translation initiation factors (eIFs). In the final step, the protein family masks the replication compartment with a phosphoribose to prevent recognition by the host autophagy pathway. The model will be tested by manipulating polyUb pools in the cell, performing electron microscopy on mutants deranged in this pathway, and reconstructing tubular ER rearrangements in a cell- free system. To determine the role that targeting of eIF proteins plays in these processes, the targets will be verified using purified components and the regulated initiation step will be identified. In addition, it will be determined if eIF proteins are inactivated to remove ribosomes from rough ER and allow close docking of ER sheets to the replication compartment, and if eIF manipulation skews the host translational response to favor bacterial replication. Extensive preliminary studies support the proposed experiments, providing a solid underpinning for this work. Throughout these studies, the objective is to identify a weak link in the microbial strategy of manipulating ER tubules to promote intracellular growth, with an eye toward developing antimicrobials that target this process.

许多病原体通过构建膜结合的复制区室在宿主细胞内生长，这对复制至关重要。 生长。这些隔室提供了重要的保护，免受宿主先天免疫细胞质监视 系统并干扰将生物体贩运到降解区室中。车厢内的生长 由微生物蛋白质驱动，促进隔室构建，防止先天免疫 监视并提供对宿主细胞的调节控制。病原体的这些不同分支有何不同 协调性尚不清楚。使用这种策略的细菌是嗜肺军团菌，它 在肺炎疾病期间，它在巨噬细胞内的液泡中生长。一组超过 300 种军团菌蛋白 被转移到宿主细胞中，控制细胞内生活方式的各个方面，包括形成 复制区室，阻断宿主细胞翻译，并防止细胞溶质识别 复制室。该生物体劫持宿主管状内质网（ER）作为其中之一 复制区室形成的最早步骤，这是由细菌 Sde 蛋白促进的事件。 拟议的研究将测试 Sde 功能的两个模型。首先，它将测试 Sde 蛋白的模型 通过促进内质网小管形成、复制室形成和免疫回避来控制 三步路径。其次，它将测试主翻译启动操作发挥作用的模型 在协调复制液泡构建和免疫回避中发挥作用。第一个模型提出 Sde 去泛素酶活性从复制区室的多聚泛素 (Ub) 中释放出游离泛素 (Ub)。 这将作为 ADPribosyltransferase 和磷酸二酯酶结构域的池来促进 Ub 肾小管 ER 蛋白 Rtn4 和宿主翻译起始因子 (eIF) 的修饰。在最后一步中， 蛋白质家族用磷酸核糖掩盖复制区室，以防止被宿主识别 自噬途径。该模型将通过操纵细胞中的多泛素池、执行电子 显微镜观察该途径中紊乱的突变体，并重建细胞中的管状 ER 重排 免费系统。为了确定 eIF 蛋白靶向在这些过程中所起的作用，目标将是 使用纯化的组分进行验证，并且将确定受监管的引发步骤。此外，还将 确定 eIF 蛋白是否失活以从粗糙 ER 中去除核糖体并允许紧密对接 ER 片到复制室，并且如果 eIF 操作使宿主翻译反应偏向 有利于细菌复制。广泛的初步研究支持所提出的实验，提供了 为这项工作奠定了坚实的基础。通过这些研究，目标是找出其中的薄弱环节 操纵内质网小管促进细胞内生长的微生物策略，着眼于 开发针对这一过程的抗菌药物。