GBPs as new inflammasome regulators during mammalian host defense

GBP 作为哺乳动物宿主防御过程中新的炎症小体调节剂

• 批准号: 10307127
• 负责人: John David MacMicking
• 金额: $ 41.88万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-19 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10307127
• 关键词: Acute; Bacteria; Bacterial Infections; Binding Proteins; Biochemical; Blood; CRISPR/Cas technology; Caspase; Cell Death; Cell Wall; Cell membrane; Cells; Chromosomes; Coatomer Protein; Complement; Complex; Dependence; Detection; Engineering; Event; Family; Family member; Gastroenteritis; Gene Cluster; Genes; Genome engineering; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Grant; Guanosine Triphosphate Phosphohydrolases; Host Defense; Human; Immune; Immune response; Immune system; Immunity; In Vitro; Individual; Infection; Inflammasome; Integration Host Factors; Intensive Care Units; Knockout Mice; Lesion; Life; Lipopolysaccharides; Lytic; Macromolecular Complexes; Membrane; Modeling; Molecular; Mus; Natural Immunity; Outcome; Output; Pathogenesis; Pathway interactions; Population; Process; Property; Proteins; Role; Salmonella; Salmonella typhimurium; Sensory; Sepsis; Signal Transduction; Surface; Testing; Therapeutic; Tissues; VDAC1 gene; Variant; cytokine; design; experimental study; foodborne; gain of function; gram-negative sepsis; guanylate; human disease; in vivo; insight; loss of function; macrophage; member; microbial; mutant; pathogen; pathogenic bacteria; pathogenic microbe; prevent; protein complex; reconstitution; recruit; response; sensor; trafficking

PROJECT SUMMARY/ABSTRACT Inflammasomes alert the mammalian immune system to the presence of infection and tissue damage. These cytosolic protein complexes detect danger signals or microbial products released by a wide variety of intracellular pathogens. In the case of bacterial pathogens, a number of prokaryotic signatures are recognized including the major cell-wall constituent of most Gram-negative species, lipopolysaccharide (LPS). Detection of LPS inside host cells activates a “non-canonical“ inflammasome pathway where caspase-11 (Caspases 4 and 5 in humans) act as upstream sensors to stimulate inflammasome complex assembly and processing of the pore-forming protein, Gasdermin D (Gsdmd), further downstream. Gsdmd pores release protective cytokines and contribute to a lytic form of cell death termed pyroptosis that may help eliminated infected host cells. How these sequential events are co-ordinated and the host factors involved remains a major question in the field of innate immunity and host defense. Here, we focus on members of a new 65-73kDa immune GTPase family termed Guanylate- Binding Proteins (GBPs) that control distinct steps in the non-canonical pathway. Preliminary results suggest Gbp2 may target cytosolic bacteria to help liberate LPS for caspase-11 detection whereas Gbp3 acts further downstream to regulate Gsdmd trafficking to the plasma membrane. GBPs thus offer a unique opportunity to understand how this sequential hierarchy unfolds. In Aim 1, we will test the respective contributions of Gbp2 and Gbp3 to immunity against Gram-negative Salmonella typhimurium (Stm) infection via the non-canonical inflammasome in vitro and in vivo. CRISPR-Cas9 deleted human and mouse cells as well as newly-created Gbp2-/-, Gbp3-/- and GbpDchr.3H1 mice will be infected with Stm variants designed to interfere with GBP recruitment or responsiveness to LPS. Thereafter, we will dissect the molecular mechanisms enlisted by these GBPs to confer their intracellular functions as part of Aim 2. Here gene-deficient macrophages complemented with GBP mutants with distinct biochemical lesions will reveal how GBPs direct the inflammasome core machinery to LPS- positive bacteria or control downstream events such as Gsdmd trafficking and assembly on the plasma membrane. Cell-free studies will also attempt to reconstitute the GBP “coatomer” on the bacterial outer membrane that serves as a platform for inflammasome assembly. Collectively, our proposal examines a new set of host factors that act at different stages within the non-canonical signaling cascade as part of a unique functional hierarchy, helping choreograph these events with major implications for the treatment of sepsis and Gram-negative bacterial infections.

项目摘要/摘要 炎症体提醒哺乳动物的免疫系统注意感染和组织损伤的存在。这些 胞浆蛋白复合体检测由多种细胞内释放的危险信号或微生物产物 病原体。在细菌病原体的情况下，许多原核生物特征被识别，包括 大多数革兰氏阴性菌的主要细胞壁成分是脂多糖。室内细菌内毒素的检测 宿主细胞激活caspase-11(人类的caspase 4和5)的“非规范”炎症体途径 作为上游感受器，刺激炎性小体复合体的组装和成孔加工 蛋白质，Gasdermin D(Gsdmd)，进一步下游。Gsdmd毛孔释放保护性细胞因子并有助于 一种称为上睑下垂的溶解形式的细胞死亡，它可能有助于清除受感染的宿主细胞。这些顺序是如何 事件是协调的，所涉及的宿主因素仍然是先天免疫领域的一个主要问题 和东道主防守。在这里，我们关注一个新的65-73 kDa免疫GTP酶家族的成员，该家族被称为Guanylate- 控制非规范途径中不同步骤的结合蛋白(GBP)。初步结果表明 GBP2可能以胞浆细菌为靶标，帮助释放内毒素，用于caspase-11检测，而GBP3作用更远 下游调节Gsdmd向质膜的运输。因此，Gbps提供了一个独特的机会 了解这种按顺序排列的层次结构是如何展开的。在目标1中，我们将测试GBP2和GBP2各自的贡献 GBP3对革兰氏阴性鼠伤寒沙门氏菌(STM)感染的非规范免疫作用 体外和体内炎症小体。CRISPR-Cas9删除的人和小鼠细胞以及新创建的 GBP2-/-、GBP3-/-和GbpDchr.3H1小鼠将感染旨在干扰GBP募集的STM变体 或对内毒素的反应性。之后，我们将剖析这些GBP征募的分子机制，以 将它们的细胞内功能作为目标2的一部分赋予它们。 具有不同生化损伤的突变体将揭示GBPS如何将炎症体核心机制定向到内毒素- 或控制下游事件，如Gsdmd在血浆上的运输和组装 薄膜。无细胞研究还将尝试在细菌的外层重建GBP的“辅助器” 作为炎症小体组装平台的膜。总而言之，我们的提案审查了一套新的 在非规范信号级联中的不同阶段起作用的宿主因子 功能层次，帮助编排这些事件，对脓毒症的治疗和 革兰氏阴性细菌感染。