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: 10535452
• 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/10535452
• 关键词: Acute; Bacteria; Bacterial Infections; Binding Proteins; Biochemical; CRISPR/Cas technology; Capsid Proteins; 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; Macrophage; Membrane; Modeling; Molecular; Mus; Natural Immunity; Outcome; Output; Pathogenesis; Pathway interactions; Population; Process; Property; Protein Deficiency; 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; member; microbial products; 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.

项目总结/摘要 炎性小体提醒哺乳动物免疫系统感染和组织损伤的存在。这些 胞质蛋白复合物检测由多种细胞内蛋白质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，识别出许多原核签名，包括 大多数革兰氏阴性菌的主要细胞壁成分脂多糖（LPS）。内毒素检测 宿主细胞激活“非经典”炎性体途径，其中半胱天冬酶-11（人类中的半胱天冬酶4和5） 作为上游传感器刺激炎性小体复合物的组装和孔形成的加工 蛋白质，Gasdermin D（Gsdmd），更下游。GSDMD孔释放保护性细胞因子， 细胞死亡的裂解形式称为pyroptosis，这可能有助于消除受感染的宿主细胞。这些连续的 事件是协调的，涉及的宿主因素仍然是先天免疫领域的主要问题 宿主防御在这里，我们专注于一个新的65- 73 kDa的免疫GTdR家族的成员，称为鸟苷酸- 控制非经典途径中不同步骤的结合蛋白（GBP）。初步结果表明 Gbp 2可以靶向胞质细菌，以帮助释放LPS用于caspase-11检测，而Gbp 3进一步起作用 下游调节Gsdmd运输到质膜。因此，GBP提供了一个独特的机会， 理解这个顺序层次结构是如何展开的。在目标1中，我们将测试Gbp 2和 Gbp 3通过非经典途径对革兰氏阴性鼠伤寒沙门氏菌（Stm）感染的免疫作用 炎性小体的体外和体内研究。CRISPR-Cas9缺失的人类和小鼠细胞以及新创建的 Gbp 2-/-、Gbp 3-/-和GbpDchr.3H1小鼠将感染设计用于干扰GBP募集的Stm变体 或对LPS的反应性。此后，我们将剖析这些GBP的分子机制， 赋予其作为目标2的一部分的细胞内功能。在这里，基因缺陷的巨噬细胞补充GBP 具有不同生化损伤的突变体将揭示GBP如何将炎性小体核心机制导向LPS- 阳性细菌或控制下游事件，如血浆上的Gsdmd运输和组装 膜的无细胞研究也将试图在细菌的外层重新构建GBP的“被膜体”。 膜作为炎症小体组装的平台。总的来说，我们的建议审查了一套新的 宿主因子在非经典信号级联中的不同阶段起作用， 功能层次，帮助编排这些事件，对脓毒症的治疗有重要意义， 革兰氏阴性细菌感染。