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

基本信息

批准号：
10083168
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
项目类别：
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/10083168
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

项目摘要

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/摘要炎性症警告哺乳动物免疫系统的存在和组织损伤。这些胞质蛋白复合物检测危险信号或微生物产物，由多种细胞内释放病原体。在细菌病原体的情况下，确认了许多核特征大多数革兰氏阴性物种，脂多糖（LPS）的主要细胞壁一致。在内部检测LPS 宿主细胞激活了一种“非经典”炎性途径，其中caspase-11（人类的caspase 4和5）充当上游传感器，以刺激炎性体复合物组装和孔形成的加工蛋白质，加油D（GSDMD），进一步下游。 GSDMD孔释放受保护的细胞因子并贡献细胞死亡的裂解形式称为凋亡，可能有助于消除受感染的宿主细胞。这些顺序如何事件是协调的，涉及的主机因素仍然是先天免疫领域的主要问题和主持人防御。在这里，我们专注于新的65-73KDA免疫群酶家族的成员，称为鸟苷酸 - 结合蛋白（Gbps）控制非经典途径中的不同步骤。初步结果表明 GBP2可能靶向胞质细菌，以帮助释放LPS进行CASPASE-11检测，而GBP3进一步起作用下游以调节GSDMD贩运到质膜。 Gbps因此提供了独特的机会了解该顺序层次结构如何展开。在AIM 1中，我们将测试GBP2和 gbp3通过非典型的人性病感染革兰氏阴性沙门氏菌伤寒沙门氏菌（STM）体外和体内炎症。 CRISPR-CAS9删除了人和小鼠细胞以及新创建的 GBP2 - / - ，GBP3 - / - 和GBPDCHR.3H1小鼠将被旨在干扰GBP募集的STM变体感染或对LP的响应能力。此后，我们将剖析这些Gbps入伍的分子机制赋予其细胞内功能作为目标2的一部分。在这里，基因缺陷巨噬细胞用GBP完成具有不同生化病变的突变体将揭示Gbps如何将炎性体核心机械引导到LPS- 阳性细菌或对照下游事件，例如GSDMD运输和等离子体上的组装膜。无细胞的研究还将尝试重建细菌外的GBP“涂层器” 膜，可作为炎症组件的平台。我们的提案考试总的来说是一套新的作为独特的一部分，在非规范信号级联反应内在不同阶段起作用的宿主因素功能层次结构，帮助编排这些事件，对败血症的治疗产生了重大影响革兰氏阴性细菌感染。