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.

项目概要/摘要炎症小体提醒哺乳动物免疫系统存在感染和组织损伤。胞质蛋白复合物检测各种细胞内释放的危险信号或微生物产物就细菌病原体而言，可识别许多原核特征，包括大多数革兰氏阴性菌的主要细胞壁成分，脂多糖 (LPS) 检测内部的 LPS。宿主细胞激活“非典型”炎症小体途径，其中 caspase-11（人类的 Caspases 4 和 5）作为上游传感器刺激炎性复合物的组装和成孔加工蛋白质 Gasdermin D (Gsdmd)，Gsdmd 孔进一步释放保护性细胞因子并做出贡献。一种称为细胞焦亡的裂解形式的细胞死亡可能有助于消除这些连续的感染宿主细胞。事件的协调以及所涉及的宿主因素仍然是先天免疫领域的一个主要问题在这里，我们重点关注新的 65-73kDa 免疫 GTP 酶家族的成员，称为鸟苷酸- 初步结果表明，控制非规范途径中不同步骤的结合蛋白（GBP）。 Gbp2 可能以胞质细菌为目标，帮助释放 LPS 用于 caspase-11 检测，而 Gbp3 的作用更进一步因此，下游调节 Gsdmd 向质膜的运输提供了独特的机会。了解这个顺序层次结构如何展开在目标 1 中，我们将测试 Gbp2 和 Gbp2 各自的贡献。 Gbp3 通过非典型机制对革兰氏阴性鼠伤寒沙门氏菌 (Stm) 感染产生免疫力 CRISPR-Cas9 删除了人类和小鼠细胞以及新创建的炎症小体。 Gbp2-/-、Gbp3-/- 和 GbpDchr.3H1 小鼠将感染旨在干扰 GBP 募集的 Stm 变体此后，我们将剖析这些 GBP 所涉及的分子机制。作为目标 2 的一部分，赋予其细胞内功能。此处基因缺陷型巨噬细胞与 GBP 互补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引导至LPS- 阳性细菌或控制下游事件，例如血浆上的 Gsdmd 运输和组装无细胞研究还将尝试在细菌外层重建 GBP“涂层”。总的来说，我们的提案研究了一套新的膜作为炎性体组装的平台。作为独特的信号级联的一部分，在非规范信号级联中的不同阶段发挥作用的宿主因子功能层次结构，帮助编排这些对败血症和脓毒症治疗具有重大影响的事件革兰氏阴性细菌感染。