Human GBPs in cell-autonomous immunity to intracellular bacterial pathogens

摘要--作为细胞内细菌病原体免疫调节剂的人球蛋白 鸟苷结合蛋白(GBP)是一类寄主防御蛋白，在细胞周期中发挥着重要的作用。 对细胞内细菌病原体的细胞自主免疫。这些蛋白质是主要的调节因子。 基本的宿主防御模块，并参与炎症小体激活和 执行直接杀菌活动。然而，几乎所有这些工作都是在小鼠身上完成的 人GBP(HGBPs)的模型和抗菌功能仍未得到很好的描述。在我们的 最近的研究发现人类GBP的不同宿主防御功能，并将特定的人类 GBP是针对一类重要的细菌病原体的新型抗性机制。 特别是，我们发现人GBP1(HGBP1)支持两种新的宿主防御机制 胞浆革兰氏阴性杆菌：1)hGBP1蛋白与胞浆革兰氏阴性杆菌相关 宿主细胞胞浆中的阴性细菌，如福氏志贺氏菌或泰兰伯克霍尔德氏菌。 一旦与细菌结合，hGBP1直接干扰细菌基于肌动蛋白的细胞内运动 从而阻止细菌扩散；以及2)hGBP1介导趋化因子的产生 被感染的上皮细胞所致。因此，hGBP1的激活似乎是两个不同但 协同免疫功能：一种细胞固有防御程序，防止病毒在细胞间传播 病原体，而旁分泌免疫信号的产生促进激活和募集 免疫细胞到达感染部位。在这项工作中，我们将定义这些基础的分子机制 我们已经发现了hGBP1的两个重要角色。在Aim1中，我们将结合细胞培养和 用生化方法确定hGBP1特异性检测革兰氏阴性杆菌的机制 宿主细胞胞浆中的细菌，这是抑制基于肌动蛋白的运动性的关键过程。在AIM2中，我们将 描述hGBP1与细菌结合阻止基于肌动蛋白的运动的机制。在Aim3中，我们将 探索hGBP1作为免疫传感通路新的调节因子的第二个独立功能 利用细菌遗传学、宿主遗传学和细胞产生免疫调节性趋化因子 生物学方法。在所有这些研究中，我们将利用我们最近的发现 福氏志贺氏菌效应蛋白IpaH9.8拮抗hGBP1功能。因此，IpaH9.8的使用- 缺陷的福氏志贺氏菌株将使我们能够监测和功能解剖由hGBP1驱动的可操作的 主机响应。总体而言，这里提出的工作将使我们从根本上理解 HGBP1和其他人类GBP在细胞自主免疫中对细胞内细菌病原体的免疫。 好了！

SUMMARY - Human GBPs as regulators of immunity to intracellular bacterial pathogens Guanylate binding proteins (GBPs) are host defense proteins that play diverse and critical roles in cell-autonomous immunity to intracellular bacterial pathogens. These proteins are major regulators of fundamental host defense modules and involved in processes such as inflammasome activation and the execution of direct bactericidal activities. However, almost all of this work has been performed in mouse models and antimicrobial functions of human GBPs (hGBPs) are still poorly characterized. In our recent studies we identified divergent host defense functions of the human GBPs and linked specific human GBPs to novel resistance mechanisms that protect against an important class of bacterial pathogens. Specifically, we found that human GBP1 (hGBP1) underpins two novel host defense mechanisms against cytosolic Gram-negative bacterial pathogens: 1) hGBP1 protein associates with cytosolic Gram- negative bacteria such as Shigella flexneri or Burkholderia thailandensis in the host cell cytosol. Once bound to bacteria, hGBP1 directly interferes with the actin-based intracellular motility of bacteria and thereby blocks bacterial dissemination; and 2) hGBP1 mediates activation of chemokine production by infected epithelial cells. Thus, hGBP1 activation appears to serve as a central node for two distinct but synergistic immune functions: a cell-intrinsic defense program prevents cell-to-cell spread of the pathogen while the production of paracrine immune signals promotes the activation and recruitment of immune cells to the site of infection. In this work, we will define the molecular mechanisms underlying these two important roles for hGBP1 that we have uncovered. In Aim1 we will combine cell culture and biochemical approaches to define the mechanism by which hGBP1 specifically detects Gram-negative bacteria in the host cell cytosol, a process critical for the inhibition of actin-based motility. In Aim2 we will characterize the mechanism by which hGBP1 bound to bacteria blocks actin-based motility. In Aim3 we will explore a second, independent function of hGBP1 as a novel regulator of an immune sensing pathway leading to the production of immune-modulatory chemokines using bacterial genetics, host genetics and cell biological approaches. In all of these studies we will take advantage of our recent discovery that the S. flexneri effector protein IpaH9.8 antagonizes hGBP1 function. Therefore, the use of IpaH9.8- deficient S. flexneri strains will enable us to monitor and functionally dissect an operational hGBP1-driven host response. Overall, the work proposed here will provide a fundamental understanding of the role of hGBP1 and other human GBPs in cell-autonomous immunity to intracellular bacterial pathogens. !