Intracellular sensing of flagellin and innate immunity to Legionella pneumophila

描述（由申请人提供）：本提案的总体目标是了解哺乳动物细胞如何检测和响应细胞内细菌病原体的存在。尽管使用了抗生素，但细菌感染仍然是一个重大的公共卫生挑战。我们的研究利用革兰氏阴性细菌嗜肺军团菌（Legionella pneumophila）作为了解细菌病原体如何与巨噬细胞相互作用的模型，嗜肺军团菌是一种严重肺炎（称为军团病）的病原体。军团菌的毒力取决于其在巨噬细胞内存活和生长的能力。以前的工作已经确定，两个基因（Naip 5和Ipaf）有助于协调细胞防御，保护巨噬细胞免受军团菌感染，但Naip 5/Ipaf赋予军团菌抗性的分子机制在很大程度上仍然是个谜。Naip 5和Ipaf表现出与称为Nod样蛋白的胞质病原体检测蛋白的大家族的同源性。我们的初步研究结果表明，军团菌的耐药性取决于快速触发的Naip 5/Ipaf-含有炎性小体，发生在检测到细菌鞭毛蛋白在巨噬细胞胞质溶胶。炎性小体激活与多种人类疾病有关，因此使用军团菌作为理解炎性小体激活的模型将对我们理解人类健康和疾病具有广泛的意义。我们还意外地观察到Naip/Ipaf不足以保护巨噬细胞免受军团菌感染，此外，还需要通过肿瘤坏死因子受体进行信号传导。TNF已经是临床治疗疾病如关节炎和克罗恩病的重要治疗靶点。因此，更深入地了解Naip/Ipaf和TNF合作限制军团菌生长的分子基础可能对人类健康和疾病具有重要意义。因此，本资助提案的具体目标是：1。测试细菌鞭毛蛋白的细胞内存在足以触发限制细菌生长的Ipaf/Naip 5依赖性信号传导途径的假设;绘制触发Ipaf/Naip 5所需的鞭毛蛋白内的决定簇;并使用此信息，测试鞭毛蛋白与Naip 5和/或Ipaf物理相互作用的假设。2.通过在小鼠中靶向缺失Naip 5来检验Naip 5对于巨噬细胞对军团菌的抗性至关重要的假设。3.检验Naip 5/Ipaf信号通过与TNF信号协同作用保护巨噬细胞的假设。公共卫生相关性：预计从上述工作中获得的结果将允许更深入地了解细菌如何引起疾病以及哪些因素导致对这些细菌的成功免疫应答。这些知识应该有助于设计新的抗菌疗法的合理方法。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand how mammalian cells detect and respond to the presence of intracellular bacterial pathogens. Despite antibiotics, bacterial infections continue to present a significant public health challenge. Our studies utilize the gram-negative bacterium Legionella pneumophila, the causative agent of a severe pneumonia called Legionnaires' Disease, as a model for understanding how bacterial pathogens interact with macrophages. The virulence of Legionella depends on its ability to survive and grow within macrophages. Previous work has established that two genes (Naip5 and Ipaf) are instrumental in orchestrating cellular defenses that protect macrophages from Legionella infection, but the molecular mechanism by which Naip5/Ipaf confer resistance to Legionella has remained largely mysterious. Naip5 and Ipaf exhibit homology to a large family of cytosolic pathogen- detector proteins called the Nod-like proteins. Our preliminary results suggest that resistance to Legionella depends on rapid triggering of a Naip5/Ipaf-containing inflammasome that occurs upon the detection of bacterial flagellin in the macrophage cytosol. Inflammasome activation is connected to a variety of human diseases, and thus use of Legionella as a model for understanding inflammasome activation will have broad implications for our understanding of human health and disease. We have also made the unexpected observation that Naip/Ipaf are not sufficient to protect macrophages from Legionella, and that in addition, signaling via the tumor necrosis factor receptor is also required. TNF is already an important therapeutic target in the clinical treatment of diseases such as arthritis and Crohn's Disease. Thus, a deeper understanding of the molecular basis by which Naip/Ipaf and TNF collaborate to restrict Legionella growth could possibly be of great relevance to human health and disease. Thus, the specific aims of this grant proposal are: 1. Test the hypothesis that the intracellular presence of bacterial flagellin protein is sufficient to trigger the Ipaf/Naip5-dependent signaling pathways that restrict bacterial growth; map the determinants within flagellin required to trigger Ipaf/Naip5; and using this information, test the hypothesis that flagellin physically interacts with Naip5 and/or Ipaf. 2. Test the hypothesis that Naip5 is critical for macrophage resistance to Legionella by targeted deletion of Naip5 in mice. 3. Test the hypothesis that Naip5/Ipaf signaling protects macrophages by synergizing with TNF signaling. PUBLIC HEALTH RELEVANCE: It is anticipated that results obtained from the above work will permit a deeper understanding of how bacteria cause disease and of what factors lead to successful immune responses to these bacteria. Such knowledge should contribute to rational approaches to designing novel antibacterial therapies.