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。尽管有抗生素，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杆菌嗜肺军团菌，这是一种名为军团病的严重肺炎的病原体，作为了解细菌病原体如何与巨噬细胞相互作用的模型。军团菌的毒力取决于它在巨噬细胞内生存和生长的能力。以前的工作已经证实，两个基因(Naip5和ipaf)在协调保护巨噬细胞免受军团菌感染的细胞防御中起着重要作用，但Naip5/ipaf赋予对军团菌耐药性的分子机制在很大程度上仍然是个谜。NAIP5和IPAF与一个称为NOD样蛋白的细胞质病原体检测蛋白大家族同源。我们的初步结果表明，对军团菌的耐药性取决于快速触发含有Naip5/IPAF的炎症体，该炎症体在巨噬细胞胞浆中检测到细菌鞭毛蛋白时发生。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的侵袭，此外，还需要通过肿瘤坏死因子受体传递信号。在关节炎和克罗恩病等疾病的临床治疗中，肿瘤坏死因子已经成为重要的治疗靶点。因此，深入了解NAIP/IPAF和肿瘤坏死因子协同抑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，这项拨款提案的具体目标是：1.测试细菌鞭毛蛋白在细胞内的存在足以触发依赖于IPAF/Naip5的信号通路以限制细菌生长的假设；绘制触发IPAF/Naip5所需的鞭毛蛋白内的决定因素；并利用这些信息，测试鞭毛蛋白与NAip5和/或IPAF物理相互作用的假设。2.在小鼠体内通过靶向缺失Naip5来验证Naip5在巨噬细胞对军团菌耐药中起关键作用的假设。3.验证NAIP5/IPAF信号与肿瘤坏死因子信号协同保护巨噬细胞的假说。公共卫生相关性：预计从上述工作中获得的结果将使人们能够更深入地了解细菌是如何致病的，以及哪些因素会导致对这些细菌的成功免疫反应。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.