Probing the Innate Response to 3', 5'-cyclic Diguanylate (c-diGMP)

描述(申请人提供)：由细菌引起的传染病，特别是抗药性病原体，已经成为美国医疗保健系统日益沉重的负担，医院相关感染的直接医疗费用估计在每年300-450亿美元之间。此外，新抗生素的发展已经跟不上越来越多耐药细菌的快速出现。幸运的是，越来越多的复杂的与生俱来的“传感器和响应系统”已经被描述出来，这些系统与它们的宿主共同进化。在脊椎动物中，这包括几个不同的固有传感器家族，它们识别专门的细菌分子或“分子模式”。这些传感器启动众所周知的促炎信号级联反应，最终导致免疫效应物的表达，如肿瘤坏死因子-1和干扰素(IFN)。这些传感器和响应系统的特性为开发同样复杂的新治疗策略提供了一个重要的机会。为了研究对嗜肺军团菌的天然免疫应答，我们确定了一种新的细菌调节分子3‘，5’-环二鸟苷酸酯(c-diGMP)在刺激自分泌干扰素的表达中起重要作用，从而有效地抑制细菌的生长。最近也报道了对单核细胞增多性李斯特菌3‘，5’-环二腺苷(c-diamp)的类似反应。我们提出了生化和遗传学的方法来表征哺乳动物传感器系统中对c-diGMP作出特殊反应的组件。为此，我们建立了生物素修饰的c-diGMP和c-diGMP报告细胞系。我们预计，我们识别的传感器组件或密切相关的同系物，将指导对c-diamp的反应。具体地说，我们建议：1.利用亲和层析法鉴定哺乳动物c-diGMP受体。2.利用遗传筛选来识别哺乳动物传感器系统中直接对c-diGMP的炎症反应的组件。 公共卫生相关性：I型干扰素(IFN-IS)以其抗病毒活性而闻名，它还能对兼性细胞内细菌(如图拉氏方济氏菌和嗜肺军团菌)产生有效的先天反应。本研究探讨了嗜肺乳杆菌的重要调节分子c-diGMP刺激核因子-B活化和干扰素-I表达的机制。这些研究集中在寻找一种新的先天免疫反应途径，这对抗菌治疗和疫苗开发具有重要意义。

DESCRIPTION (provided by applicant): Infectious diseases caused by bacteria, especially antibiotic resistant pathogens, have become an increasing burden on the US health care system, with direct medical costs for hospital associated infections estimated to range between 30-45 billion dollars annually. Moreover, the development of new antibiotics has been unable to keep pace with the rapid emergence of ever more resistant bacteria. Fortunately, a growing number of sophisticated innate "Sensor and Response Systems" that co-evolved with their hosts have been described. In vertebrates this includes several distinct families of innate sensors that recognize specialized bacterial molecules or "molecular patterns". These sensors initiate well-known pro-inflammatory signaling cascades that culminate in the expression of immune effectors, like TNF-1 and Interferons (IFNs). Characterization of theses Sensor and Responses Systems provides an important opportunity for the development of equally sophisticated new therapeutic strategies. In an effort to characterize the innate responses that direct an effective response towards Legionella pneumophila, we have determined that a novel bacterial regulatory molecule, 3',5'- cyclic diguanylate (c-diGMP) plays an important role in stimulating the expression of autocrine IFNs, which effectively suppress bacterial growth. An analogous response to Listeria monocytogenes derived 3',5'-cyclic diadenylate (c-diAMP), has also recently been reported. We propose biochemical and genetic approaches to characterize the components of the mammalian sensor system that specifically responds to c-diGMP. To this end we have developed a biotin modified c-diGMP and a c-diGMP reporter cell line. We anticipate that the sensor components we identify, or closely related homologs, will direct the response to c- diAMP. Specifically we propose to: 1. Exploit affinity chromatography to identify the mammalian receptor for c-diGMP. 2. Exploit a genetic screen to identify the components of the mammalian sensor system that direct inflammatory response to c-diGMP. PUBLIC HEALTH RELEVANCE: Type I Interferons (IFN-Is), known for their antiviral activity, also direct an effective innate response towards facultative intracellular bacteria, like Francisella tularensis and Legionella pneumophila. The proposed studies explore the mechanism by which c-diGMP, an important L. pneumophila regulatory molecule, stimulates NF-:B activation and IFN-I expression. These studies focus on identifying a new innate immune response pathway, which has important implications on antibacterial therapy and vaccine development.