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

描述（由申请人提供）：由细菌，特别是抗生素耐药病原体引起的传染病，已成为美国医疗保健系统日益沉重的负担，每年医院相关感染的直接医疗费用估计在 30-450 亿美元之间。此外，新抗生素的开发无法跟上耐药细菌迅速出现的步伐。幸运的是，越来越多与宿主共同进化的复杂的先天“传感器和响应系统”已被描述。在脊椎动物中，这包括几个不同的先天传感器家族，它们识别特殊的细菌分子或“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 TNF-1 和干扰素 (IFN) 等免疫效应物的表达。这些传感器和响应系统的表征为开发同样复杂的新治疗策略提供了重要机会。 为了描述针对嗜肺军团菌有效反应的先天反应，我们确定了一种新型细菌调节分子 3',5'- 环二鸟苷酸 (c-diGMP) 在刺激自分泌 IFN 的表达方面发挥着重要作用，从而有效抑制细菌生长。最近还报道了对单核细胞增生李斯特氏菌衍生的 3',5'-环二腺苷酸 (c-diAMP) 的类似反应。我们提出了生物化学和遗传方法来表征专门响应 c-diGMP 的哺乳动物传感器系统的组件。为此，我们开发了生物素修饰的 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 的响应。具体来说，我们建议： 1. 利用亲和层析来鉴定 c-diGMP 的哺乳动物受体。 2. 利用基因筛选来识别哺乳动物传感器系统中引导 c-diGMP 炎症反应的组件。 公共健康相关性：I 型干扰素 (IFN-Is) 以其抗病毒活性而闻名，还可针对兼性细胞内细菌（如土拉弗朗西斯菌和嗜肺军团菌）产生有效的先天反应。拟议的研究探讨了 c-diGMP（一种重要的嗜肺军团菌调节分子）刺激 NF-:B 激活和 IFN-I 表达的机制。这些研究重点是确定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.