cGAS-STING依赖的鱼类新型天然免疫信号通路鉴定及其功能研究

The nucleotidyl transferase cGAS, its second-messenger product cGAMP, and the cGAMP sensor STING are the newly defined DNA sensing system in the cytoplasm of mammalian cells. However, the comprehensive antimicrobial roles of this system still need to be further explored, and data on their occurrence and functions in teleost fish are limited. To this end, this project focuses on the molecular identification and functional characterization of the cGAMP-mediated cGAS-STING signaling pathways in interferon and antimicrobial-peptide innate immunity in teleost fish. The structural features, tissue distribution patterns, subcellular localizations and the possible evolutionary history of the family members are to be elucidated. To understand the DNA recognition patterns of cGAS regarding to different aquatic pathogens, the genome DNA of various aquatic bacteria and viruses will be used to induce the synthesis of cGAMP. The configuration of this cGAMP second-messenger is monitored to see whether it shares the same pattern with the mammalian counterparts. Besides, the cGAMP recognition motif of teleost STING and its affinity to various cGAMP molecules will be studied. As an pivotal producer of type I interferon in the cytoplasm, the cGAS mediated STING-TBK1-IRF3-IFN signaling pathway is of great importance in the cytoplasm antiviral process, we sought to analyze whether this signaling pathway is conserved in teleost fish and hence deduce the evolutionary pattern of this signaling. Furthermore, IL-6 has been reported as an immune effector molecule of the cGAMP dependent cGAS-STING antimicrobial system through the cGAS-STING-NF-κB signaling, we also attempt to propose for the first time that this system may also involves in the production of hepcidin, an important antibacterial peptide, for the antibacterial effects of the organism. Thus, a more comprehensive anti-pathogen network based on the cGAMP dependent c-GAS-STING signaling pathway will be delineated. Based on those precedent mechanism studies, we further devote to studying the possibility of cGAMP as immune adjuvant, as it plays an essential role in the signaling that produce immune cytokines, type I interferons and antibacterial peptide. We hope to find that cGAMP will effectively improve the survival of fish against aquatic intracellular pathogen infection (eg. Listeria monocytogenes, Aeromonas hydrophila, Vibrio alginolyticus and iridovirus, etc.) with the production of the relative immune effector molecules. This project is anticipated to enrich and improve the antimicrobial mechanism of the innate immune system. Most importantly, the study will provide an innovative approach for the development and usage of an immune adjuvant, which is of great application values.

本项目拟首次在鱼类中系统开展新型胞质DNA模式识别受体cGAS及其介导的天然免疫信号通路研究。阐明cGAS催化合成cGAMP的酶学机制；鉴定cGAS依赖的干扰素和抗菌肽信号通路的分子组成、信号识别与转导机制、免疫学功能及其在脊椎动物天然免疫中的保守性与起源进化规律；探索研究一条新的cGAS介导的STING-IL-6-Hepcidin信号途径，揭示抗菌肽合成调控新机制。通过上述研究，阐明cGAS具有识别不同病原DNA感染信号并启动抗病毒和抗细菌双重天然免疫调节功能，是一种在进化中保守的普适性病原模式识别受体，为深入揭示包括鱼类在内的脊椎动物天然免疫调控机制提供科学指导。在此基础上，研究开发新型的cGAMP第二信使免疫抗病制剂，拓展环二核苷酸的应用领域，为鱼类病害防治提供新的思路和策略。项目立题富有开拓和创新性，研究结果可以丰富鱼类免疫学内容，对阐明天然免疫识别与调控的分子机制具有重要的意义。

cGAS (Cyclic GMP-AMP Synthase) 是一种重要的胞质DNA识别受体，具有催化合成cGAMP第二信使分子以启动先天和适应性免疫等功能，并在诸多疾病的形成中发挥重要作用，成为近年免疫学研究的热点。目前有关cGAS的研究主要见诸人类和部分哺乳动物，低等脊椎动物中尚缺少系统的研究。鱼类作为早期脊椎动物的代表性物种，利用鱼类开展cGAS研究，对揭示cGAS及其信号通路的分子与功能演化机制以及丰富先天免疫理论等都具有重要的意义。本项目在鱼类中分离鉴定出cGASa和cGASb两个同源分子，对其结构特征、组织分布、亚细胞定位、合成产物、催化机制、分子演化规律以及在先天免疫信号通路和粘膜免疫中的功能开展了系统的探索研究。发现cGASa和cGASb具有Mab21、NTase core和锌囊等保守的核心功能结构域以及非保守的富含碱性氨基酸的氨基末端和包含跨膜区的羧基末端等特征，广泛表达于多种免疫组织并定位于细胞质。功能分析显示，cGASa和cGASb都能通过寡聚化而合成2’3’-cGAMP，其中cGASa的合成能力大于cGASb，而cGASb能通过与cGASa形成异寡聚体而抑制cGASa合成2’3’-cGAMP的活性。cGASa和cGASb都能通过STING激活Ⅰ型IFN和NF-κB信号通路，并参与斑马鱼先天免疫防御。通过构建系列突变体研究了cGASa/b的功能位点和作用机制。通过基因敲减等技术揭示了cGASa-STING介导的IFNφ1对IgZ粘膜免疫球蛋白的产生发挥调控作用。本项目揭示了cGAS在硬骨鱼类中的起源及其在早期脊椎动物中的功能机制，不仅丰富了鱼类免疫学内容，为cGAS研究提供了新的实验动物模型，而且为深入阐明以cGAS信号通路为代表的先天免疫系统的演化规律提供了重要科学依据。同时对于我国渔业生产中的病害免疫防治也具有重要潜在应用价值。