半滑舌鳎gasdermin E介导的细胞焦亡及其在抗感染免疫中的作用研究

Pyroptosis is a highly inflammatory form of programmed cell death that protects multicellular organisms from invading pathogenic bacteria and viruses, which plays an essential role in the innate immune responses against microbial infections. Gasdermin family members share novel and functionally important gasdermin N terminal domains, which are identified to be the direct executioner for the cell pyroptosis. In mammals, several gasdermin members are proved to possess the intrinsic pyroptosis-inducing activity. Gasdermin D and gasdermin E are found to be specifically cleaved by caspase-1/4/5/11 and caspase-3 respectively, the released N terminal domain exhibited membrane pore-forming activity, which initiated cell pyroptosis. Although mammals harbor several gasdermin members that exhibited pyroptotic activity, teleosts only harbor gasdermin E. We have discovered that teleost gasdermin E can mediate pyroptosis, but the molecular mechanism of pyroptosis and the effect of pyroptosis on the immune defense against microbial infection remain unclear. Based on these previous findings, we will examine the gasdermin E-mediated pyroptosis in an important economic fish species Cynoglossus semilaevis. We will investigate the pyroptosis-inducing activity of gasdermin E, the cleavage modulation of gasdermin E by caspase, and the immunological role of gasdermin E-mediated cell pyroptosis in the immune defense against microbial infection. The results of this study will provide a theoretical guidance to disease control and prevention in fish.

细胞焦亡作为一种新的促炎程序性细胞死亡方式，在宿主抗感染免疫中发挥至关重要的作用。研究发现，gasdermin家族蛋白是细胞焦亡的直接执行者。哺乳动物拥有多个gasdermin成员，其中gasdermin D和gasdermin E分别被caspase-1/4/5/11和caspase-3剪切，释放的N端结构域能够破坏细胞膜，介导细胞焦亡。与哺乳动物不同，硬骨鱼类仅含有gasdermin E。申请人在前期研究中发现硬骨鱼gasdermin E能够介导细胞焦亡，但是其调控机制及其在鱼类抗感染免疫中的作用不清楚。本项目拟在前期工作基础上，研究重要海水养殖鱼类半滑舌鳎gasdermin E介导的细胞焦亡，鉴定gasdermin E介导细胞焦亡的活性，揭示caspase对gasdermin E的剪切机制，明确gasdermin E介导的细胞焦亡在抗感染免疫中的作用，为鱼类病害防控提供理论指导。

本项目围绕重要海水养殖鱼类半滑舌鳎等开展了细胞焦亡研究，鉴定了半滑舌鳎GSDME N端结构域具有介导细胞焦亡的免疫功能，发现caspase（CASP）1/3/7能够剪切GSDME铰链区的同一位点，产生完整的N端片段。CASP1剪切GSDME诱导细胞焦亡，而CASP3/7剪切GSDME诱导细胞凋亡向细胞焦亡转换。脂多糖和尼日利亚菌素刺激巨噬细胞能够诱导CASP1激活及GSDME剪切，从而导致细胞焦亡。在大菱鲆中，发现CASP3/7与CASP6能够剪切GSDMEa两个不同位点，其中，CASP3/7剪切GSDMEa产生完整的N端片段，诱导细胞焦亡；而CASP6剪切GSDMEa破坏了N端结构域的完整性，抑制细胞焦亡。抑制CASP3/7介导的GSDMEa激活能够促进病原菌感染及大菱鲆死亡。该研究发现了鱼类中存在不同于哺乳动物的独特的细胞焦亡机制，为水产养殖鱼类疫病防控提供了重要的理论依据。此外，本项目系统研究了GSDM在无脊椎动物中的演化动态与谱系分化，首次在无脊椎动物中发现了功能性GSDME。发现CASP3能够特异性剪切珊瑚GSDME，产生两种形式的N端片段，二者均具有介导细胞焦亡功能。病原菌感染能够诱导CASP3激活及GSDME剪切，珊瑚发生坏死；而抑制CASP3剪切GSDME能够阻止感染造成的珊瑚死亡。该研究突破了GSDM起源于低等脊椎动物的旧有认知，为无脊椎动物抗感染免疫研究提供了新的切入点。研究成果在国际知名学术期刊Science Immunology（封面文章），Journal of Immunology及Cell Death and Disease等发表SCI论文4篇。