Activation Mechanism of CcGSDMEb-1/2 and Regulation for Bacterial Clearance in Common Carp (Cyprinus carpio).

Gasdermin E (GSDME), to date, is considered the only direct executor of the pyroptosis process in teleost and plays an important role in innate immunity. In common carp (Cyprinus carpio), there contains two pairs of GSDME (GSDMEa/a-like and GSDMEb-1/2), and the pyroptotic function and regulation mechanism of GSDME still remain unclear. In this study, we identified two GSDMEb genes of common carp (CcGSDMEb-1/2), which contain a conserved N-terminal pore-forming domain, C-terminal autoinhibitory domain, and a flexible and pliable hinge region. We investigated the function and mechanism of CcGSDMEb-1/2 in association with inflammatory and apoptotic caspases in Epithelioma papulosum cyprinid cells and discovered that only CcCaspase-1b could cleave CcGSDMEb-1/2 through recognizing the sites 244FEVD247 and 244FEAD247 in the linker region, respectively. CcGSDMEb-1/2 exerted toxicity to human embryonic kidney 293T cells and bactericidal activity through its N-terminal domain. Interestingly, after i.p. infection by Aeromonas hydrophila, we found that CcGSDMEb-1/2 were upregulated in immune organs (head kidney and spleen) at the early stage of infection, but downregulated in mucosal immune tissues (gill and skin). After CcGSDMEb-1/2 were knocked down and overexpressed in vivo and in vitro, respectively, we found that CcGSDMEb-1/2 could govern the secretion of CcIL-1β and regulate the bacterial clearance after A. hydrophila challenge. Taken together, in this study, it was demonstrated that the cleavage mode of CcGSDMEb-1/2 in common carp was obviously different from that in other species and played an important role in CcIL-1β secretion and bacterial clearance.