Synergism of duplicated Gasdermin Eb in Carassius auratus gibelio unveiling a regulatory amplification loop for pyroptosis.

The Gasdermin (GSDM) family drives pyroptosis via membrane pore formation, regulating immunity and disease. In teleosts, Gasdermin E (GSDME) is the sole pyroptosis executor but diverged into paralogs (GSDMEa/b) in cyprinids. We identified four GSDME paralogs (CaGSDMEa1/a2/b1/b2) in Carassius auratus gibelio and characterized functional divergence between CaGSDMEb1/b2. Both paralogs show broad tissue expression and are upregulated during Aeromonas hydrophila infection or LPS stimulation, indicating the involvement of antibacterial immune responses. However, only CaGSDMEb2 executes pyroptosis by being cleaved by Cacaspase-19 (caspase-4/5 homolog) upon cytosolic LPS detection, although CaGSDMEb1-NT and CaGSDMEb2-NT form pores. Evolutionary analysis identified a key divergence that CaGSDMEb1 lacks the conserved aspartic acid (ASP) in the caspase cleavage motif, rendering it resistant to proteolytic activation. Notably, full-length CaGSDMEb1 undergoes subfunctionalization by binding the N-terminal domain of Cacaspase-19, enhancing caspase activity and amplifying pyroptosis. Thus, CaGSDMEb1 may regulate pyroptosis through a full-length protein-dependent mechanism, bypassing its NT domain. These findings suggest functional divergence between duplicated GSDME genes-CaGSDMEb2 retains ancestral executioner activity, while CaGSDMEb1 may have evolved a regulatory role-and suggest a unique amplification loop for pyroptosis. This study provides insights into the evolutionary plasticity of the ancient GSDME family.