Porcine Gasdermin D Is a Substrate of Caspase-1 and an Executioner of Pyroptosis

Gasdermin (GSDM) family proteins were recently identified as the executioner of pyroptosis. The mechanism of pyroptosis mediated by gasdermin D (GSDMD) (a member of GSDM family) in humans and mice is well understood. In pyroptosis, mouse and human GSDMDs are cleaved by activated proinflammatory caspases (caspase-1, 4, 5, or 11) to produce anamino-terminal domain (GSDMD-NT) and a carboxyl-terminal domain (GSDMD-CT). The GSDMD-NT drives cell membrane rupture, which leads to the pyroptotic death of the cells. The expression of porcine GSDMD (pGSDMD) has recently been determined, but the activation and regulation mechanism of pGSDMD and its ability to mediate pyroptosis are largely unknown. In the present study, the activation of porcine caspase-1 (pcaspase-1) and cleavage of pGSDMD occurred in the duodenum and jejunum of a piglet challenged with enterotoxigenic Escherichia coli were first determined. Then the capability of pcaspase-1 to cleave pGSDMD was determined in a cell-free system and in human embryonic kidney cells. The pGSDMD cleavage by pcaspase-1 occurred after the pGSDMD molecule's (276)Phenylalanine-Glutamine-Serine-Aspartic acid(279) motif. The pGSDMD-NT generated from the pGSDMD cleavage by pcaspase-1 showed the ability to drive cell membrane rupture in eukaryotic cells. When expressed in E. coli competent cells, pGSDMD-NT showed bactericidal activity. These results suggest that pGSDMD is a substate of pcaspase-1 and an executioner of pyroptosis. Our work sheds light on pGSDMD's activation mechanisms and functions.

Automated summary

Gasdermin family proteins, including GSDMD, have been identified as the key players in pyroptosis. While the mechanism of GSDMD-mediated pyroptosis is well understood in humans and mice, the activation and function of porcine GSDMD (pGSDMD) remain largely unknown. This study demonstrated the activation of porcine caspase-1 (pcaspase-1) and cleavage of pGSDMD in the small intestine of piglets infected with enterotoxigenic Escherichia coli. The cleavage of pGSDMD by pcaspase-1 resulted in the production of pGSDMD-NT, which exhibited the ability to induce cell membrane rupture and showed bactericidal activity. These findings suggest that pGSDMD plays a role in pyroptosis and provide insights into its activation mechanisms and functions.