Porcine caspase-8 and 13 cleave GSDMD to generate products can cause cell death.

Pyroptosis is a form of programmed cell death mediated by gasdermin family proteins (GSDMs), characterized by the disruption of the cell membrane and the release of cytoplasmic contents. Protease-mediated cleavage is essential for activating the pyroptosis-mediating function of GSDMs. Therefore, identification of proteases that are capable of processing GSDMs is crucial for elucidating the mechanisms of pyroptosis in different hosts. To date, it has been determined that porcine caspase-1 (pCASP1) can cleave porcine GSDMD (pGSDMD) to activate the pyroptosis-mediating ability of pGSDMD. However, it remains unknown whether other proteases can recognize and activate pGSDMD in porcine cells. We first cloned the gene encoding porcine caspase-8 (pCASP8) and porcine caspase-13 (pCASP13) from tissues of a piglet infected with Enterotoxigenic Escherichia coli (ETEC). We then assessed the ability of pCASP8 and pCASP13 to cleave pGSDMD. The results showed that both pCASP8 and pCASP13 cleaved pGSDMD after the 279th amino acid residue, which is an aspartate (D), in the pGSDMD molecule, thereby generating an amino-terminal domain (NT) of pGSDMD (pGSDMD-NT) is capable of damaging the cell membrane and leading to cell death. Recombinantly expressed and purified pGSDMD-NT protein (rpGSDMD-NT) could kill ETEC when incubated with the bacteria, whereas rpGSDMD-CT and rpGSDMD could not. Our study provides evidence that pCASP8 and pCASP13 can induce pyroptosis in porcine cells by cleaving pGSDMD. Our findings provide essential insights into the molecular mechanisms of pyroptosis in porcine cells.