Teleost GSDMEc regulates GSDMEa-mediated pyroptosis

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INTRODUCTION: Gasdermin (GSDM) is a family of proteins that execute pyroptosis after being activated by caspase (CASP) cleavage. Mammals possess five GSDM members (A - E) with pyroptotic ability. Teleosts possess only one pyroptotic GSDM, GSDME, that exists in three orthologs, GSDMEa, b, and c. GSDMEa and GSDMEb are known to induce pyroptosis, but the function of GSDMEc is unknown. OBJECTIVES: The present study aimed to elucidate the function of teleost GSDMEc and examine the interplay among teleost GSDME orthologs by using snakehead Channa argus as a representative species. METHODS: Pyroptosis was assessed via microscopy and biochemical assays. GSDME cleavage, oligomerization, and membrane translocation were examined via immunoblotting. The interactions of GSDME products were examined using confocal microscopy and co-immunoprecipitation. GSDME knockdown in fish and in vivo bacterial infection were performed. RESULTS: C. argus possessed three GSDME variants (CaGSDMEa, CaGSDMEc1, and CaGSDMEc2). CaGSDMEa was cleaved by C. argus CASP (CaCASP) 1/8 to produce an N-terminal fragment (NT), NT261, that induced pyroptosis. CaGSDMEc1 and CaGSDMEc2 were also cleaved by CaCASP1/8, but the resulting NTs, NT123 and NT108, respectively, were unable to induce pyroptosis. However, both NT123 and NT108 could bind and promote the pyroptotic activity of NT261 by facilitating NT261 oligomerization and membrane translocation. The interaction between NT261 and NT123/NT108 depended on a positively charged motif that is conserved in the metazoan GSDME and is essential to the membrane localization of NT123 and the pyroptotic activity of NT261. Bacterial infection induced CaGSDMEa/CaCASP8 activation and CaGSDMEc1/c2 cleavage in snakehead cells, resulting in pyroptosis, IL-1β/18 maturation cleavage, and extracellular DNA-net formation. CaGSDMEa/c1 knockdown significantly increased bacterial dissemination in fish tissues and reduced fish survival. CONCLUSIONS: Our results revealed the functions and interactive mechanism of teleost GSDME orthologs, and provided new insights into the regulation of pyroptosis in lower vertebrates.

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