ChCaspase-1 cleaves ChGasdermin-E, inducing pyroptosis, and participates in the bacterial defence of the oyster Crassostrea hongkongensis

Abstract

Oysters have evolved an effective and powerful innate immune system to recognize foreign substances and eliminate invaders through orchestrated immune responses such as programmed cell death (PCD), including apoptosis, autophagy, necroptosis, and pyroptosis. Both apoptosis and autophagy have been considered to be crucial mechanisms for oyster defence. However, the role of pyroptosis, a gasdermin (GSDM)-dependent programmed cell death pathway that is critical for defence against infection, in oysters remains unclear. In this study, we investigated the activity of Crassostrea hongkongensis GSDME (ChGSDME), which is cleaved by inflammatory C.hongkongensis caspase-1 (ChCASP1). In vivo, qRT–PCR analysis showed that ChCASP1 and ChGSDME were widely expressed in oyster developmental stages and tissues, and responded to Vibrio coralliilyticus challenge. Meanwhile, hemocyte death was reduced after knockdown of ChCASP1 or ChGSDME by RNA interference (RNAi). Following injection of V. coralliilyticus, the cell death rate of the knockdown ChCASP1 or ChGSDME group was significantly lower than that of the control group. In vitro, coexpressing ChCASP1 and ChGSDME in HEK293T cells resulted in pyroptotic morphology, a bubble-like membrane, and a a truncated protein (ChGSDME N-terminal). Subsequently, immunofluorescence revealed that ChGSDME-NT oligomerized at the cell membrane, inducing bubble-like membrane formation, thereby causing low cytotoxicity. Luciferase reporter gene analysis revealed that ChCASP1 significantly upregulated Nuclear factor kappa-B (NF-κB) and modestly upregulated the p53 signalling pathway. Taken together, our study demonstrated that the pyroptosis-related genes ChCASP1 and ChGSDME mediate pyroptosis in HEK293T cells and play important roles in cell death during V. coralliilyticus infection in C. hongkongensis.