| Pyroptosis is a form of programmed cell death that was first discovered to be involved in the process of immune cell resistance to pathogenic bacterial infection.Immune cells that have undergone pyroptosis rupture their cell membranes and release their cytoplasmic contents,which in turn trigger an even more intense immune response known as an inflammatory response.Pyroptosis plays an important role not only in the inflammatory response to pathogenic infections,but also in many diseases such as some genetic disorders,autoimmune diseases and cancer.The study of the molecular mechanism of pyroptosis and its immune function has become a frontier in the field of cell death research.The discovery and subsequent mechanistic studies of pyroptosis have almost exclusively been carried out in vertebrates.There is a paucity of research on the phenomenon,molecular mechanisms and immune function in invertebrates.In this study,bioinformatics,molecular immunology and cell biology were used to identify the key molecule CgGSDME in the pyroptosis pathway from oysters,and to investigate its activation mechanism and mediated immune function.The following conclusions are drawn:1.Structural features and expression characteristics of key molecules of the in the pyroptosis signaling pathway in C.gigasOne GSDM family gene was screened from the transcriptome data of transcriptome data in C.gigas,and its gene sequence number was LOC10533619/CGI＿10022772,which was named CgGSDME.The open reading frame(ORF)of CgGSDME contains 1398 bp and encodes 465 amino acid residues.Its relative molecular weight was predicted to be 51.59 k Da and its isoelectric point was5.05.CgGSDME consists of a Gasdermin domain.The three-dimensional structures of CgGSDME and Hs GSDME were predicted by SWISS MODEL,and it was found that the three-dimensional structures of CgGSDME and Hs GSDME were similar.The recognition and activation sites of Hs GSDME were compared with those of CgGSDME,and it was found that CgGSDME had a conserved recognition and activation site DXXD(XX is any hydrophobic amino acid).The tissue distribution and immunostimulatory expression patterns of CgGSDME and CgCaspase-8-2 were detected by real-time PCR.The results showed that CgGSDME was mainly highly expressed in gills,and CgCaspase-8-2 was mainly highly expressed in haemocytes.Under the stimulation of Vibrio splendidus,the expression level of CgGSDME m RNA in haemocytes was significantly increased compared with the control group at 6,12 and48 h(p < 0.05).CgCaspase-8-2 m RNA in haemocytes,compared with the control group,the expression level was significantly increased at 3,6 and 24 h(p < 0.05).The expression level of CgCaspase-8-2 m RNA in the gills was significantly increased at 3,6,12 and 24 h compared with the control group(p < 0.05),indicating that CgGSDME and CgCaspase-8-2 responded to V.splendidus stimulation.Under LPS stimulation,CgGSDME m RNA expression levels were significantly increased at 3,12 and 24 h compared with the control group(p < 0.05),indicating that CgGSDME responded to LPS stimulation.Western blotting showed that CgCaspase-8-2 protein was significantly cleaved in haemocytes 3 h after V.splendidus stimulation.Flow cytometry analysis showed that CgCaspase-8-2 protein was expressed in all three types of haemocytes in C.gigas and the expression level was the highest in granulocytes,which was higher than that in semi-granulocytes and agranulocytes.2.Caspase-8-2/GSDME signaling pathway mediates pyroptosis of haemocytes in C.gigasScanning electron microscope and fluorescence microscope showed that haemocytes of C.gigas were swollen and ruptured and the cell membrane bulged at 3h after stimulation by V.splendidus and the western blotting experiment found that Significant cleavage of CgGSDME protein in haemocytes 3 h after V.splendidus stimulation.Immunocytochemistry found that CgGSDME was translocated from the cytoplasm to the cell membrane after V.splendidus stimulation and CgGSDME colocalized with both CgCaspase-8-2 and the cell membrane.The changes of CgGSDME under the stimulation of V.splendidus were detected by the treatment of Caspase-8 inhibitor Ac-IETD-CHO.The results showed that the protein expression of CgGSDME in haemocytes was significantly decreased at 3 h.3.Effect of CgGSDME and CgCaspase-8-2 on inflammatory factor expression and histopathological changesAfter respectively knockdown of CgGSDME and CgCaspase-8-2,the changes of inflammatory factors under the stimulation of V.splendidus were detected by RNA interference experiments.The results showed that in the CgGSDME-RNAi experimental group,the expressions of CgIL17-5,CgTNF and CgIFNLP m RNA were significantly decreased at 3 h(p < 0.05).At the same time gills tissue swelling was reduced.In CgCaspase-8-2-RNAi experimental group,the expressions of CgIL17s(CgIL17-1,-2,-3,-4,-6),CgTNF,CgIFNLP and CgBig Def1 m RNA were significantly decreased after 12 h(p < 0.05).The changes of inflammatory factors under the stimulation of V.splendidus were detected by treatment with Caspase family inhibitor Z-VAD-FMK.The results showed that in the experimental group treated with the Caspase family inhibitor Z-VAD-FMK,the expressions of CgGSDME,CgIL17-5,CgTNF and CgIFNLP m RNA were significantly decreased at 3 h(p < 0.05).The expression of both decreased significantly at 3 h(p < 0.05).The changes of inflammatory factors under the stimulation of V.splendidus were detected by the treatment of Caspase-8 inhibitor Ac-IETD-CHO.The results showed that the protein expressions of CgTNF and CgIFNLP in serum were significantly decreased at 3 h and the swelling of gill tissue was significantly reduced compared with the control group from tissue sections.4.Recombinant proteins of the CgGSDME gasdermin-N domain are bacteriostatic and bactericidal effectsThe recombinant protein of the gasdermin-N domain of CgGSDME molecule was expressed in prokaryotic cells in vitro.Through the in vitro bacterial binding experiments,it was found that the recombinant protein could bind to Gram-negative bacteria,Gram-positive bacteria and different degrees of binding activity.The binding activity to V.splendidus,Micrococcus luteus and Yarrowia was higher than that to Staphylococcus aureus,Escherichia coli and Pichia pastoris.Bacterial clearance experiments showed that the recombinant protein of the gasdermin-N domain of CgGSDME molecule had significant clearance activity against V.splendidus,E.coli,M.luteus and S.aureus.In vitro antibacterial activity(colony counting method)experiments show that it has a certain inhibitory effect on V.splendidus,E.coli,M.luteus and S.aureus.In vitro antibacterial activity(growth curve method)experiments showed that the recombinant protein of the gasdermin-N domain of CgGSDME molecule had a certain inhibitory effect on V.splendidus and S.aureus.The above results indicated that haemocytes could undergo pyroptosis,and CgCaspase-8-2 and CgGSDME were key molecules in the pyroptosis pathway of haemocytes in C.gigas.And identified Caspase-8-2/GSDME cell pyroptosis signaling pathway in C.gigas.Proved the existence of biologically active GSDME in invertebrates.Revealed GSDME activation mechanism and the occurrence of pyroptosis in C.gigas.Discovered GSDME involved in the occurrence of pyroptosis and inflammatory responses induced by pathogenic bacteria in C.gigas.In addition,the recombinant protein of the gasdermin-N domain of CgGSDME can recognize and bind a variety of pathogenic bacteria and can inhibit the survival and growth of bacteria.This discovery enhances the understanding of the evolution and function of the programmed death-associated gasdermins proteins and adds to the gap in research on pyroptosis in invertebrates,as well as providing a theoretical basis for future research on programmed death in invertebrates by identifying for the first time the mechanism of cellular scorch death in bivalves. |
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