Mechanism Of Pyroptosis Pathway In Teleost And Hydra

Pyroptosis is a type of programmed cell death.It participates in the process of immune cells to resist pathogen infection.The pyroptotic immune cells exhibit cell membrane blebbing and the release of cytoplasmic content,which leads to a stronger immune response namely inflammation.Also,pyroptosis plays an important role in the inflammatory response caused by pathogen infection,as well as in many human diseases,such as some genetic diseases,autoimmune diseases and cancer.At present,the research on the mechanism and physiological function of pyroptosis has become a research highlight in the field of cell death.However,unlike apoptosis,the research of pyroptosis is almost carried out in mammals from discovery to follow-up mechanism research,but not from nonmammals such as nematodes,flies and zebrafish.Therefore,the phenomenon,molecular mechanism and physiological function of pyroptosis in nonmammalian cells are scarcely investigated.In addition,Gasdermin(GSDM)proteins are widely found in nonmammalian,but the number of GSDM protein in nonmammalian is decreased sharply,which is very different from the discovery in mammals.These clues indicate that the pyroptosis pathway in nonmammalian is significantly different from that of mammalian.In view of this,this work tried to understand the mechanism of pytoptosis in lower animals including Paralichthys olivaceus,Scophthalmus maximus and Danio rerio.Using bioinformatics,biochemistry,cell biology and genetic manipulation,this work revealed the molecular mechanism of JfNLRP3 inflammasome mediated pyroptosis pathway in Paralichthys olivaceus and its preliminary significance in antiinfection.Moreover,we found the molecular mechanism of Scophthalmus maximus SmCaspase mediated pyroptosis signal pathway,which is involved in the activation of both canonical and noncanonical inflammasome.At the same time,we revealed the biochemical mechanism of zebrafish GSDME protein,which is cleaved and activated by a variety of inflammatory and apoptotic caspase proteins.Firstly,we found canonical inflammasome may play an important role in the pyroptosis model of Paralichthys olivaceus caused by Edwarsiella piscicida infection.Then we searched the upstream signal molecule of canonical inflammasome in Paralichthys olivaceus by bioinformatics screening.A series of bioinformatic analysis and biochemical experiments confirmed that JfNLRP3 inflammasome activated JfCaspase-1,then the active JfCaspase-1 cleaved JfGSDME which inducing pyroptosis.Meanwhile,two key genes of pyroptosis pathway,JfNLRP3 and JfCaspase-1,were knocked down or overexpressed in vivo to investigate the role of this pathway in the resistance of Edwarsiella piscicida infecting.Secondly,we found that noncanonical inflammasome played a decisive role in the pyroptosis model of Scophthalmus maximus.Subsequently,through similar bioinformatics analysis and biochemical experiments,we revealed the dual function of Scophthalmus maximus SmCaspase protein in pyroptosis pathway.SmCaspase could be activated by directly combining with LPS,meanwhile,it was activated by canonical inflammasome through binding with SmNLRP3 and SmASC.The active SmCaspase then cleaved SmGSDMEa and SmGSDMEb to liberate the N termini,and however only the N-terminal of SmGSDMEb triggered pyroptosis.Moreover,after knocking-down of SmNLRP3,SmCaspase and SmGSDMEb in vivo,turbot los the resistance to the infection of pathogen.Finally,in the exploration of the activation mechanism of zebrafish GSDME,we expressed and purified zebrafish caspase protein and GSDME protein and then mixed them to detect the cleavage of GSDME.The results showed that zebrafish GSDMEa/b was cleaved by inflammatory caspase protein caspase-19b,apoptotic effector protein caspase-3a/b and caspase-7,and apoptotic initiation protein caspase-8a/b.The pyroptosis pathway in teleost,based on our data,exhibited more variety.Whether there are more mechanisms of pyroptosis in lower invertebrates is worth exploring.Thus,we analyzed the evolutionary origin of GSDM protein family,the executor of pyroptosis.Our results showed the distribution of GSDM protein in lower animals,and found an ancient GSDM protein with potential cytotoxic function in cnidaria.Given that,Hydra vulgaris,the representative species of cnidaria,was selected as the research object to explore the mechanism of pyroptosis pathway in ancient invertebrate.In the evolutionary analysis of GSDM protein family,we found that GSDM protein not only exists in mammals,but also widely distributed in lower animals,and the most primitive ancestor of GSDM protein is PJVK protein on Placozoa.Similar to mammalian PJVK protein,the PJVK protein on Placozoa has only 250 amino acids and lacks the C-terminal self-inhibitory domain,thus it is considered to possess no ability to induce pyroptosis.However,Cnidaria,the relatives of Placozoa,harbor another GSDM protein except PJVK,and this GSDM show potential cytotoxicity due to the complete N-terminal and C-terminal domains.Therefore,Cnidaria may be the first species with the ability of pyroptosis.According to these clues,this paper aimed to explore the molecular mechanism of pyroptosis in primitive lower species using Hydra vulgaris,a representative species of Cnidaria.Our results showed that the N-terminal fragment of HvGSDME could significantly induce pyroptosis and bactericidal activity.Four different apoptotic caspase proteins,namely caspase-B,caspase-C,CardCaspase-1 and CardCaspase-2,can directly cleave GSDME and liberate its active N-terminal.In addition,the apoptotic inducer trigger pyroptosis like cell death in hydra cells including cell swelling and the cleavage of GSDME protein.These results confirm that the ancient invertebrates with a history of 680 million years ago possess the ability of pyroptosis,and the signaling pathway of pyroptosis may be more complex.Taken together,this work revealed the molecular mechanism of pyroptosis signaling pathway in lower animals including teleost and ancient invertebrate,and preliminarily explored the physiological functions of pyroptosis signaling pathway in some species.In this study,we found that the molecular mechanism of pyroptosis signaling pathway in lower animals are both conservative and diverse.Lower animals are mechanism similar to mammals in the inflammasome mediated pyroptosis pathway,however some species,such as turbot,show a little uniqueness.The same caspase of turbot simultaneously participates in canonical and noncanonical inflammasome activation.In the activation mechanism of GSDM protein,lower animals showed more diversity,for instance,the same GSDM protein was cleaved by both inflammatory and apoptotic caspases at the same site.In addition,in ancient invertebrate,GSDM protein could not be directly cleaved by inflammatory caspase.The unique features revealed in the lower animal pyroptosis signaling pathway expand the molecular mechanism of cell pyroptosis pathway,and also provide reference for the study of mammalian pyroptosis pathway.