The Mechanism Of GSDME Mediated Cell Pyroptosis In Pathogenesis Of EV71 Infection

Hand-foot-and-mouth disease(HFMD)is a common childhood illness caused by enteroviruses.It is prevalent and reported worldwide,especially in Asia region.Children under five years are the main susceptible population of HFMD.Since the outbreak of hand-foot-mouth disease caused by enterovirus 71 in Fuyang,Anhui province in 2008,the annual number of cases has exceeded million for ten years,ranking first among the C-class notifiable disease in Mainland China.Enterovirus 71(EV71)is the main pathogen causing severe hand-foot-and-mouth disease.EV71 can invade the central nervous system and lead to serious nervous system diseases,including aseptic meningitis,brainstem encephalitis,acute flaccid paralysis and neurogenic pulmonary edema.However,the pathogenesis of EV71 is still unclear,this has seriously hindered the research and development of clinical treatment and antiviral drugs.Currently,there is no specific antiviral drug,which has become one of the urgently public health problems to be solved in China.Therefore,the study on the mechanism of EV71 infection and pathogenesis is important for the prevention and treatment of hand-foot-mouth disease.Viruses need to proliferate in host cells.The interaction between viruses and host cells determines the outcome of virus infection.Different ways of cell death have complicated interactions with virus infection.Studies have shown that cell apoptosis,pyroptosis and autophagy play important roles in the virus infection and replication.Recent reports have shown that influenza virus,Zika virus and enterovirus infection can induce cell pyroptosis.Under a range of microbial infections,the pattern recognition receptor NOD-like receptor recognition the signal in the cell and forms inflammasome,activates caspase-1,the activated caspase-1 cleaves GSDMD and liberates the N-terminal effector domain from the C-terminal inhibitory domain.The N-terminal domain oligomerizes in the cell membrane and forms pore to induce cell pyroptosis.Gasdermin family another member GSDME can also mediate pyroptosis,but converting apoptosis induced by chemotherapy drugs into pyroptosis.This plays an important role in normal tissue damage induced by chemotherapy drugs.We previous study showed that EV71 infection can activate NLRP3 inflammasome,inducing secretion of IL-1? and IL-18,and virus encoding 3C or 2A in turn cleavage NLRP3 and thus inhibit activation of NLRP3 inflammasome.At the same time,3C protease can also cleavage GSDMD,destroying the function of N-terminal 1-275 fragment that induces cell pyroptosis.However,we found that EV71 infected can still induce cell pyroptosis.Based on this discover,this topic further studies the mechanism of cell pyroptosis caused by EV71 and the role of pyroptosis in replication and pathogenesis of EV71 infection.EV71 infected HeLa,RD and SK-N-SH cells.Through LDH release,ATP activity and microscopic observation of cell morphology,we found that EV71 can induce cell pyroptosis.Western blot results showed that EV71 infection could induce cleavage of endogenous GSDME.In order to further verify the cleavage mechanism of GSDME mediated by EV71,we constructed GSDME expression plasmids with Myc and FLAG-tag.Through exogenous transfection experiments at 293T,it was found that EV71 infection could induce cleavage of GSDME,while exogenous transfection with EV713C or 2A protease could not induce cleavage of GSDME.Some studies show that GSDME can be cleaved by caspase-3,and the cleavage site is D270.we constructed GSDME D270E mutant plasmids and overexpressed it in 293T cells.It was found that EV71 infection could not cause cleavage of GSDME D270E mutant,suggesting that EV7I infection may cause cleavage of GSDME at D270 site.Further study found that EV71 infection can activation of endogenous caspase-3,and in caspase-3 knock-out cell,EV71 infection could not induce cleavage of GSDME,indicating that EV71 infection caused cleavage of GSDME at D270 site through activation of caspase-3.In order to further study the role of caspase-3 and GSDME in EV71-induced cell pyroptosis,we constructed caspase-3,Gsdme gene single and double knock-out cell lines.The results showed that compared with wild-type cells,the proportion of cell pyroptosis caused by EV71 infection was significantly reduced in knock-out cell lines,further proving that EV71 infection caused GSDME cleavage by activating caspase-3 to induce cell pyroptosis.The role and pathogenesis of GSDME-mediated cell pyroptosis in viral infection have not been reported.We infected wild-type and Gsdme knockout mice with EV71 and found that the survival rate of Gsdme knockout mice was higher than that of wild-type mice,suggesting that GSDME plays an important role in the pathogenesis of EV71.And we found that the release of inflammatory factors IL-6,TNF-? and MCP-1 in serum of Gsdme knockout mice decreased.Extraction of bone marrow macrophages from wild-type and Gsdme knockout mice,EV71 infection,found that the release of inflammatory factors in Gsdme knockout cells decreased,and the proportion of cell pyroptosis decreased.In addition,we previous research found that Golgi apparatus protein ACBD3 participates in enterovirus replication,so whether other proteins in ACBD3 complex participate in virus replication,we found a member of this complex PEF1,also affects EV71 replication.PEF1 belongs to penta-EF-hand protein family and is an important host factor.It participates in a variety of cell life activities,such as vesicle transport between endoplasmic reticulum and Golgi apparatus,protein ubiquitination,proteolysis,etc.Our results showed that the replication of EV71 is significantly reduced in PEF1 knocked down or knocked out cells.EV71 infection does not affect the expression of PEF1,but it can affect the intracellular distribution of PEF1 and reduce its co-localization with ACBD3.The specific mechanism needs further study.In conclusion,EV71 infection can activate caspase-3 to cleavage GSDME and inducing cell pyroptosis.At the same time,knockout of Gsddme in mice alleviates EV71 caused diseases,suggesting that GSDME plays an important role in the pathogenesis of EV71 infection.These study results can provide new theoretical basis for further elucidating the pathogenesis of EV71.In addition,we found that PEF1 is also an important host factor involved in EV71 replication.