| Innate immunity is the first line against bacterial infection,which not only plays critical role in maintaining homeostasis,but also critical for activation of the adaptive immunity.The Inflammasome activation is one of the important inflammatory response,which could induce the newly defined programmed cell death,pyropotosis.In mammalian,the cononical inflammasome consists of three part,the Nod-like receptors(NLRs),apoptosis associated speck like protein containing CARD(ASC)and the effector proteins.Among them,ASC is an adapter protein that contains both PYD and CARD domain.During the NLRs sense the PAPMs or DAMPs,they can recruit the ASC through PYD-PYD interaction,then the CARD domain will condense the PYD filament into speck and recruit the dowmstream pro-caspase-1,to form the inflammasome complex,and activate the caspase-1 to induce the maturation and releasing of cytokines,like IL-lbeta and IL-18.However,in teleost,the components of the imflammasome still need to be identified,and their molecular mechanisms during bacterial infection remains poorly understood.Based on the identified inflammasome molecules in mammals,we cloned the related genes which mediate the inflammasome activation,including SmGSDME,caspase-3 and SmASC in turbot through RACE technology.After multiple sequence alignment and evolution analysis,we found that the N-terminal sequences of GSDME in different species are relatively conserved,and the sequence of iSmGSDME in turbot and large yellow croaker are most homologous due to an N-terminal transmembrane domain.The sequence of caspase-3 in turbot is conserved during evolutionary process and is close to Sebastes schlegelii which do not possess transmembrane domain.Moreover,there are PYD and CARD domains in SmASC and the structure is highly conserved compared with mammalian ASC,while the sequence of SmASC is most homologous with ASC in Japanese flounder.Considering the crucial role of ASC in inflammasome assemble,we aim to explore the function of SmASC.Firstly,we found that SmASC could cross-link to form oligomer after DSS treatment.Moreover,overexpression of SmASC could result into obvious speck in HEK293T cells.Oligomer and speck were impaired in the absence of PYD or CARD domain.Secondly,we analyzed the tissue-specific expression of SmASC in vivo,and found that SmASC mainly expressed in liver and muscle.Finally,by utilizing E.piscicida and V.anguillarum to infect turbot via enterocoelia injection,we found the expression of SmASC significantly increased,which reveal that the SmASC might play a critical role in anti-bacterial infection.To further exploring the role of SmASC during bacterial infection,we administrated siRNA plasmid to knockdown the expression of SmASC in vivo,and found that the bacterial burden was significantly increased in SmASC-knockdown fish,compared with the control groups.In addition,when we injected the SmASC-overexpressed plasmid into turbot in vivo,we found that that the bacterial colonization dramatically reduced complared with the control groups.Taken together,we identified the important roles of SmASC during E.piscicida infection,and laid a foundation for further studies to identify the Nod-like receptors and the downstream effector proteins in innate immune response. |
