Functional Characterization And Mechanism Studies Of BmCaspase-8-like, A Negative Regulator Of Immunity In Silkworm, Bombyx Mori

Insect immunity consists of humoral and cellular immunity.The humoral immunity mainly depends on two NF-?B signaling pathways: Toll and IMD signaling pathways.Upon activation by invading pathogens,the immune signals lead to translocation of NF-?B family transcription factor from cytoplasm to the nucleus to activate the expression of antimicrobial peptide genes.However,excessive activation of NF-?B signaling pathway results in impaired physiology,including shortened lifespan,intestinal flora disorders or neurodegeneration.Therefore,immune negative regulator is critical for maintaining the homeostasis.Several immune negative regulators have been identified in Drosophila,but few related studies have been reported in other insects,so the study on the immune-negative regulation of silkworm,a lepidoptera model insect,is of great importance.In this study,in order to characterize BmCaspase-8-like(BmCasp8L)as a negative regulator of silkworm immunity,we first performed sequence analysis,domain prediction,phylogenetic analysis,spatio-temporal expression profile and immune-induced expression profile analysis,then examined the effect of over-expression or knock-down of BmCasp8 L in cells as well as in larvae to the expression of antimicrobial peptides.To understand its molecular mechanism,we over-expressed BmCasp8 L and evaluated the cleavage of BmIMD and BmRelish under the stimulation of DAP-PGN or co-expression of BmDREDD.We also used ThT staining,SDD-AGE(semi-denaturing detergent agarose gel electrophoresis)and confocal microscopy to identify the amyloid aggregates formed by BmCasp8 L alone or with BmDREDD,Finally,we performed co-immunoprecipitation to verify the interaction between BmCasp8 L and BmDREDD.The main results are as follows: 1.BmCasp8 L is a negative regulator of innate immunity of silkwormBmCasp8L is homologous to BmDREDD,DmDREDD and human Caspase-8.The similarity between BmCasp8 L and N-terminal of BmDREDD and DmDREDD is 61% and 42%,respectively.Domain analysis showed that it contains two tandem DED domains,but lacks the C-terminal Caspase domain.Bmcasp8 l was highly expressed in immune tissues such as fat body and hemocyte in the 3rd day of 5th-instar larvae.But in the prepupa stage,it was mainly expressed in the silk gland.Spatio-temporal expression profile showed that in molting larvae Bmcasp8 l level was higher than in newly exuviated ones.The expression level in the developmental metamorphosis period,such as the prepupa,the 7th day of pupa,and the 1st day of moth,was relatively higher than the other stages.Bmcasp8 l expression level increased within 1 h post infection by Bacillus bombyseptieus or Serratia marcescens,and then gradually returned to normal.Over-expression of Bmcasp8 l in BmE cells led to a significantly decrease of the anti-microbial peptides BmCecropinA1,BmCecropinB,and BmAttacin,while down-regulation of Bmcasp8 l by RNAi led to an increase of BmCecropinA1,BmCecropinB,BmAttacin.Knock-down of Bmcasp8 l by dsRNA injected into the 3rd day of 5th-instar larvae also led to an up-regulation of BmCecropinA1.2.BmCasp8 L inhibit the IMD pathway by formed aggregates with BmDREDDWestern blotting was used to detect the cleavage of BmIMD and BmRelish when BmCasp8 L was over-expressed,and revealed that BmCasp8 L suppressed the cleavage of BmIMD and BmRelish performed by BmDREDD.TUNEL staining showed that BmCasp8 L dose-dependently inhibited BmDREDD-induced apoptosis.Furthermore,SDD-AGE,ThT fluorescence and confocal microscopy demonstrated that BmCasp8 L formed amyloid aggregates both in cells and in vitro.BmCasp8 L can also form amyloid aggregates with BmDREDD,which inhibited the activation of BmDREDD.Although BmFADD did not form amyloid aggregates alone,it can form amyloid aggregates with BmDREDD or BmCasp8 L.Co-IP experiment finally proved that BmCasp8 L interacted with BmDREDD as well as BmFADD.