BmMLs-assisted BmToll9 Directly Recognizes Lipopolysaccharide-induced Innate Immunity Of Bombyx Mori Larvae

Toll-like receptors(TLRs)are one of the most important pathogen recognition receptors(PRRs)in the natural immune signaling of animals.As the oldest family of receptors,TLRs are widely found in coelomate,named after Tolls in invertebrate insects,and TLRs in vertebrates.Tolls and TLRs are key transmembrane receptor protein families that transmit different immune response signals in insects and vertebrates,and their functional differentiation and differential evolution are hot topics at home and abroad.Insect Tolls and vertebrate TLRs recognize pathogen-associated molecular patterns(PAMPs)in different ways.In insects,the pathogen PAMPs activate the serine/threonine protease system and then Toll via the intercellular Sp?tzle.Vertebrate TLRs,as pathogen recognition receptors,can directly identify specific PAMPs.In addition,vertebrate TLRs are only involved in immunity,and insect Toll receptors not only participate in immune response but also play an important role in embryonic development,which indicated insect Tolls and vertebrate TLRs recognize pathogen-associated molecular patterns(PAMPs)in different ways.Imler analysed phylogenetic tree on insect Toll and mammalian TLRs,which shows that the evolution of TLRs belong to two branches: Tolls from Drosophila melanogaster and Anopheles gambiae formed a branch;The TLRs of Mus musculus and Homo sapiens belong to the second branch,except the insect Toll9,which is in the same branch as the mammalian TLR4,and suggested that the insect Toll9 is similar to the vertebrate TLR4 in evolutionary relationship.In addition,we analyzed the molecular structure of Bm Toll9 and TLR4 and found that the structures are remarkably similar.So does insect Toll9 have the same function and mechanism of action as vertebrate TLR4?TLR4 is a central receptor for lipopolysaccharide(LPS)in mammals,and LPS is a common component of the cell wall located at the outermost layer of Gram-negative bacteria.In our previous studies,we found that Bombyx mori was induced by high-purityLPS,and the antibacterial activity of hemolymph was enhanced.At the same time,the transcriptional activity of Bm ML(mammalian MD2 similar protein)superfamily member Bm Esr16 and antibacterial peptide genes Moricin and Cecropin B in the fat body was enhanced by RT-q PCR.These results suggest that LPS can trigger the innate immune pathway of B.mori.The previous literatures and our previous experimental results indicate that the innate immune response mechanism of the Toll pathway in Bombyx mori may be related to Bm Toll9 and Bm MLs proteins.Combining the evolutionary relationship between Toll9 and mammalian TLR4,we hypothesized that the Toll9 function of insects may be similar to that of vertebrate TLR4,which directly identifying the pathogen PAMPs and triggering the innate immunity of the B.mori.In order to verify this hypothesis,the B.mori Bm Toll9 and Bm MLs proteins were introduced.Molecular biological methods such as RT-q PCR,Western-blotting,Co-immunoprecipitation and construction of chimeric vector Bm Toll9-TLR4 were used to investigate whether Bm Toll9 is involved in B.mori innate immunity and whether Bm Toll9 can directly recognize pathogen PAMPs.The major findings were as following:(1)After the B.mori induced by LPS,the lipid-binding protein Bm Esr16(one of the Bm MLs proteins)was obtained.Bioinformatics analysis revealed that Bm Esr16 is similar to mammalian myeloid differentiation protein-2(MD-2),with four conserved cysteines forming two disulfide bonds and abundant ?-Folded structures.Bm Esr16 is capable of binding to LPS.Only the LPS-induced but not E.coli-or PGN-induced antibacterial activity in the hemolymph and AMP gene expression in B.mori larvae were blocked when endogenous Bm Esr16 protein was neutralized by a polyclonal antibody specific to Bm Esr16,indicating that Bm Esr16 plays an important role in LPS recognition and immune pathway.(2)Bioinformatics analysis revealed that Bm Toll9 contains 745 amino acids with a transmembrane domain and a conserved sctllr motif flanking the C-terminus of the extracellular domain,belonging to the V-type TLR.Bm Toll9 is expressed in the midgut,fat body and blood cells of the immune organs.Importantly,only the LPS-induced but not E.coli-or PGN-induced antibacterial activity in the hemolymph and AMP gene expression in B.mori larvae were blocked when endogenous Bm Toll9 protein was neutralized by a polyclonal antibody specific to Bm Toll9.It indicated that Bm Toll9 is involved in the innate immune pathway of LPS-induced B.mori.(3)Bm Toll9 extracellular domain was found to bind to Bm Esr16 and Bm PP by Co-immunoprecipitation and immunofluorescence;Western blot analysis showed that the temporal and spatial expressions of Bm Toll9,Bm Esr16 and Bm PP in the midgut,fat body and blood cells were consistent,suggesting that these three proteins have a coordinated effect in the development of silkworm;Furthermore,Bm Toll9 was overexpressed in Bm N and S2 cells,and Bm Esr16 and Bm PP were added as accessory proteins,respectively.The transcriptional expression of the antibacterial peptide genes Moricin,Cecropin B6 and Lebocin3 was detected under LPS induction.These three experimental evidences suggest that Bm Esr16 and Bm PP can help Bm Toll9 form a dimer in response to LPS-induced immune responses,similar to MD2-assisted TLR4 in response to LPS-induced immune responses in mammals.(4)Construction of the Bm Toll9-TLR4 chimeric receptor and transfection of mammalian RAW264.7.After blocking the TLR4 in RAW264.7 cells,the chimeric receptor Bm Toll9-Hs TIR4 can be induced by LPS with the help of the accessory proteins Bm Esr16 and Bm PP,which leads to the up-regulation of the immune-related factors IL6,TNF? and NO in the cell supernatant.The results showed that Bm Toll9 can directly recognize PAMPs to stimulate innate immunity under LPS induction,which is similar to TLR4 in mammals.In summary,the results of the study lead to the following conclusions: Bombyx mori Bm Esr16 or Bm PP assisted Bm Toll9 in response to LPS-induced B.mori larvae innate immunity,which was similar to mammalian TLR4.This conclusion suggests that there may be a molecular signaling mechanism in the B.mori that directly recognizes the pathogen PAMPs independent of the classical insect Toll pathway.The results of this study will deepen the current understanding of the molecular mechanism of insect immunity,enrich the understanding of the functional differentiation and differential evolution of insect Tolls and vertebrate TLRs in immune signaling,and provide reference for explaining the origin of animal innate immunity and providereference for silkworm resistant varieties.