The Function On Signaling-regulation And Characterization Analysis Of Toll9 In The Black Tiger Shrimp(Penaeus Monodon)

The black tiger shrimp(Penaeus monodon)is one of the three prawn shrimps in the world and it has an important role in the economic system of Chinese aquatic products.This fishery industry suffers from great challenges due to the environmental degradation and the break-out of various bacteria and virus.As invertebrate,shrimps depend on the innate immune system to defend against pathogens,and Toll-like receptors(TLRs)can be the key component of shrimp innate immunity.Toll-like receptors play the vital role in defending against exogenous microorganisms,which act as the PRRs(pattern recognition receptor,PRR).Actually,TLRs can recognize PAMPs(Pathogen Associated Molecular Patterns,PAMPs)to transduct the invasion signaling into cell nucleus through intracellular signal-cascade,and promote the translation,expression and release of various immune factors followed by the stimulation of innate immunity.The researches on Toll-receptor of P.monodon are the foundation of discovering the immune mechanism against pathogens.This paper taken the ORF full-length of PmToll9 as template and based on mammalian cell line and Drosophila S2 cell line to construct the cell-immunity models,which researched the function on signaling-regulation and characterization of Toll9.Bioinformatic analysis was also conducted to analyze the protein features.Combining with pathogens in-vitro experiments showed that PmToll9 showed a broad spectrum of activities in recognizing PAMPs,and could recognize some kinds of gram-negative bacteria.Microbial infection in-vivo experiments indicated that gram-positive bacteria could up-regulate the expression of PmToll9,but the gram-positive bacteria showed the inhibiting effect.In the cell-immune model,kinds of experiments found that PmToll9 can truly take part in the immune system,because it can regulate the signal-transduction of TLR/Toll pathway through the activation of NF-?B or the antimicrobial peptide(AMPs).The pathogens challenge with PIC,PIH,ODN2006,LPS,R848,CL097,Rec FLA-ST and PGN confirmed that these ligands can't be directly recognized by PmToll9,which might need the participation of Spatzle.The construction of LRR-deletion mutants confirmed that these mutants could impair the ability in activating NF-?B of PmToll9.Among them,the deletion mutants of LRR4 and LRR10/11/12 showed the most significant down-regulation,which indicated that LRR4 and LRR10/11/12 might be the key domain in recognizing PAMPs of PmToll9.The specific research achievements were described as follows:1.The prokaryotic expression and binding properties of PmToll9Compared with the prokaryotic expression system induced by IPTG,the auto-induction expression system was more suitable to extract and pure the recombinant protein of PmToll9 and Toll9-ED.ELISA detection showed that Toll9-ED has strong activities in binding to PIC,PIH,ODN2006,LPS and Resoquimod,which confirmed that Toll9-ED has a broad spectrum of activities in recognizing PAMPs.Toll9-ED could combine with gram-negative bacteria,but fail to Aeromonas hydrophila and Staphylococcus aureus,which indicated that Toll9-ED might act as PRRs to recognize and defense some gram-negative bacteria.Bacteria stimulation in vitro showed that Streptococcus agalactiae can significantly activate the expression of Toll9,while Vibrio harveyi could inhibit it.It revealed that gram-positive bacteria can promote Toll9 to start the Toll signal pathway,but gram-negative bacteria inhibits it.2.The induction effect of mammalian TLR signal-pathway by PmToll9The structural analysis showed that PmToll9 has 12 LRRs in the extracellular domain,has no signal peptide,but has 12 N-glycosylation and 119 phosphorylation prediction sites.The PmToll9-TIR domain showed the highest similarities with DmToll9 and AgToll9.We also found that the concave surface of PmToll9 presents a smooth,curved ?-sheet structure,which is similar with the horseshoe shape as mammals.We found PmToll9 could successfully express a recombinant eukaryotic protein in HEK293 T cells using western blot technology.Dual-luciferase reporter system found PmToll9 could significantly activate NF-?B reporter gene,but fail to activate ISRE and IFN-? when transfected with 200 ng DNA amounts,which is the most efficient transfection does in HEK293 T cells.qRT-PCR data also identified that PmToll9 can activate TLR signaling pathway and promote the high expression levels of the downstream factors,MyD88,TRAF6,I?B-?,IL-8 and IL-10.Six LRRs-deletion mutants were constructed and the data showed these mutants had obvious declines in luciferase activities and the mutant pCMV-De LRR4 showed the most significant decline.qPCR data indicated that LRRs-deletion mutants efficiently impaired the activities of the downstream immune factors IL-8,I?B-?,and TRAF6.It demonstrates that LRRs-deletion mutants could result in the weaken abilities of PmToll9 in signaling transduction.Overexpression of PmToll9-GFP fusion protein in Hela cells revealed the primary cellular localization of PmToll9 is in the cytoplasm.3.The activation and expression of Drosophila Toll signal pathway by PmToll9We constructed the in-vivo expressional model based on Drosophila S2 cells,dual luciferase reporter assay showed that PmToll9 could obviously activate the promoters of antimicrobial peptides(AMPs),the penaeidin 453/536 from P.monodon and Drosomycin from Drosophila melanogaster respectively.And the overexpression of PmToll9 could up-regulate the mRNA expression of Tube/Cactus/Dorsal/Dif/Drosomycin,suggesting that these immune-related factors are involved in the PmToll9-mediated signal pathway.The PAMPs stimulation experiments showed that reporter genes could be activated and downstream factors presented differential expressional levels in S2 cells after challenged with PGN,PIC and PIH.According to these results,we speculated that the signaling transduction pathway activated by PmToll9 is Toll/Sp?tzle/NF-?B/Tube/Cactus/Dorsal/Dif/Drosomycin.LRRs-deletion mutants experiment showed that the deletion of LRR4 and LRR10/11/12 significantly impaired the activities of PmToll9,while the deletion of LRR3 increase the activities,indicating that important functional or recognized region were exist in LRR3/4/10/11/12 and they might play important role in the ligand recognition process of PmToll9.This paper discovered the function on signal-regulation of TLR signaling pathway in mammals and Toll signaling pathway of Drosophila,and the immune characteristics on pathogens defense of PmToll9.We have obtained a lot of achievements and they will lay a great theory foundation for the researches on the mechanism of disease-resistance furtherly.