Study On The Mechanism Of Innate Immune Pattern Recognition, Signal Transduction And Antimicrobial Peptide Genes Expressional Regulation In Silkworm, Bombyx Mori

Bombyx mori, domesticated from wild silkworm, is a fusule insect with great economic value. However, the annual losses of China caused by silkworm disease are about millions, and seriously affect the sustainable development of the industry. Researches show that when insects are infected with microbes, various of antimicrobial peptides are induced and produced to kill the invading microorganisms. Thus the expression of these antimicrobial peptide genes is mainly regulated by Toll and Imd signal pathways. We focused on the immune-related genes such as short type peptidoglycan recognition protein 3(BmPGRP-S3) andβ-glucan recognition protein 3,4 (BmβGRP3,4), as well as the key genes BmSpzl and Bmlmd located in the Toll and Imd pathways, in order to explore the signal transduction of humoral immune and the expressional regulation mechanism of antimicrobial peptide genes in B. mori. The below are results obtained.1. The cloning and expression analysis of genes coded by pattern recognition receptorsThe pattern recognition receptors, which participate in pattern recognition in the upstream of humoral immune-Toll and Imd pathway, meanwhile regulate the activation of signal factors downstream and the expression of antimicrobial peptide genes. We do researches on the pattern recognition receptors of peptidoglycan recognition proteins andβ-glucan recognition proteins.10 PGRPs and 4βGRPs are cloned, and we obtain the complete coding sequences. All theβGRPs have signal peptide. BmPGRPl-3 are located in the chromosome 11 closely, the glucan-binding region is not detected, whereas BmPGRP4 is located in the chromosome 22 independently, including typical glucan-binding domain. For gene family of PGRPs, typical amidase activity domain is predicted regardless of long-and short- subfamily. Short type subfamily has signal peptide, but long type subfamily hasn't. Five genes of long type subfamily are located in chromosome 1 closely. Of short type subfamily,2 and 3 located in chromosome 9 and 16, respectively. RT-qPCR analysis of the silkworm larvae samples which were collected 3,6,12 and 24 hours after challenged with Escherichia coli, Bacillus bombysepticus, Beauveria bassiana, respectively, showed that two long-type BmPGRP-L1,-L3 and three short-type BmPGRP-S1-3 could be up-regulated by gram-positive bacteria, gram-negative bacteria and fungi infection, respectively. Meanwhile, the induced expression profile among different tissues indicated that all the 13 receptor genes shared different expression patterns in head, integument, midgut and fat body challenged by gram-positive bacteria, gram-negative bacteria and fungi, respectively, many of them were induced in head, less were induced in integument and midgut, least in fat body.2. Study on the function of BmβGRP3There are three Gram-negative bacteria binding protein genes contained in the genome of Drosophila, two of them play a role in pattern recognition in the upstream of Toll signal pathway. It was confirmed that BmβGRP1 and BmβGRP2 can bindβ-1,3-glucan and active cellar immune response in B. mori. However, whether they participate in pattern recognition in upstream of Toll or IMD pathway is not detected.BmβGRP3 has an open reading frame of 1470bp, encoding a protein with 489 residues. The 3'UTR contained a consensus polyadenylation signal (aataaa) upstream from the poly(A) tail. The deduced protein contained a putative signal peptide predicted by SignalP software in the N-terminal region. The calculated molecular mass of the mature peptide was about 53KD and theoretical pI was 6.25. To find pattern recognition receptors in the upstream of humoral immune pathway, we make focus on the study of BmβGRP3. We found the function region of bindingβ-1,3-glucan was conserved in amino acid sequence. BmβGRP3 can be upregulated (the level of individual and issue) by infecting with gram-positive, gram-negative bacteria and fungi, respectively. It shows that BmβGRP3 has different expression patterns and certain issue specificity responding to the induction of different miroorganisms. The RNAi knock-down BmβGRP3 indicates that it plays an important role in the innate immune response in B. mori. Further study suggests that Toll signal pathway can regulate the expression of some antimicrobial peptide genes. While RNAi knock-down BmβGRP3, the expressions of Toll pathway signal factors, such as BmSpzl and antimicrobial peptide genes CecAl,CecE,Att2 and Enbl decrease, which shows Toll signal pathway regulates the expression of antimicrobial peptide genes CecA1,CecE, Att2 and Enbl. On the other side, the expression of Bmlmd, IMD signal pathway signal factor, and antimicrobial peptide genes CecB, CecD and Glv2 was not affected, which suggests that there is other immune pathway, for example IMD signal pathway, may regulate the expression of antimicrobial peptide genes CecB, CecD and Glv2.According to the induction expressions of BmSpzl and BmImd, Toll and Imd signal pathways can be activeted by gram-positive bacteria, gram-negative bacteria and fungi, respectively, which is significant different to Drosophila. The Toll signal pathway is only activated by gram-positive bacteria and fungi, the Imd signal pathway can be activated by gram-negative bacteria and certain gram-positive bacteria. It is reasonable that Bombyx mori obtain more efficient immune response mechanism when face to unique growth and development environment in long-standing evolution.3. Prokaryotic expression and function analysis of BmβGRP4 and BmPGRP-S3In this research, we found that BmβGRP3 is located the up-stream of Toll signal pathway and regulate the expression of some antimicrobial peptides genes, such as CecAl, CecE, Att2 and Enbl. However, it is yet to be studied further about the Imd pathway in immune activation and regulation of expression of antimicrobial peptides.We look forward to studying on the BmβGRP4 and BmPGRP-S3, to clarify their specific biological functions in humoral immune response. We constructed BmβGRP4 and BmPGRP-S3 prokaryotic expression vectors, purified the two proteins, and obtained the polyclonal antibodies. The expression of BmβGRP4 and BmPGRP-S3 can be obviously increased by gram-negative bacteria, gram-positive and fungi induced, respectively, in both transcriptional and translational levels. RNAi knock-down BmβGRP4 causes that the expression of BmPGRP-S3 is repressed in both transcriptional and translational levels. However, RNAi knock-down BmPGRP-S3 does not affect the expression of BmβGRP4 in both transcriptional and translational levels. So we speculate that BmPGRP4 is located in the upstream of BmPGRP-S3 to exercise their functions. RNAi knock-down BmβGRP4 or BmPGRP-S3 causes repression of signal transduction factor BmImd and antimicrobial peptides genes, including CecB, CecD, Moricin and Glv1,2, which indicates that the expressions of these antimicrobial peptide genes are mediated by Imd pathway. On the other hand, repressions of Toll pathway signal factor BmSpzl and antimicrobial peptides CecAl, CecE, Att2 and Enbl are not observed. Interestingly, the expression level of Enbl is even raised, which proves that Toll and Imd signal pathways independently mediate the expression of antimicrobial peptide genes.Moreover, RNAi knock-down BmβGRP4 or BmPGRP-S3 causes repression of BmClip15, BmFrep3, BmGaletin3 and BmSpn12. BmFrep3 is a fibrinogen-related protein family member which related with invertebrate innate immunity, it contains a fibrinogen domain in the C-terminal with the ability of bacteria and protozoan recognition. Three family members have been found in B.mori; BmGaletin3 belongs to Galactose lectin gene family, which contains a conserved carbohydrate recognition domain with the ability ofβ-galactosidase sugar binding. Galactose lectins are deemed to microbe recognition and phagocytosis. BmClip15 and BmSpnl2 are serine protease and serine protease inhibitor, respectively, which involved in cascade control of innate immune signal transduction by amplifying or reducing the signal. So we can speculate that BmPGRP4 and BmPGRP-S3 also may participate in the cellar immune response except for mediating Imd pathway.4. Functional research of BmSpzl and BmlmdWe identified the function of pattern recognition receptors-BmβGRP3,BmβGRP4 and BmPGRP-S3 in innate immune response in B.mori. BmβGRP3 mediates Toll pathway, Bm(3GRP4 and BmPGRP-S3 mediate Imd pathway. The expression of antimicrobial peptide genes CecAl, CecE, Att2, Enbl and CecB, CecD, Moricin, Glvl,2 are regulated via Toll and Imd pathway, respectively. In order to identify the antimicrobial peptide genes regulated via Toll and Imd pathways, and further confirming the transduction mechanism and antimicrobial peptide genes expression pattern, we performed RNAi knock-down BmSpzl and Bmlmd, which are the key genes in Toll and Imd pathways.Both BmSpzl and BmImd can be induced be Gram-negative bacteria, positive bacteria and fungi, respectively. Western blotting analysis shows that the precursor and mature proteins of BmSpzl also can be induced be Gram-negative bacteria, positive bacteria and fungi, respectively. RNAi knock-down BmSpzl represses the expressions of signal factors, BmTube and BmRel in Toll pathway, and the antimicrobial peptide genes of BmCecropin-A1, BmCecropin-E, BmAtt2 and BmEnbl. Simultaneously, RNAi knock-down BmImd represses the expressions of signal factors, BmDreed, BmRelish in Imd pathway, and the antimicrobial peptide genes of BmCecropin-B, BmCecropin-D, BmMoricin and Bmgloverinl,2.The fact that Toll pathway regulates antimicrobial peptide genes CecAl, CecE, Att2 and Enb1, the Imd pathway regulates antimicrobial peptide genes CecB, CecD, Moricin and Glvl,2 has been proved via studying on BmSpzl and Bmlmd. So we speculate that Toll and Imd pathway work separately in regulation of interrelated antimicrobial peptide genes.5. The mechanism of antimicrobial peptide gene expression in B. moriAntimicrobial peptides are kinds of minor polypeptides which are expressed during the insect self-defence, and which have played an important role in insect immunity. In the whole genomes, there are a lot of antimicrobial peptides have been expressed in the silkworm compared with other insects, and a great majority of them are exist in multi-gene family forms, most of them are coordinated expression. Yamakawa et al had analyzed 7 species of antimicrobial peptides in silkworm, which have one or more KB-like and GATA components. Moreover, some have CAATW components, which may be helpful in the starting frequency of gene transcription. In addition, the antimicrobial peptides in silkworm have much higher activity of bacteria induction and self-defence. But so far, the mechanism of antimicrobial peptide genes expression is still unclear. In this study, we propose a possible mechanism about antimicrobial peptide genes expression and regulation.Firstly, Toll signal pathway is stimulated by pattern recognition receptor BmβGRP3 which recognizes the different pathogens and activates the other signal factors in this pathway, such as extracellular factor BmSpzl, transmembrane factor Toll receptor and intracellular factors BmMyd88,BmTube, to result in BmRel ingress intranuclear and then binding with the expression regulation region of antimicrobial peptide genes (KB-like and GATA components), so that the Toll pathway regulates the expression of those antimicrobial peptide genes:CecAl, CecE, Att2 and Enb1.Secondly, Imd signal pathway is stimulated by pattern recognition receptors BmβGRP4 and BmPGRP-S3 which recognize the different pathogens and activate the related signal factors in Imd pathway, to result in BmRelish ingress intranuclear and then binding with the expression regulation region of antimicrobial peptide genes (KB-like and GATA components), so that the Imd pathway regulate the expression of those antimicrobial peptide genes:CecB, CecD, Moricin and Glvl,2. While Toll and Imd signal pathways independently mediate the expressional regulation of antimicrobial peptide genes. So here we only provide a preliminary mechanism of silkworm antimicrobial peptide genes expressional regulation, deeper and more specific understanding of the mechanisms need further studies to confirm.