A Serine Protease Inhibitor (BmSpn-5)regulates The Immune System Of Silkworm, Bombyx Mori

Insects play vital roles in biologic chain as they have a wide variety of species, and survive in quite different environments. Frequently, insect larvae grow and develop in decaying organic matter, and insect adults often serve as vectors for microorganisms that cause plant and animal diseases. To survive, insects have evolved effective immune system to defend themselves against attacks from pathogens, besides their tough cuticle. Insect immune system is mainly composed of humoral responses and cellular responses. The humoral responses refer to the immune reactions in hemolymph such as recognition of pathogens,protealytic cascades, antimicrobial peptides(AMPs), intermediate products of melanization,etc. The cellular defenses refer to responses like phagocytosis, encapsulation, and nodulation that are directly mediated by heamocytes.AMPs are major effecters of insect humoral responses. They kill pathogen directly and are induced mainly through Toll and IMD pathways. Melanization is another indispensable immune reaction of insect. It is responsible for encapsulating multicellular pathogens,repairing tissues, and defending against pathogens. Phenoloxidase(PO) is the key enzyme of melanization. Upon the recognition of pathogens, Toll pathway and prophenoloxidas(PPO)are activated through a serine protease cascade. Serpin super family represents the largest family of serine protease inhibitors. In insect immunity, Serpins mainly inhibit the serine protease cascade that activating Toll pathway and melanization. In the silkworm, Bombyx mori, the study of serpins were early initiated by Japanese scientists. However, seldom study was carried out there after. It was until the sequencing of silkworm genome that attention was refocused on silkworm serpins. However, these studies are preliminary and the functions of silkworm serpins are still unclear so far. Here, we cloned and expressed silkworm Serpin-5(BmSpn-5) in vitro, investigated its functions in the silkworm immune system. Our results are as follows:(1) We set up a model of encapsulational melanization in the larval silkworm. To examine whether Bm Spn-5 affects encapsulational melanization, we constructed a model using Q-Sepharose Fast Flow(QFF) beads in the silkworm. QFF beads were encapsulated immediately after injecting into hemocoel of silkworm larvae. At 1 hours post injection(hpi),the beads were surrounded by a large number of hemocytes. At 8 hpi, the process of encapsulation was completed with a clear three layers structure. Most of the encapsulated beads are attached with tissues in the hemocoel of silkworm. Some of the encapsulated beads were melanized. Melanin deposited on the surface of the beads but not on the hemocytes around them. The critical period of encapsulation was 1 to 8 hpi. The percentage of melanized beads increased significantly during this stage, and stayed stable afterwards.(2) We analyzed the sequence of BmSpn-5, expressed it in E. coli and successfully purified the recombinant protein. Sequence and phylogenetic analysis indicated that BmSpn-5was orthologous to Manduca sexta serpin-5, suggesting that they might share similar functions. Protein structure prediction indicated that BmSpn-5 was an inhibitory serpin protein. We cloned and expressed BmSpn-5 using pET32 a plasmid in E. coli BL21(DE3)cells. The fusion protein was purified by nickel-affinity chromatography from the soluble fraction, followed by digestion with enterokinase, a second nickel column purification and a Q column for further purification. We obtained the recombinant BmSpn-5 with high purity and raised its antiserum with a high titer.(3) BmSpn-5 is induced by bacterial infection and down-regulates the induction of AMPs. Bacterial infection caused significant up-regulation of BmSpn-5 gene in the fat body of silkworm larvae. We injected recombinant BmSpn-5 into the silkworm hemocoel, followed by bacterial infection. 24 hours after, the plasma antibacterial activity was reduced in the BmSpn-5 injected group. Further, the intensity of protein bands of Gloverin and Cecropin on a tricine-SDS-PAGE decreasd significantly in the plasma samples from BmSpn-5 injected group. RT-qPCR analysis showed the expression levels of gloverin-3, cecropin-D and-E decreased significantly in the silkworm larvae injected with recombinant BmSpn-5 protein.(4) BmSpn-5 down-regulates the melanization by inhibiting the PPO activation. We injected recombinant Bm Spn-5 into silkworm larvae, followed by injection of QFF beads. 4hours later, the percentage of melanized beads in Bm Spn-5 injected group was significantly lower than that in the control group, suggesting that BmSpn-5 is involved in down-regulation of beads encapsulational melanization. Injection of isopropanol-killed Yersinia pseudotuberculosis serotype O: 3 caused systematic melanization of larval silkworm. When pre-injected with BmSpn-5, the silkworm larvae would be avoided of the systematic melanization. BmSpn-5 inhibited PO activation and its complex formation caused by E.coli-PG, LPS and Curdlan. These results indicate that BmSpn-5 is a negative regulater of PPO activation.(5) At last, we identified the target proteases of BmSpn-5 in silkworm hemolymph.Thanks to the property of serpin that it can form a covalent complex with its target protease,by means of immunoaffinity purification and mass spectrometry, we identified that the silkworm clip-domain serine proteases BmHP6 or Bm SP21 formed a complex with BmSpn-5.These results suggest that BmHP6 and SP21 are the cognate proteases of BmSpn-5 and are essential in the serine protease cascade that activates the Toll and PPO pathways.In summary, our study presents a comprehensive characterization and functions of BmSpn-5, and provides clues of the multiple pathways leading to PPO activation and AMPs induction.