The Role Of Bombyx Mori Hemocytin In Bombyx Mori Infected By Nosema Bombycis

Silkworms are the key of sericulture industry,Nosema bombycis(N.bombycis)is one of the most important pathogens that could infect silkworms.N.bombycis belongs to a large group of microsporidian pathogens that belong to the fungal world.Their host range is extremely wide and could basically infect all animals,including humans.Since the discovery of N.bombycis in 1857,more than 1,400 microsporidian pathogens have been identified so far.Silkworms recognize pathogens through immune recognition molecules.Among them,a class of immunorecognition protein,Hemocytin,may play key role in combating N.bombycis infection.Hemocytin is expressed in the granules of silkworm blood cells(granulocytes),and can recognize pathogens and initiate host immune responses.Here in this study,we found that after the silkworm infected by N.bombycis,the transcription and expression levels of Hemocytin were significantly increased,indicating that Hemocytin participates in the immune responses against N.bombycis.We showed that Hemocytin directly interacted with N.bombycis spores by immunofluorescent assay.We also proved that a spore wall protein,SWP25,may be the key protein during Hemocytin and N.bombycis interaction.We then demonstrated that Hemocytin promoted hemocytes aggregation and melanization.We also proved that Hemocytin can inhibit the proliferation of N.bombycis.The main results are as follows:1.Hemocytin transcription and expression levels are up-regulated after N.bombycis infectionIn order to confirm that Hemocytin can respond to the invasion of N.bombycis,we prepared silkworm hemolymph of fifth instar silkworms.The experimental group is challenged by N.bombycis(5×10~4 spores/head).Hemolymph was sampled from the first day to the sixth day after infection.Relative quantitative PCR analysis showed that the transcription level of Hemocytin was up-regulated after N.bombycis infection.In order to further verify the results,we constructed a pET23b-Hemocytin expression vector to express fragmented Hemocytin protein.The recombinant Hemocytin protein was purified and the mouse-anti-Hemocytin polyclonal antibody by injection of this protein.The following Western blot analysis showed that the protein expression level of Hemocytin in the hemolymph sample was up-regulated.The above results indicated that Hemocytin responds to the invasion of N.bombycis and may participate in the immune response against N.bombycis.2.Hemocytin can directly interact with N.bombycisTo verify whether Hemocytin can identify invading N.bombycis,we observed through IFA that N.bombycis can directly adhere to Hemocytin in the hemolymph sample.To consolidate the results,we co-incubated the prokaryotic expressed recombinant Hemocytin protein and N.bombycis.The results also showed that Hemocytin can bind to N.bombycis and can aggregate the pathogen.We speculated that Hemocytin may recognize N.bombycis through certain proteins on the surface of the pathogen.We sent Hemocytin-treated N.bombycis samples and untreated N.bombycis samples for quantitative proteomics analysis and identified several differentially expressed proteins.The results showed that the spore wall protein 25(SWP25)was one of the most differentially expressed proteins,indicating that SWP25 may be our potential target.Then we verified the interaction between Hemocytin and SWP25through yeast two-hybrid analysis.It proved the direct interaction between Hemocytin and SWP25.The results indicate the molecular basis of host-pathogen interaction.3.Hemocytin facilitates host cellular immune responsesIn order to explore the immune response that Hemocytin involves,the recombinant Hemocytin protein was coupled with agarose gel beads.First,we incubated the beads with hemolymph,and we observed that the Hemocytin-conjugated beads can accelerated the hemocytes agglutination and melanization.We speculated that the immune response initiated by Hemocytin may be related to encapsulation and melanization.Secondly,the Hemocytin-conjugated beads were co-incubated with hemolymph and N.bombycis.IFA results showed that Hemocytin protein could significantly promote the encapsulation and melanization of N.bombycis in hemolymph of silkworm.To further verify the importance of Hemocytin during N.bombycis infection,we used RNAi technology to interfere the expression of Hemocytin in the silkworm,and we demonstrated that the N.bombycis proliferation was significantly increased.Meanwhile,after injecting recombinant Hemocytin protein into silkworm,the proliferation of N.bombycis in the host was significantly decreased.The above results indicated that the downstream response initiated by Hemocytin is essential for the host in combating N.bombycis.Our results also shed lights on the further exploration of novel resistant material against N.bombycis infection.