The Expression Characteristic And Function Analysis Of The Secreted Protein NbSPN9 Of Nosema Bombycis

Nosema bombycis is the pathogen of pebrine disease of silkworm,which is also the only quarantine object in silkworm egg production.As an obligate unicellular and intracellular parasitic eukaryotic microorganism,N.bombycis is a good model for studying the interaction between pathogen and host.Microsporidia parasitize in host cells and relies on the nutrition and energy of the host cells for reproduction.The secreted proteins of microsporidia play an extremely important role in infecting the host,resisting host immunity and regulating the host’s physiological metabolism.Preliminary research in the laboratory identified 19 Serpin(serine protease inhibitors)family genes(NbSPNs)from the N.bombycis,among which 8 Serpins genes encode proteins with signal peptides,including NbSPN9.Serpin is a superfamily of serine protease inhibitors containing a SERPIN domain,which is widespread in various organisms such as humans and N.bombycis.Serpin is mainly involved in the regulation of important biological processes such as blood coagulation,cell apoptosis,and immunity.It has a strong inhibitory effect on the activity of serine proteases such as trypsin and chymotrypsin.From the perspective of the interaction between pathogen and host,this study explored the localization characteristic and biological effects of the secreted protein NbSPN9 of N.bombycis,the main results are as follows.1.Functional analysis of NbSPN9 signal peptide of N.bombycisThe NbSPN9 gene of N.bombycis has a full length of 1179 bp,which encodes a protein containing 392 amino acid residues,with a molecular weight of 46.04 k Da.The amino acids 1-21(position)of the NbSPN9 protein are the predicted signal peptide sequence,and the amino acids 72-378(position)are the conserved serine protease inhibitor domain(SERPIN domain).NbSPN9 is predicted as a secreted protein in the Eu Sec Pred online platform.In order to verify the functionality of the NbSPN9 signal peptide,the signal peptide gene sequence of the NbSPN9 gene was cloned into the yeast signal peptide trap vector p SUC2T7M13 ORI,and the recombinant plasmid was transformed into the sucrase-deficient yeast YTK12.Compared with the positive control and the negative control,it is found that the signal peptide sequence of NbSPN9 can induce the secretion of the target protein sucrase into the yeast cells,and promote the growth of the transformed yeast on the YPRAA medium.The sucrose is hydrolyzed into monosaccharides,and the monosaccharides react with TTC to produce red waterinsoluble TTF.The results show that the signal peptide of NbSPN9 is functional,and the NbSPN9 protein may be secreted out of the cell during the proliferation of N.bombycis,and act as an effector molecule.Indirect Immunofluorescence Assay(IFA)analysis was performed on the infected cells with the prepared NbSPN9 antibody,and it was found that NbSPN9 was localized in the cytoplasm of the infected cells.In addition,in order to clarify the localization characteristics of NbSPN9 in infected cells,we constructed the p SL1180-NbSPN9-V5-His eukaryotic expression vector,overexpressed the fusion protein in Bm E cells,and found that NbSPN9 was also localized in the Bm E cytoplasm using IFA.The above results indicate that NbSPN9 can be secreted into the host cytoplasm to play a regulatory role,further confirming the secretion characteristics of NbSPN9.2.Expression and localization analysis of the protein NbSPN9 of N.bombycisThrough RT-q PCR analysis,it was found that the NbSPN9 gene was transcribed in the silkworm hemolymph infected with N.bombycis at 2-6 dpi.The NbSPN9 gene was continuously transcribed from 6 h to 96 h after N.bombycis infecting the cells,and the transcription amount was the highest at 24 hpi.The transcription level gradually rose then fall down.It is speculated that NbSPN9 may play an important regulatory role in the process of N.bombycis infection of host cells.In order to further determine the functional tissues of NbSPN9 in the silkworm infected with N.bombycis,we collected the midgut,fat body and hemolymph of the infected silkworm,and detected the expression of NbSPN9 by Western blot.It was found that in the midgut,the expression of NbSPN9 was continuously detected 1 to 6 days after infection.In the fat body,the expression of NbSPN9 can be detected 2 to 5 days after infection.In the hemolymph,the expression of NbSPN9 can be detected 5-6 days after infection.Among them,the fat body and hemolymph are important tissues for the silkworm immune defense.It is speculated that NbSPN9 may participate in the serine protease cascade,inhibiting the melanization of the silkworm hemolymph or inducing the expression of antimicrobial peptides and other immune-related pathways,thereby regulating the host immune response.3.Analysis of the the regulatory effect of NbSPN9 on host immunityThe Bm E transgenic cell line stably overexpressing NbSPN9 was successfully constructed.The analysis of NbSPN9 transgenic cells using RNA-seq technology revealed that the expression of 1417 genes has changed significantly,of which 649 genes are up-regulated and 768 genes are down-regulated.After enriching the differentially expressed genes by KEGG pathway,41 genes related to the immune system are differentially expressed.After verifying these immune-related differential genes by RTq PCR,it was found that after overexpression of NbSPN9,the expression levels of Bm MBP and BGRP2,Bm HP21 in the PO cascade reaction pathway,and antimicrobial peptide genes Gloverin1 and Cecropin A were significantly down-regulated,Which is consistent with the RNA-seq results.Furthermore,under the conditions of infection with Staphylococcus aureus,the host immunity was activated.The analysis of specific genes by RT-q PCR revealed that the serine protease genes(Bm HP14,Bm HP6 and Bm HP21),Bm Toll9-1 and antibacterial genes(Cecropin A and Gloverin1)in NbSPN9 transgenic cells are still significantly down-regulated.These results further indicate that NbSPN9 has the function of regulating the prophenoloxidase activation cascade and the Toll signaling pathway,which effectively inhibit the host immune response.The NbSPN9 protein was prokaryotically expressed,the recombinant soluble protein was purified,and the inhibitory phenoloxidase activity of the recombinant protein was determined.The results show that NbSPN9 can effectively inhibit the blood phenoloxidase activity of the silkworm,indicating that NbSPN9 can effectively regulate the prophenoloxidase activation cascade of the silkworm and inhibit the hemolympha melanization of the silkworm.Yeast two-hybrid experiment also verified the interaction between NbSPN9 protein and PPAE(Prophenoloxidase-activating enzyme).In summary,NbSPN9 is a secreted protein of N.bombycis,,which can be targeted to the host cytoplasm and has the effect of regulating the host’s immune defense.However,the specific molecular mechanism of regulation needs to be further studied.