Studies On The Spore Wall Protein 26 (SWP26) From Microsporidia Nosema Bombycis

Microsporidia are obligate intraceUular,unicellular eukaryote.They can infect almost all of the phyla of the animal kingdom including humans,and constitute important pests for sericulture,fisher or shrimp farms.Recently,in studies of other pathogens,their surface proteins have been shown to play important roles in host tissue infection and invasion,and the pathogenesis of host.For example,the surface proteins of Plasmodium sporozoites and Plasmodium schizozoites could be used as ligands and receptors-binding surface of target cells and participated in the invasion.In addition, more recent research reported that the spore wall protein EnP1 of Encephalitozoon spores was shown to bind host cell surface glycosaminoglycans(GAGs) by way of the heparin-binding motif and be involved in modulating in vitro host cell adherence and infection.Moreover,the in vitro adherence of Encephalitozoon spores to host cells can significantly increase host cell infection.However,the effective isolation and identification of the SWPs is very difficult due to hard extraction of the SWPs,low sequence homology and the finit genetic information.Until now,only eleven SWPs were reported with submitted gene sequences.Of these,eight SWPs have been found in members of the family Encephalitozoonidae and three ones came from Nosema bombycis.In the present study,we first developed monoclonal antibodies(MAbs) against Nosema bombycis spore wall proteins.Subsequently,the immunoprecipitation and Western blot analysis combinding MADLI-TOF-MS analysis were used to isolate and identify the target proteins that reacted specifically with these antibodies.And sequence analysis,gene cloning,prokaryotic expression and immunoelectron microscopy(IEM) analysis were employed.Finally,function analysis of the spore wall protein SWP26 was conducted,thus initially discusses is made on the interactions of SWP26 and host midgut proteins.The results are as follows:1.Preparation and identification of monoclonai antibodies against the spore wall proteins of Nosema bombycisThe BALB/c mice were immunized four times with the total proteins of Nosema bombycis,then the spleen cells of the hyperimmunized mouse and SP2/0 cells were fused with PEG-1500.The cell fusion rate of 13.3%was first obtained,55.4%second obtained.An indirect enzyme-linked immunosorbent assay(ELISA) was developed to detect monoclonal antibodies(MAbs)secreted by hybridoma cell lines.And positive rate of 4.68%were first obtained,5.26%second obtained.Two hybridoma cell lines secreting monoclonal antibodies,named 2G10 and 2B10 respectively,were developed by three subclones.Subsequently,the specificity of McAb against N.bombycis' spore wall proteins was identified by means of IFAT and Western-blot(WB).The MAb 2G10 IFA showed that intense flavor-green fluorescence was evident on the surfaces of the purified mature spores but little to none in the spores extracted by SDS,suggesting the antibody could recognize one or more spore wall proteins.And the MAb 2B10 IFA demonstrated that there was intense fluorescence detected in the surfaces of the spores extracted by SDS but no in the purified spores,which suggested that the MAb 2B10 could recognize the endospornal proteins or protein antigens of the plasma membrane. In addition,two MAbs were tested by Western blot,respectively.Of these,MAb 2G10 recognized 26,54 and 76 kDa proteins from the total proteins of Nosema bombycis spores while a single protein of 26 kDa from the spore wall proteins extracted by SDS. The WB data showed that the MAb 2G10 could react with three different proteins of N. bombycis spores including SWP26.And MAb 2B10 acknowledged a single protein of 50 kDa from the same fraction extracted by two different methods.In conclusion,the obtained MAbs add to the existing microsporidian antibody bank and should play a vital role in researching spore wall proteins of N.bombycis.Meanwhile,this work also may help to develop the useful diagnostic reagent for N.bombycis.2.Isolation and identification of the target proteins of the two MAbs against the Nosema bombycis spore wall proteins by immunoprecipitation(IP) and mass spectrometry(MS) analysisUsing the obtained MAbs against the spore wall proteins in the previous chapter, we separated and enriched and identified the corresponding target antigen proteins from N.bombycis lysates by IP and WB combinding MS analysis.First.the target proteins were separated and purified from the antibody-antigen complexes by the IP method.The MAb 2G10 was incubated with the N.bombycis lysate proteins,and antibody-antigen complexes were absorbed using Protein A+G-Agarose and analyzed by SDS-PAGE and WB.Then the three protein bands(26,54 and 76 kDa) examined by IP were cut out of the SDS-PAGE gel and in-gel digested with trypsin,respectively.Peptide mass fingerprints(PMFs) were obtained from MALDI-TOF MS analysis.Then,mascot or GPMAW6.10 soft was employed for the analysis of PMFs data using the N.bombycis protein database(unpublished data).The most matching results are as follows:the 76 kDa protein is the heat shock related 70 kDa protein;the 54 kDa protein is the elongation factor 1 alpha protein;the 26 kDa protein is SWP26 which will confirmed by the further experiments.Likewise,using the same methods,the target protein against MAb 2B10 was separated and identified.The MS result showed that the target protein is a new protein with a molecular weight of 55 kDa which was not reported before.Thus,we successfully obtained the corresponding target proteins against two MAbs,which may lad a foundation for further identification of the spore wall proteins. Moreover,the IP-WB-MS method is simple and easy to be operated,intending to provide new approaches for the research of the spore wall proteins.3.Gene cloning,sequence analysis and prokaryotic expression of the spore wall protein SWP26 from Nosema bombycisBased on the Nosema bombycis genome data,we had performed gene cloning, sequence analysis and prokaryotic expression of the spore wall protein SWP26 obtained from the previous two chapters in this study.At first,the SWP26 gene encoding a 223-amino acids protein was cloned and the size of the corresponding CDS is 672 bp in length.Sequence analysis showed that the SWP26 has a predicted N-terminus signal peptide and a C-terminal heparin-binding motif(HBM),which suggested that it is a possible secreted protein and involved in spore adherence through a HBM.So,we designed two pairs of primers and used to amplify two different sequence fractions of the SWP gene and the mutant SWP26 gene(△SWP26,without HBM),respectively. Then we constructed the recombinant expression plasmid pGEX-SWP26 and pGEX-△SWP26 lacking HBM,respectively.The resulting recombinant plasmids were transformed into Escherichia coli BL21 cells and expression of recombinant SWP26(or△SWP26) was induced.The heterologously expressed proteins(an～52 kDa fusion protein(rSWP26) and an～50 kDa fusion protein(r△SWP26)) were purified by affinity chromatography using a His.Bind Purification Kit(Novagen),which is preparing for the function analysis of SWP26.Meanwhile,the SWP26-specific polyclonal antibody was generated by immunizing mice with the purified fusion proteins and used in Western blot analyses.A single 26-kDa band was detected from the N.bombycis spore protein lysates,which is in agreement with the size calculated from the sequence.The result clearly demonstrated that the antiserum has strong reactivity to SWP26 and can be used in the subcellular location of SWP26.Besides,this result also supported the reliability and validity of the IP-WB-MS method in the anterior study.4.Ultrastructurai localization and developmental expression of SWP26In order to assess the expression of two proteins(SWP26 and the target protein of MAb 2B10) during the different developmental stages and to further determine the cellular location of the protein,intracellular parasites were examined by IFA and IEM. IEM of ultrathin sections of N.bombycis-infected silk glands demonstrated that SWP26 is a spore wall protein and present in immature and mature spores.In particular,SWP26 was expressed at high levels in the developing endospore.Additionally,in the immature spores,a number of gold particles were distributed along the plasma membrane but few in the endospore regions.However,in premature or mature spores,a few particles were present in the exospore or endospore region of the spore wall and not the plasma membrane,which is in agreement with the IFA data.Likewise,the stage specificity and cellular location of the target protein of MAb 2B10 were performed by IFA and IEM.IEM data showed that the target protein of MAb 2B10 is present throughout all stages of intracellular development and a microsporidian cell wall protein involved in endospore formation.At the stage of merogony,a number of the target proteins are present in the sporoplasm of spore cells at different developmental stage.Moreover,binding of MAb 2B10 to the sporoplasm increased greatly as the plasma membrane thickened.In the sporogony phase,the massive target proteins accumulated in the forming endospore region.It is significant that the target proteins increased in the endosporal region but decreased in the sporoplasm region during the sporogony phase.However,in mature spores,little Ab reliably decorated only the inner electron-lucent layer of the spore wall,which is in agreement with the IFA data.Thus,the identification of the two SWPs of N.bombycis may be enriched with the composition of the spore wall proteins,and will profit for researching the biosynthesis and organization of the spore wall.This work may also improve our understanding of the biological process of this microsporidia.In addition,spore wall proteins could be used as a good prospective target for diagnostic research and drug design in controlling the silkworm,Bombyx mori,pebrine disease in sericulture.5.Function analysis of the spore wall protein 26(SWP26) from microsporidia Nosema bombycisIn this chapter,using rSWP26 and r△SWP26 proteins as inhibitors and the expressed GST protein as the control,host cell binding assay and the spore adherence and host infection assays were conducted,as well as the assay regarding isolation and identification of host proteins which will interact with SWP26,thus indicating the function of SWP26 to some extent so as to study on the relationship between parasites and host.First,the purified recombinant proteins(rSWP26 or r△SWP26) were placed onto BmN-SWU1 monolayers in 12-well plates.Then the monolayers were collected, lysed and subjected to Western blot analyses.The results showed that the wild type protein rSWP26,but not the mutant r△SWP26,bound to the host cells,suggesting that rSWP26 can attach to host cells through the C-terminal HBM.Secondly,the spore adherence assay was conducted using rSWP26 and r△SWP26 proteins as inhibitors and the expressed GST protein as the control,the results are as follows:adherence was reduced by approximately 10%with 5μg/ml of rSWP26;there was almost no difference in the total number of bound spores when in the presence of the r△SWP26 protein and in the presence of the expressed GST protein.These data indicate that rSWP26 has the ability to reduce N.bombycis spore adherence to some extent but the blocking effect was not extraordinary,while r△SWP26 lacking the HBM is not effective in preventing spore adherence to host cells.These results also demonstrate that the HBM may play an important role in the protein-glycan interactions.Also,we examined the corresponding relationship between spore adherence and host cell infection using the rSWP26 protein.This experiment signifies that the two are directly linked. Nevertheless,in the adherence and infection assays,we also found the inhibition was not obvious compared with the control samples.All experimental data above proved that SWP26 can have a direct interaction with host cell proteins at cellular level.At last, with the rSWP26 as the bait protein,the GST-pull down assay was performed to separate the host target proteins which have the interaction with SWP26,and five hypothetical interacting proteins were obtained.Thus,we know that SWP26 can recognize and interact with the host cell proteins,and the interaction mechanism is quite complex.But,the interaction mechanism and the particular biological function of SWP26 will be studied subsequently.Taken as a whole,this research will have laid a foundation for further identification of the interacting proteins.