Research On The Spore Wall Protein SWP26of Nosema Bombycis And Its Interactive Protein Of Host

Microsporidia are unicellular, obligate intracellular, eukaryotic parasites. Its hostranges from protists to invertebrates and vertebrates. The species Nosema bombycis, whichis the pathogen of pebrine disease in silkworms, can be transovarially transmitted to the nextgeneration. So it is defined as the only disease needed for quarantine in sericulture. Atpresent, Pebrine disease used to prevail in the countries and areas practicing sericulture, andstill causes serious financial loss to the sericulture industry in China and India today. Sincethe N. bombycis has been discovered in the middle of the nineteenth century, it has morethan150years of history about the research on the microsporidia. The microsporidian sporestage, not the vegetative stages, is the only stage that lives outside the host, which is animportant characteristic of the phylum. The spore coat is separated from the cell by a plasmamembrane and consists of an electron-dense proteinaceous exospore and an electron-lucentendospore that contains chitin and proteins. The microsporidian spore wall plays animportant role in protection against environmental stresses and permits the long-termsurvival of the parasite after its release from the host cell. Indeed, recent studies havesuggested that some spore wall proteins are involved in the complex infection mechanism,and are responsible for spore adherence, which is an integral part of activation and host cellinfection. Previous studies showed that SWP26of N. bombycis contains a C-terminalheparin-binding motif (HMB) which is known to interact with extracellularglycosaminoglycans (GAGs) and may be involved in modulating in vitro host celladherence and infection. Transmission immunoelectron microscopy revealed that theSWP26protein is expressed at high levels in the endospore and plasma membrane duringendospore development, and is sparsely distributed in the exospore of mature spores.Furthermore, a few particles were detected in the endospore and exospore of the spore wallduring the late spore-forming phase and were not detected on the plasma membranes,thereby suggesting that SWP26is involved in endospore formation, host cell adherence, andinfection in vitro.In the present study, analysis of expression patterns revealed that the transcripts ofSWP26can be detected at24h p.i in the infected midgut of silkworm by N. bombycis.Therefore, it has been suggested that this spore wall protein is expressed at an early stageduring the development of N. bombycis. This proposal is congruent with the results of immuno-electron microscopy. However, little is known about the molecular interactionsbetween SWP26and host proteins. So, we want to comprehend that whether SWP26couldrecognize and interact with the host cell proteins.In this study, we used Bac-to-Bac baculovirus expression system to express the SWP26fused enhanced green fluorecence protein (EGFP) in BmN cells. The SWP26gene andEGFP gene were inserted into the baculovirus transfer vector pFastBac1. The transfervector pFastBac1-SWP26-EGFP was transformed into Escherichia coli DHl0Bac toconstruct recombinant vBmSWP26-EGFP. The vBmSWP26-EGFPwas then used to transfect BmNceIIs to obtain the recombinant bacuIovirus. Sodium dodecyl sulfate–polyacrylamide gelelectrophoresis (SDS-PAGE) and western blotting detected a protein band of about51kDain BmN cells infected by the recombinant virus, which was correspondent to the deducedmolecuIar weight of SWP26fused EGFP. In addition, fluorescence were observed withinthe cytoplasm and within the nucleoplasm. These results indicated that the SWP26had beensuccessfully expressed in BmN ceIIs. This study laid the foundation for screening theinteractive proteins of Bombyx mori with the SWP26of N. bombycis at the cellular level.Then, we obtained a potential interacting protein--turtle like protein of B. mori(BmTLP) with SWP26using co-immunoprecipitation method. Yeast two-hybrid analysisalso indicated that the SWP26protein interacted, in vivo, with BmTLP protein. Thecomplete open reading frame (ORF) of BmTLP was obtained using PCR with thecorresponding primers. The ORF is1.344kb in size and encodes a polypeptide of447amino acids with a calculated molecular weight of about49.6kDa and an isoelectric pointof5.55. The deduced BmTLP protein sequence has an N-terminal signal peptide of27amino acids with a cleavage site between amino acids27and28. The NCBI BLASTpsearch revealed that BmTLP has the highest similarity (98%) with Danaus plexippus(GenBank Accession No.: EHJ71750), as well as a high similarity of84%with Drosophilamelanogaster (GenBank Accession No.: ACN43741). Five N-glycosylation sites arepredicted in BmTLP using the NetNGlyc1.0server online software. It suggested thatBmTLP may be a secreted glycoprotein. In addition，the protease domain of the BmTLPprotein was identified using the SMART program to analyze the amino acid sequence.BmTLP contains the basic features of the immunoglobulin superfamily (IgSF) such as IGdomains at the residual positions34–145and342–428, and IGc2-type domains at residues161–226and252–321. Interestingly, we found that the same4domains exist in both theBmTLP protein and the hemolin protein of silkworm (coding sequence:BGIBMGA008736-TA; protein: BGIBMGA008736-PA; GenBank Accession No.: DQ096295). However, these domains are differently distributed in the2proteins. In contrastto the hemolin protein, two IGc2-type domains are located in the middle of the two IGdomains.Fluorescent quantitative real-time polymerase chain reaction (qRT-PCR) analysisrevealed that the relative expression level of BmTLP was significantly higher in the infectedmidgut than that of silkworm at12h postinfection (hpi). However, the relative expressionlevel of BmTLP was notably down-regulated in the infected midgut of silkworm after12h.The results suggested that BmTLP protein may play an important role at early stage ofinfection by N. bombycis.