Studies On The Functional Genome Of Nosema Bombycis

Microsporidia are a large group of obligate intracellular eukaryotic parasites, which infect almost all animals ranging from invertebrates to vertebrates, including human. They could resist various environmental pressure which benefit from a thick layer of spore wall on their surface. The spore wall of microsporidia is composed of exospore, endospore and plasma membrane. Recently, although several microsporidia genomes were published, the molecular biologic study on microsporidia has not achieved great not only spore wall proteins are not deeply researched, only twelve of which have been identified and reported, but also molecular mechanism of the infection is still unknown. Recently researchers have discovered the phenomenon that microsporidia will first attach to host cells in the process of invasion, and spore wall of microsporidia, which first touch with the host cells, plays a great role in the infection process. Like the surface proteins of other pathogenic microorganisms（Plasmodium sporozoites and Toxoplasma gondii）, spore wall proteins of microsporidia are considered to participate in the process of attachment and infection. The first microsporidia, Nosema bombycis, which was discovered from infected Bombyx mori in1857, is the pathogen of pebrine, cause vertical transmission via eggs and huge loss in sericulture. The research on spore wall proteins is helpful to explain infective mechanism and provide a tool to prevent pebrine. To date, just a total of five spore wall proteins have been characterized in Nosema bombycis, thus it is time for us to identify new ones. Based on the previous study that zhengli wu identified14hypothetical spore wall proteins from Nosema bombycis, we choose two of these genes in this research, named as hswpll and hswpl3. We firstly investigated the sequences of hswpll and hswpl3, and the characteristics of transcription by RT-PCR. Secondly, we carried out heterologous expression and produced polyclonal antibodies. Thirdly, indirect immuno-fluorescence assay was conducted to detect the localization of two proteins in the Nosema bombycis. Subsequently, we utilized the yeast expression system to localize NbHSWP13. Meanwhile, we use cell-binding assay and anti-SWP11serum pretreatment of mature spores assay to confirm the role of SWP11during attachment and infection. The main results are as follow:1. Prokaryotic expression and polyclonal antibodies production of hswpll gene of N. bombycisAs supported by genome date of N. bombycis, the open reading frame of hswpll is a1346-bp single-copy gene, which codes for a463-amino acid protein （52.4kDa, pI9.71）. This sequence of protein displays the characteristics of six heparin-binding motifs （HBMs）,32phosphorylation sites and one consvered Dna J domain, but the lack of a signal peptide, glycosylation sites, transmembrane domains and GPI-anchoring sites. Syntenic distribution of hswp11and Dna J among N. bombycis and Nosema ceranae displays the genes loci are conserved in their genomes. RT-PCR analysis showed that Nbhswp11can be amplified from first day to seventh day post-infection. Based on such results above, we constructed the heterologous expression vector pET30a-Nbhswp11, subsequently, recombinant vectors was transformed into E. coli BL21and induced by0.5mM IPTG at16℃.This fusion protein is expressed as inclusion body and is consistent with predicted molecular weight. Using nickel-nitrilotriacetic superflow columns, the recombinant proteins were purified and used as antigen to immunize mouse for preparing polyclonal antibody, respectively. Immunoblot analysis showed that NbHSWPll was expressed in mature spore and spore coat of N. bombycis.2. Localization and functional analysis of NbHSWPll from Nosema bombycisIndirect immunofluorescence assay was preformed to determinate the distribution of NbHSWP11in Nosema bombycis. IFA indicated NbHSWP11was located on the spore surface and spore coat of Nosema bombycis. Thus, we identified another new spore wall protein renamed NbSWP11from Nosema bombycis. Based on this result, the native NbSWP11was used in host cell binding assay in vitro. The result showed that this protein could bind with the host cells testified by IFA. Anti-SWP11polyclonal antibodies pretreatment of mature spore could reduce spore adherence to host cells significantly. The inhibition rate of spore adherence was up to20%. All results above suggested that SWP11may play a role during the process of adherence and infection.3. Hetero-expression and polyclonal antibodies production of hswpl3gene of N. bombycisIn our genome date of N. bombycis, Nbhswpl3has two-copy genes and we chose the copy of NBO₄62g0007as tartget gene for research. NBO₄62g0007open reading frame is a2499-bp gene which codes for an833-amino acid protein（94.3kDa,pI7.42）. Sequences analysis shows that HSWP13has a N-terminal signal peptide with a predicted cleavage site between positions17and18, a near C-termina130-amino acid motif with a majority of hydrophobicity amino acids, two heparin-binding motifs （HBMs）,11glycosylation and phosphorylation sites, but lack transmembrane domain, GPI-anchoring site and conserved domain. Except for signal peptide cleavage site, there are two other cleavage sites of this sequence. On-line prediction of subcellular location indicates that Nbhswpl3is located in nucleus. Based on the genomic data of Nosema antheraea we found that there is a78%homology between NbHSWP13of N. bombycis and NaHSWP13of N. antheraea. RT-PCR results indicated Nbhswpl3was transcribed at12hour post infection, which suggests it plays a key role in early stage of life cycle of N. bombycis. After analyzing the predicted functional domain, we cut two fragments from N and C terminal, named Nbhswp13n, which codes a382AA peptide, and Nbhswpl3c which codes a201AA peptide, respectively. We constructed two Prokaryotic expression vectors:pET30a-hswp13n and pET30a-hswp]3c and transmitted into E. coli Rosetta. SDS-PAGE analysis of induced expression showed recombinant proteins of HSWP13N is about5kDa bigger than predicted molecular weight, while HSWP13C is nearly12kDa bigger. The fusion proteins of HSWP13N and HSWP13C were purified using Ni-NTA affinity chromatography. After verification of Western blotting and MALDI-TOF/TOF-MS, the recombinant proteins were used as antigen to raise polyclonal antibodies in Kunming mice, respectively. Western blotting analysis indicated that there is a hybrid signal bind in total proteins of mature spores unmature spore of N. bombycis, and protein of infected midgut of N. bombycis, which is about24kDa smaller. According to predicted cleavage site, we supposed that NbHSWP13became smaller because of some N-terminal cleavage sites in this full sequence, the intramolecular cleavage may occur as the post-translational modification..4. Localization analysis of the HSWP13First, IFA was conducted to detect the distribution of HSWP13in N. bombycis spores. The result demonstrated that there was no fluorescence signal in mature spore, that is, HSWP13did not localize exospore of spore wall. Thus the purified unmarure spores treated with TritonX-100were used to determinate localization of HSWP13. The green fluorescence was observed on the spore wall. Then the germination of spore on the slides was carried out to future analyse the localization of this protein. We could see fluorescence signal on the spore coats. This results suggested that HSWP13was localized on the enospore. In order to access the localization of HSWP13in model organism Saccharomyces cerevisiae, the400-amina acid length of extreme N-terminal, including a signal peptide, and the382-amina acid length of N-terminal, which did not possess a signal peptide were cloned into the expression vector pUG35, and then transformed pUG35-nbhswp13n-sp and pUG35-Nbhswpl3n-Asp into the yeast strain CEN.PK2. We screened the positive recombinant yeast on the SC-Ura plate, then induced for expression of GFP labeled fusion proteins. After PCR analysis of genome of recombinant S. cerevisiae, and Western blotting analysis of total protein of the recombinant model organism, the yeast cells were stained with the DAPI. The result showed that both recombinant proteins of pUG35-NbHSWP13N-SP and pUG35-NbHSWP13N-ASP were targeted to the nucleus of S. cerevisiae, which confirm the predicted subcellular localization of this protein. Meanwhile, it displayed that NbHSWP13was targeted to the nucleus of S. cerevisiae not depending on theN-terminal signal peptide.