Study On The Spore Wall Protein5(SWP5)from Microsporidia Nosema Bombycis

Microsporidia are a group of obligate intracellular, fungus-like, unicellular eukaryotic animal pathogens with an extensive host range, including almost all vertebrates and invertebrates. In the past10years, it promoted the research on the mechanism of invasion, diagnostic and drug design in pathogenic microsporidia since the discovery of patients with AIDS infected by microsporidia. Nosema bombycis, a silkworm （Bombyx mori） parasite, was firstly described in1857. It is the etiological agent of the deadly protozoan disease pebrine cause severe worldwide economic losses in regions where sericulture is practiced including China and India, and Thailand, etc. Therefore, to reveal the mechanism of invasion N. bombycis, and control of pebrine, are the main two objectives of research in sericulture regions. Microsporidian spore wall was composed by chitin and proteins. However, it is difficult to isolate, identify and characterize the function of spore wall protein （SWP） successfully, due to the complicated spore wall structure and bottleneck effect of research technology of microsporidia. To date, five spore wall proteins have been identified from Encephalitozoon. Among them, two exospore proteins, SWP1and SWP2, were identified from Encephalitozoon cuniculi and Encephalitozoon intestinalis, respectively. Three endospore proteins, Enpl, Enp2, and the chitin deacetylase-like protein EcCDA, were found in E. cuniculi. Recently, four spore wall proteins, SWP32, SWP30, SWP26and SWP25, were identified from N. bombycis. For the roles of these SWPs durining the process of infection, most of them are involved in endospore and exospore formation, only Enpl and SWP26were reported as an adherence ligand allowing the parasite to attach to host cells and potentially modulate infection. In addition to spore wall, polar tube played a crucial role in microsporidia infection. Upon appropriate environmental stimulation, the polar tube can suddenly extrude and penetrate the plasma membrane of the host cell, and then the sporoplasm is transferred into the cytoplasm of the host cell, where the spores develop and complete their life cycle. Three previously identified major polar tube proteins （PTP1, PTP2, and PTP3） interact with each other and form the major protein components of the polar tube.In this study, the expression and localization of SWP5were investigated by reverse transcription-PCR （RT-PCR）, Southern blotting, indirect immunofluorescence assay （IFA）, and immunoelectron microscopy （IEM）. The interaction between SWP5and polar tube proteins was also explored using immunoprecipitation, mass spectroscopy （MS）, immunofluorescence, and germination assay. The result shows as follows:1. Bioinformatics identification and characterization of SWP5gene of Nosema bombycisSequence analysis showed that SWP5has a calculated molecular mass of20.3kDa and a theoretical pI of4.54. A23-amino-acid signal peptide was predicted, but no transmembrane domain or GPI anchor sequence signal. In addition, potential posttranslational modification sites were identified in SWP5, including phosphorylation sites, O-glycosylation sites, and sites of glycation of ε-amino groups of lysines. The structure of SWP5is simple, only a few of alpha-helix and beta-collapse were found by secondary structure and crystal structure prediction, failed to find the typical functional domain. Similarity analysis showed that SWP5shares25%identity with a protein in Nosema ceranae （GenBank accession no. XP₀02996352.1）. Scan of gene collinearity among microspordia that genomes were known shows that SWP5did not fall in any synteny blocks. In addition, SWP5has been identified as a protein without homologs in reported spore wall proteins. Taken together, our results show that SWP5protein is unique, could be used as a good prospective target for diagnostic research in sericulture.2. Chromosome localization and transcription of SWP5in N. bombycisTo investigate the localization of SWP5gene on the chromosome, we practised the southern blot and bioinformatic analysis. Genome sequence analysis showed that SWP5 has another copy （GenBank accession number HQ881497）. Southern blotting revealed two chromosome regions with strongly hybridized signals in the N. bombycis genome in agreement with the results of genome analysis. To survey the expression pattern of SWP5at different development stages, RT-PCR was performed using cDNAs of the midguts of silkworms from1to10days postinfection. The results showed that SWP5was transcripted at a low level at24hours post-infection. With the development process continued, SWP5expressed significantly from2to10days postinfection compared with the level at1day postinfection. Taken these data together, SWP5maybe involved from spore wall formation at the sporogony to mature spore.3. Heterologous expression and immunoblot analysis of recombinant SWP5from Nosema bombycisTo characterize SWP5, the SWP5（GenBank accession number EF683105） was cloned. SWP5PCR amplified fragment and the expression vector p-Cold I were digested with EcoR I and Sal I, then ligated together, and transformed into E. coli Rosetta Gami cells. Recombinant SWP5was expressed in E. coli and purified using a Ni-NTA superflow cartridge. An anti-SWP5polyclonal antibody was successfully prepared in mice. Western blot analysis indicated that a unique positive band was detected in the spore total proteins.4. Immunolocalization of SWP5Subcellular localization analysis predicted SWP5to be an extracellular protein. To explore its cellular localization, SWP5was analyzed by Western blot, IFA and IEM with the anti-SWP5serum. Western blot analysis results showed that a unique positive band was detected in the spore coat proteins, which suggested that the SWP5may localize on the spore wall. In order to further identify SWP5whether localized on the endspore or exospore, IFA was performed with anti-SWP5serum. The results showed spores of pre-incubated with the anti-SWP5serum were displayed a bright green fluorescence signal, while no spores were observed in those treated with SDS and negative control. IFA analysis results suggested that SWP5was localized on the exospore. For further clarifying the localization of SWP5, IEM was performed using anti-SWP5serum too. IEM assay found the gold particles were distributed mainly on the exospore and also in the polar tube region of spores, but absent on the chitin-rich layer between the exospore and the plasma membrane. Taken the results together, we confirmed SWP5is a spore wall protein involved in spore wall structural integrity. 5. Immunoprecipitation and co-localization analysis of protein interaction between SWP5and PTPFor further explore the roles of SWP5, we try to find out the target protein which interacts with SWP5by using immunoprecipitation and immunofluorescence with anti-SWP5serum. Two different proteins, which were polar tube protein PTP2and PTP3with apparent molecular masses of31and150kDa, were identified by immunoprecipitation and mass spectrometry. In addition, the discharged spores were treatment with anti-SWP5serum, and then examined with a fluorescence microscope. The results showed the polar tubes were displayed a bright green fluorescence signal, which suggested that SWP5may interact with the polar tube. Taken together, immunoprecipitation, mass spectrometry and IFA analysis indicated that SWP5interacts with PTP2and PTP3, and the polar tube probably interacts with the spore wall in N. bombycis.6. SWP5plays an important role in microsporidian polar tube discharge and infectionIn order to reveal the roles of SWP5in germination and infection, mature spores were pre-incubated with anti-SWP5serum and mouse negative serum. Firstly, spores were induced to germinate treatment with K2CO3. Average germination rates were50.63%,35.36%, and35.95%for negative serum, and anti-SWP5serum （dilution1:100and1:200）, respectively. Statistical results showed that the spores could extrude their polar tubes under alkaline conditions, and the anti-SWP5serum could inhibit spore germination by30%compared with control. Secondly, the spores pre-incubated with anti-SWP5serum were feeded to silkworm. The survival rates of spores treated with anti-SWP5serum was significant higher than control treatment from6to10days post-infection. These data suggest that SWP5play an important role during microsporidian infection, but further data are still to confirm.