Identification Of The Surface Proteins In Microsporidia Nosema Bombycis

Microsporidia are obligate intracellular parasitic, unicellular eukaryotes. More than1,300 species and 160 generas have been found. Microsporidia cause severe threat to human health, worldwide sericulture, beekeeping and aquaculture. In particular, Nosema bombycis, a microsporidian pathogen of pebrine disease, inflicts severe worldwide economic losses in regions where sericulture is practiced. The surface protein of pathogen is the first and direct contact with the host cell, which can mediate parasite-host interactions. Surface proteins of N. bombycis are exposed on the spore’s electron-dense outer layer and may play a crucial role in spore adhesion to host cells.Therefore, identify systematically the surface proteins of N. bombycis is important to revealing microsporidian infection mechanism, but also provide for the discovery of prospective diagnostic markers and drug targets for the control of microsporidian diseases. However, little research has specifically addressed surface proteome identification in microsporidia due to technical barriers. In this study, surface proteins of N. bombycis were isolated and identified by biotin- streptavidin system and proteomics approach. The main results are show as follows:1. Biotinylation of the Nosema bombycis spore surface proteinsSpores were incubated with Sulfo-NHS-SS-Biotin, then indirect immunofluorescence assay(IFA) were performed to detect the efficiency of the the Sulfo-NHS-SS-Biotin labelling. The result indicated that the strong green fluorescence signal was detected on the spore surface. In contrast, no fluorescence signal was detected in other intracellular organelles and in untreated spores. These results suggest that the spore surface proteins were successfully labeled by biotin.2. Optimization of total proteins extractionThe rich total protein is the premise of separating and purifying the more surface proteins. However, it is difficult to extract more total proteins because of the electron-dense and chitinous structural barrier of the spore wall. To solve this problem,we used the high temperature and pressure method, boiling method, grinding method, ultrasonic fragmentation, and combined with urea, SDT, Leammli, Na OH lysis buffer to explore the best conditions for extracting total protein of N. bombycis. SDS-PAGE showed that the best method of extraction proteins from N. bombycis is lysised in0.01 M Na OH and 2% SDS buffer under sonication condition.3. LC-MS/MS and bioinformatics analysis of the surface proteinsSpores were labelled with EZ-Link Sulfo-NHS-SS-Biotin. The biotinylated proteins were then extracted and enriched by streptavidin pull-down. These enriched protein samples were subject to LC-MS/MS analysis. Finally, 23 proteins were identi?ed as the hypothetical surface proteins. 5 of 23 proteins were localized on the surface of spores in previous report, which are the SWP5, SWP8, SWP12, SWP26,SWP30. Another 6 proteins contain a signal peptide, only one putative surface protein(Accession No. EOB13402.1) has transmembrace domain. In addition, 8 putative surface proteins were predicted to extracellular proteins. Moreover, 7 proteins were identified as hypothetical proteins without any known functional domain. GO analysis showed that 11 of 23 proteins could be categorized as associated with a biological process. Briefly, 9 of the 11 proteins are putatively involved in biological adhesion,responses to stimuli, development or metabolic processes. In addition, 8 of the 11 proteins possess ATP-, GTP- and nucleotide-binding capacity and catalytic or translational regulation functions.4. Protein subcellular location of the surface proteins SWP5, SWP8, SWP12,SWP26,SWP30 and EOB14207.1To further verify the proteins identified by mass spectrometry is the surface protein,Nb SWP5, Nb SWP8, Nb SWP12, Nb SWP26 and Nb SWP30 were analyzed by using western blot. The results showed that a specific hybridization signal presented in the surface protein spectrum library. This result imply that Nb SWP5, Nb SWP8, Nb SWP12,Nb SWP26 and Nb SWP30 are the surface proteins.We also performed with the indirect immunofluorescence analyzed by using polyclonal antibodies against the spore wall protein Nb SWP5, Nb SWP8, Nb SWP12,Nb SWP26, Nb SWP30 and Nb EOB14207.1, respectively. After incubated with the polyclonal antibody Anti-SWP5,-SWP8,-SWP12,-SWP26,-SWP30 and-EOB14207.1,the surface of spores showed obvious green fluorescence hybridization signal whereas without any fluorescent signal appears in the control treatment. After spores treated by nucleic acid fluorescent dye DAPI, the dual-core nucleus showed blue fluorescence.This results, to some extent suggest that the Nb SWP5, Nb SWP8, Nb SWP12,Nb SWP26,Nb SWP30 and Nb EOB14207.1 were located on the outer wall surface of N.bombycis.In short, our work provides an effective strategy for isolating microsporidian surface protein components for both drug target identify cation and further diagnostic research on microsporidian disease control.