Expression And Characterization Of Spore Wall And Anchoring Disk Complex Protein NbSWP16 From Nosema Bombycis

Microsporidia are a group of obligate intracellular and unicellular parasites.They are widely distributed in nature and can infect almost all vertebrates and invertebrates including human beings.Microsporidia infect economic insects such as silkworms,bees and aquatic animals like fish and shrimp,bringing great economic losses to breeding industry.Seventeen species of microsporidia from 9 genera can infect human beings,especially immunodeficient people.The life cycle of microsporidia is generally divided into the infective phase,the proliferative phase and the sporeforming phase.Microsporidia have a unique infection approach named germination.Under the stimulation of the environment,the spores are activated.Then permeability of the spore wall increases and a large amount of water is absorbed from the environment.The osmotic pressure in the spore is accumulated and the polaroplast expands,resulting in strong pressure,forcing the polar tube to break through the anchoring disk and eject from the thinnest polar cap at the top of the spore.The enlarged posterior vacuole pushes the compressed sporolasm into the polar tube,and sporoplasma was transported into the host cell.However,the molecular mechanism of germination is not clear.The spore wall and anchoring disk complex protein NbSWP16 was identified in Nosema bombycis in our laboratory and was believed to participate inmicrosporidia germination and stress resistance.It is hypothesized that NbSWP16 may be the substrate of subtilisin-like protease1（Nb SLP1）,the hydrolysis process facilitates the extrusion of the polar tube.Here,the soluble expression of NbSWP16 protein was achieved,the role of NbSWP16 in N.bombycis was further understood through protein characteristic analysis and interaction assay.1.Transcription and expression characteristics of NbSWP16NbSWP16 was relatively conservative in the evolution of microsporidia,and the conserved regions were mainly adjacent to the N-terminal and C-terminal.A conservative motif（PMVLYKNGALAPYDPYTNTSKYAFCVEACPCP）is predicted at the N-terminal,which may have the function of adhesion and connection.Quantitative RT-PCR analysis showned that the transcription of NbSWP16 can be detected in the whole life cycle of N.bombycis and more transcripts can be detected in the proliferative phase.Western blotting analysis showed that the expression of NbSWP16 in N.bombycis was relatively high at 2-4 days post infection and then down-regulated.The results suggested that NbSWP16 gradually accumulates in the spore wall and participates in the formation of the spore wall.2.Soluble expression and characteristic analysis of NbSWP16The soluble expression of NbSWP16 was successfully obtained by constructing E.coli [pETDsbA-NbSWP16] and E.coli [pET29-NbSWP16] strains.The soluble protein was purified and concentrated by Ni-affinity chromatography,gel filtration chromatography,enzyme digestion and ultrafiltration.Mass spectrometric analysis showed that the purified r NbSWP16 formed a complex with some unknown lipoproteins of E.coli.r NbSWP16 was incubated with commercial subtilisin.Western blot analysis showed that commercial subtilisin could hydrolyze r NbSWP16 into truncated protein.3.Interaction analysis of NbSWP16 of N.bombycisThe purified recombinant NbSWP16 was incubated with PIP Strips^TM membrane,and Western blot analysis showed that NbSWP16 could bind to phosphatidic acid.Western blot and indirect immunofluorescence showed that NbSWP16 could bind to the deproteinated chitin spore coats（DCSCs）.The yeast two-hybrid vector of NbSWP16 and polar tube proteins was cotransformed into yeast competent cells.NYM32[p GADT7-NbSWP16,p GBKT7-Nb PTP2] could grow on SD/-Trp-Leu-His screening medium and activate downstream report gene expression.But the fused yeast cell could not grow on SD/-Trp-Leu-His-Ade screening medium,indicating that there was a weak interaction between NbSWP16 and Nb PTP2.In summary,the sequence,transcription,expression and localization characteristics of NbSWP16 were analyzed in this work.The recombinant soluble protein of NbSWP16 was obtained.NbSWP16 was transcribed and expressed during the proliferative phase and participate in the formation of spore wall and anchoring disk.NbSWP16 can bind with spore chitin and lipid in sporoplasm membrane,which leading to our hypothesis that NbSWP16 participates in the spore wall assembly and maintain the stability of the spore wall by connecting chitin and sporoplasm membrane.NbSWP16 may still be hydrolyzed to facilitate polar tube extrusion during the process of germination.