Studies On The Functional Genome Of Nosema Bombycis

Microsporidia is an obligate intracellular parasite. It can infect almost all of invertebrates and vertebrates, even including humans. Nosema bombycis was firstly discovered by Nageli in 1857. It is the unique quarantine pathogen in sericulture because it can cause pebrine and bring devastating damages to sericulture countries. The prevention of pebrine has become one of the most key directions of silkworm pathogenic research.Microsporidia is a group of the spore-forming parasites. Its dense spore wall plays an important role to resist the external environment, to maintain its shape and osmotic pressure. It is thought that the spore wall which contacts directly with the external environment at first could have some important functions in the process of invasion and infection. The current research on spore wall mainly focuses on identification and function of spore wall proteins. The formation mechanism of the spore wall is less researched. The spore wall of N. bombycis is composed of the chitin-rich inner layer (endospore) with low electron density, the protein-rich outer layer (exospore) with high electron density and sporoplasm membrane. In this study, different developmental stages of N. bombycis were used to explore the dynamic formation process of chitin layer and expression of NbSWP9. Then segmented expression and site-directed mutagenesis were carried out to determine the molecular basis of binding of NbSWP9 to chitin layer. The main results are as follows:1. Dynamic secretion of NbSWP9 and chitin layer of N. bombycisWe obtained different developmental stages of N. bombycis through BmE cells infected by N. bombycis, spores purified by Percoll density gradient centrifugation and midgut digested by Trypsin, respectively. Indirect immunofluorescent was employed to analyze the secretion and formaition of chitin layer and NbSWP9 in the process of spore wall formation. The results showed that the chitin layer exist on the surface in the early period of sporoblast. Through observing the BmE infected by N. bombycis, most of the NbSWP9 expressed earlier to the spore surface than the chitin layer. In this period, the nucleus of the N. bombycis could be stained by DAPI, but the chitin layer was not observed. With the development of spores, the fluorescence signal of chitin layer enhanced, and NbSWP9 was located outside the chitin layer, however a part of them could be merged. Then fluorescence signal of NbSWP9 weakened while signal of chitin is clearer. It is speculated that NbSWP9 is easily recognized by antibody at the early period of N. bombycis than mature spores. NbSWP9 may mainly participate in maintaining the stability of spore wall in the mature spore or late developmetal stages of spore.2. Characterization of the chitin binding motif of NbSWP9Based on sequence feature of NbSWP9, the mutant of NbSWP9 without chitin binding motif (CBM) was constructed, and NbSWP9 without signal peptide was regarded as the wild type. Proteins were obtained by prokaryotic expression and affinity chromatography purification. Chitin binding capacity of mutant proteins were detected using the deproteinated chitin spore coats (DCSCs). Through the analysis of SDS-PAGE, Western blotting and IFA, results suggest that the CBM of NbSWP9 plays a key role in the binding to chitin layer, NbSWP9 cannot bind to chitin layer when its CBM is deleted.3. Identification of the key amino acid of NbSWP9 involved in the binding to chitin layerBased on the conservative analysis of NbSWP9 CBM, four site-directed mutants (swp9-C206A, swp9-C206AJG229A, swp9-C206A/N214A/G229A and swp9-C206A/ N214A/V225A/G229A) were constructed by the Overlapping PCR amplification method. The two soluble proteins SWP9-C206A and SWP9-C206A/G229A were obtained by prokaryotic expression and affinity chromatography purification, the other two proteins which meet the experimental requirement were not obtained. The chitin binding capacity of SWP9-C206A and SWP9-C206A/G229A were validated through chitin binding assay, the result showed that the binding of SWP9-C206A/G229A to chitin layer weakened, whereas SWP9-C206A could bind to DCSCs as same as the WT-SWP9. It suggests that NbSWP9-C206A/G229A will affect the binding of NbSWP9 to chitin. Our results suggest the last glycine of the CBM of NbSWP9 could play a role in the binding of NbSWP9 to chitin layer.In summary, this study conducted a preliminary exploration to chitin formation and NbSWP9 secretion of N. bombycis. Using the method of segmental expression and site-directed mutagenesis, molecular basis of chitin binding of NbSWP9 were firstly verified.