Studies On Functional Genome Of Nosema Bombycis

Extracellular proteases of pathogens including serine protease are mainly participated in regulation of both parasites' development and metabolism. In the last decades, extracellular enzymes as virulence factors have been intensively studied. Numerous enzymes have confirmed their involvement in the infection process and interaction with immune system of host. Nosema bombycis is the pathogen of pebrine which bring heavy losses to sericulture every year. To date, the pathogenic mechanism of N. bombycis is thought of plundering host's nutrition by virtue of secreted protease during schizonts stages, which ultimately result in physiological dysfunction and host cell disruption. As research moved forward on microsporidia, the genome databases including Encephalitozoon cuniculi, Encephalitozoon intestinalis, Enterocytozoon bieneusi, Antonospora locustae, Nosema ceranae have been released and provided us a powerful platform to investigate microsporidia more intensively. In our study, we characterized the subtilisin-like protease (slp) which existed in all microsporidia genomes mentioned above. Subtilisin-like proteases are widespread in prokaryotes and eukaryotes and have been well researched. In the pathogenic fungi, subtilisin proteases were involved in degrading the cuticle proteins and helped fungi penetrate host cuticle. It was reported that high concentration of subtilisin in haemolymph caused the host phenoloxidase over-expression, which was one of main reasons for insect death. Our work represented the characterization of a putative subtilisin-like serine protease NbSLPl and its predicted three dimensional structure. Subsequently, heterologous expression was conducted and the NbSLPl enzymatic activity was tested with the refolded recombinant protein. Furthermore, multiple techniques including indirect immuno-fluorescence assay, immunoelectron microscopy and immunohistochemistry were employed to probe the localization of NbSLPl. Meanwhile, antibody blocking, co-immunoprecipitation, host cell transfection and yeast two hybrid were used to screen interacted proteins of NbSLPl. The main results are summarized from three aspects as following:1. Identification of Subtilisin-like proteases from microsporidia genomes and comparative genomics analysisThe typical subtilase possesses propeptide and Peptidase_S8domain. The MEORPS database and genome databases of six microsporidia were downloaded for searching subtilisin-like proteases (slp) using BLAST software. There are three subtilisin-like serine proteases in N. bombycis genome. Comparative genomics analyses show that all microsporidia genomes contain two kinds of subtilisin proteases, one has an Inhibitor_I9domain and a Peptidase_S8catalytic domain denoted as SLP1, the other has Peptidase_S8catalytic domain and a transmembrane domain denoted as SLP2. Both of hem possess catalytic residues Asp192, His224and Ser413. By homology with other subtilases, NbSLP1contains a76-aa Inhibitor_I9domain (Asp28to Ala104, Nbslp1p) and a290-aa (about31kDa) Peptidase_S8catalytic domain (Met172to Asn461, Nbslp1c). At the N terminal, the forward30aa of NbSLPl sequence was predicted as the signal peptide. Five N-glycosylation sites and one calcium binding site in NbSLPl were predicted which suggest NbSLP1is a metal dependent protease. Multiple sequences alignment reflected the NbSLP1shares46%dentity with SLP1from N. ceranae. Syntenic distribution of sips among microsporidia genomes reveals the sips genes loci are conserved. Then three dimensional modeling of NbSLPlC exhibited a typical three-layer sandwich structure, suggested that the mechanism of proteolytic and feature of binding substrate were similar to subtilase from Bacillus subtilis. For the structure of NbSLP1, the Inhibitor_I9domain extended into the inner of catalytic domain which could block the catalytic region. It suggested NbSLP1need to be processed into mature enzyme NbSLPlC through the cleavage of Inhibitor_I9domain. The phylogenetic tree showed the subtilisins of microsporidia fall into two groups:slp1and slp1. The Nbslpl transcripts were detected from Id to5d p.i.. It seemed that NbSLP1played a key role during the process of spore development and proliferation.2. Hetero-expression and localization of the NbSLPl The fragments of Nbslpl sequence according to the predicted domains (full sequence denoted as Nbslpl, catalytic domain denoted as Nbslplc, inhibitor domain denoted as Nbslplp) were cloned into the heterologous expression vector pET30a and over-expressed in BL21(DE3). The recombinant proteins were purified using nickel-nitrilotriacetic superflow columns and were used as antigen to raise polyclonal antibodies (PAb) in rabbit and mouse, respectively. The proteolytic activity of the refolded rNbSLP1was58U/mL at30℃and pH8.0.For immunoblotting assay, the antiserum to NbSLPIC acknowledged four major spots in2-D PAGE. Based on their MW and pI we deduced that the～60kDa spot was NbSLP1, while the other three spots in group about～1kDa werethe mature enzyme forms of NbSLP1with different post-translational modification.Indirect immunofluorescence assay (IFA), immunoelectron microscopy (IEM) and immunohistochemical assay (IHC) were performed to determine the distribution pattern of NbSLP1in spores. IFA showed the mature enzyme were located at the apical of the spore, just the site of polar tube extrusion, suggested NbSLP1may carry out its activity during the germination process. Meanwhile, the signals were observed in the spores proliferated in both Sf9cells and silk gland tissues, revealed NbSLPl was probably secreted during the proliferation of spores.3. Functional analysis of NbSLPl in N. bombycis sporesBased on the apical localization of NbSLP1, the effects of serine protease inhibitors phenylmethanesulfonyl fluoride (PMSF) and antibody blocking on the germination and infection of N. bombycis were investigated. Our results indicated that PMSF significantly reduced the spore germination rate with20%, suggested that subtilisin-like proteases in N. bombycis may participate in spore germination process and behave a key role in the polar tube extrusion. However, antibody blocking assay showed the incubated spores with anti-NbSLP1did not cause reduction of adherence rate to Sf9cells.Co-immunoprecipitation (Co-IP) was used to obtain the N. bombycis proteins interacted with NbSLPl. The candidate band from the gel of co-immunoprecipitation was analyzed by linear ion trap quadrupole MS and the results showed mitochondrial glycerol-3-phosphate dehydrogenase, eukaryotic translation initiation factor2C, Polar tube protein2, M1family aminopeptidase1, Hypothetical spore wall protein7, Spore wall protein1, ATPase, transitional endoplasmic reticulum ATPase and heat shock 70kDa protein (HSP70) were precipitated. Among these, both two types of ATPase and HSP70were coincidence with the predicted substrate by motif scan. Notably, the cleavage site of ATPase was between two AAA domains. We presumed that NbSLP1may process ATPase to hydrolysis ATP for energy production.Most of subtilase are non-specific proteases, thus we also try to find the substrates of NbSLP1in the host. Firstly we expressed Nbslpl in Sf9cells by transfection. The morphology of transfected cells was irregular and the cells were suspended more easily.2D-PAGE was used to find different spots between the Nbslp1transfected cell line and the control, there are seven spots absent from the cell line of transfected Nbslpl. MALDI-TOF/MS analysis indicated N-acetyltransferase, cuticle protein and hemolymph protein maybe the substrates of NbSLPl. Secondly, by subtilase proteolytic motif scan, we found a leucine rich transmembrane protein (BmTIR) containing intracellular TIR domain, which shared highest identity with Toll like receptor, could be the candidate substrate of NbSLPl. Toll receptors play an important role in innate immune and are very conservative in both insects and vertebrates. In the co-immunoprecipitation of anti-NbSLPIC incubated with the total tissue proteins of the infected Bombyx mori, anti-BmTIR could recognize the precipitated protein band, indicated that the precipitated band contain BmTIR protein from B. mori. Proteolytic test in vitro demonstrated rBmTIR was degraded by the refolded rNbSLP1. The interaction between NbSLP1and BmTIR may interfere with the Toll mediated immunity pathway by hydrolyzing TIR domain.In conclusion, our study on the apical localization and functional analysis of NbSLP1enriched the research of SLP in microsporidia and offered some clues to investigate the NbSLP1's substrate intensively.