Studies On Functional Genome Of Nosema Bombycis

As a eukaryotic organism, Nosema bombycis is an obligate unicellular eukaryotic parasite, which can infect economically important insect Bombyx mori and cause silkworm pebrine. The spore wall of microsporidia plays key function in the invasion and host immune evasion process, as it is the first structure directly interacting with the host cells. Today much attention had been payed in the research on the microsporidian spore wall and its formation mechanism. The microsporidian spore wall consists of three layers which include an electron-dense proteinaceous exospores, an electron-lucent endospore containing chitin and proteins and plasm membrane. In the past few years, numerous scientific studies focusing on the microsporidian spore wall were performed and several spore wall proteins had been successfully identified. To date, however, the studies on the spore wall is still so limited that to elaborate the molecular formation mechanism of microsporidian spore wall, because of its complex structure that contain large quantities of sophisticated components. In order to further promote the molecular formation mechanism of microsporidian spore wall, it is essential to explore and characterize the remnant proteins localized on the spore wall of microsporidia, especially the ones with special sequence characters or important domains. Focusing on this issue, the whole genome scanning targeting to the potential spore wall proteins associating to the invasion and host immune evasion process was performed and certain candidates were obtained. Moreover, the studies on two proteins were explored. Among these two proteins, the one NBO₂9g0021 （NbHSWP16） had tandem repeats and the other NBO₅3g0008 （NbOTUl） possessed a predicted otubain catalytic domain. The results confirmed that these two proteins are truly novel spore wall proteins of N. bombycis. The main results are as following:I., Studies on the spore wall protein NbSWP16 with tandem repeats of N. bombycis1. The comparative genomics studies of proteins with tandem repeats in N. bombycis1.1 Prediction and sequence character analysis of proteins with tandem repeats in N. bombycisIt was reported that most of the identified proteins with tandem repeats were localized on the surface of the intracellular parasites. The proteins with tandem repeats in N. bombycis were filtered using two programs:XSTREAM and T-reks.396 candidate proteins, about 8.86% of the total proteins in this parasite, were obtained both with these two methods. It demonstrated that the location distribution of the tandem repeats in these proteins were random without any bias in N. bombycis. The result of amino acid analysis of these tandem repeats revealed that hydrophilic amino acids and smaller amino acid glycine tend to be selected to constitute the repeats in these proteins and it seemed that they relatively exclude the hydrophobic amino acids. Comparing the GC content of the coding sequences of the tandem repeats with the genome GC content of N. bombycis, it was noticed that the former is higher than the latter. According to the functional categories of the obtained proteins using GO database, we found that these proteins may function in several important biological processes, including cellular component, molecular binding, cellular catalytic process, cellular processes and metabolic processes. Interestingly, some proteins with tandem repeat could even be found functioning in protein localization and cellular response to stimulus. 1.2 Comparative genomics analysis of proteins with tandem repeats in N. bombycisWe downloaded 9 other microsporidian genomes from NCBI. Among these, there are four microsporidia infecting insect hosts, such as Nosema ceranae, Nosema apis, Vavraia culicis and Antonospora locustae, four microsporidia infecting animals including Encephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon intestinalis and Enterocytozoon bieneusi and also a microsporidian Nematocida parish which infect Caenorhabditis elegans as the host. The comparative genomics analysis towards the proteins with tandem repeats in these 10 microsporidia including N. bombycis was performed. The results showed that the quantities of proteins with tandem repeats in the microsporida infecting insects and N. parisii were more than the ones in microsporidia infecting animals. Among these species, the number of proteins with tandem repeats in E. bieneusi was least and the largest one was N. apis. The distribution of the tandem repeats in N. ceranae, N. apis, V. culicis and N. parisii were the same as N. bombycis, but the tandem repeats in A. locustae tended to distribute in the middle of the proteins. The distributions of the tandem repeats in other four microsporidia infecting animals were different with N. bombycis. It seemed that the tandem repeats in these four microsporidia didn’t like to located in the N-terminal of the proteins, as the number of proteins having tandem repeats in the N-terminal were much less contrast to the ones in the middle or C-terminal. Amino acid analysis of the tandem repeats demonstrated that the amino acids component in another 8 microsporidia were consistent with N. bombycis but A. locustae. GC content of the coding sequence of the tandem repeats showed that, except E. bieneusi, the GC content of the coding sequences of the tandem repeats were all higher than genome GC content in another 8 species. It suggested that the tandem repeats in these microsporidian proteins tend to choose high GC content codes to construct themselves. 1.3 Prediction of the surface proteins and TR-PCR analysis of four genes coding four potential proteins N. bombycisIn order to find the potential surface proteins in N. bombycis, a filtering analysis towards to the 396 proteins obtained above was carried out. The criteria used in filtering analysis were as following: 1) the protein with tandem repeats has signal peptide or transmembrane domain or GPI anchor motif; 2) the length of tandem repeats should be more than 5 amino acids and the identity between the tandem repeats should be more than 80%. Finally,86 potential proteins were obtained. Among these, there are 37 proteins having signal peptides,45 proteins possessing transmembrane domains and 4 proteins having both signal peptides and transmembrane domains. Using the same methods, the potential surface proteins in other 9 microsporidia were also predicted and the results showed that the number of the target proteins in microsporidia infecting animals is little smaller than other species. Furthermore, to find more insight for the function study of the predicted spore wall proteins in microsporidia, RT-PCR was performed toward to four genes in N. bombycis. The results indicated that the genes coding for NBO₂4g0018 and NBO₂9g0021 （NbHSWP16） started expression at the third days post-infection, the genes coding for NBO 490g0001 started expression at the fourth days post-infection, the genes coding for NBO₃78g0015 started expression at the fifth days post-infection. These results suggested that these four proteins may function in the life cycle of N. bombycis.2. the characterization and functional analysis of NbSWP16 in N. bombycis2.1 Sequence character analysis of hypothetical spore wall protein NbHSWP16 in N. bombycisNbhswpl6 was 1152bp and encoded a 383 amino acid protein which had a calculated molecular mass of 44kDa with a theoretical pi of 8.95.30 potential phosphorylation sites and two N-glycosylation sites were predicted in NbHSWP16.29 cysteines were found existing in this protein, suggesting that disulfide bridges may be necessary for this protein to retain its stability. A 25-amino-acid signal peptide was predicted at the N-terminal of NbHSWP16, but no GPI anchor sequence signals or transmembrane domain was predicted. Three predicted heparin binding motifs （HBMs） at the C-terminal and three proline-rich tandem repeats consisting 13 amino acids with no homology to any known protein at N-terminal were also found in NbHSWP16. The second structure prediction of NbHSWP16 showed that this protein possessed six a-helices and 12 β-sheets. Homologous sequences alignment, sequence syntenic analysis and phylogenetic analysis indicated that NbHSWP16 were comparatively conserved in the evolution process of microsporidia, implying that this protein might be indispensable for these parasites.2.2 Characterization of spore wall protein NbHSWP16 in N. bombycisAccording to bioinformatics information of NbHSWP16, the specific primers were synthesized. When the PCR finished, the amplified fragment was cloned into the pMD19-T vector （Invitrogen）. After the DNA fragment was verified by sequence, it was cloned into prokaryotic expression vector pET30a. Then, the resulting recombinant plasmids were transformed into the Escherichia coli BL21 strains. The recombinant NbHSWP16 （rNbHSWP16） was induced to express at 37℃ for 6h in the presence of 0.4 mM IPTG. The expressed fusion protein was purified with a Ni-NTA Superflow Cartridge （Qiagen）. After the polyclonal antibody against NbHSWP16 was prepared with the purified recombinant NbHSWP16 by immunizing mouses, the native protein of NbHSWP16 was detected using Western blotting and the result indicated that the mouse polyclonal antibody of rNbHSWP16 can reacted with the total protein of N. bombycis and a specific band about 44kDa consistent with the calculated molecular weight of NbHSWP16 was detected. It suggested that this protein had been expressed and may function in N. bombycis.Indirect Immunofluorescence （IFA） was employed to detect the localization of NbHSWP16 in mature spores and spore coats. The results indicated that fluorescence signals were detected not only on the surface of the mature spores but also on the germinated spore coats. To further determine the cellular location of NbHSWP16, Immunoelectron microscopy （IEM） was employed. The results of IEM demonstrated that labeled gold particles mainly distributed on the exospore region of the mature spores. These results proved that the NbHSWP16 was an exospore wall protein of N. bombycis. So, we renamed NbHSWP16 as NbSWP16. Furthermore, in order to indicate the expression stage of NbSWP16 at the development process of N. bombycis, a confocal fluorescence microscope was employed, using the purified spores at earlier developmental stage （sporonts, early sporoblasts and late sporoblasts） and the BmE cell infected by N. bombycis as experimental material. The results demonstrated that NbSWP16 started expression at the early stage of spore formation and the quantities was with a slow increment as spore development extension, which was consistent with the RT-PCR result of Nbswp16. So, we speculated that this protein may involve in spore formation of N. bombycis.2.3 Functinal analysis of spore wall protein NbSWP16 in N. bombycis2.3.1 Functional analysis of NbSWP16 in cell adhesion process in vitroNbSWP16 possessed three predicted heparin binding motifs （HBMs） at the C-terminal and had been identified as an exospores protein using IFA and IEM, suggesting that NbSWP16 protein could be involved in spore adherence. So, to test this hypothesis, spore adherence analysis was performed at cell lever in vitro. The statistical result showed that the rate of spore adherence to host cell was decreased more than 20% by anti-NbSWP16 blocking compared with the negative control in vitro, suggesting that NbSWP16 protein may related to spore adherence to host cell in vitro. But in contrast with anti-NbSWP16 used alone, no remarkable decrement inhibition was detected when antibodies of NbSWP16 and NbSWP5 were used simultaneously, indicating the anti-NbSWP16 combining anti-NbSWP5 may not have synergistic effect in this process.2.3.2 The relationship between NbSWP16 function and its tandem repeatsYeast localization system and cell stress experiment had been performed to survey the relationship between NbSWP16 function and its tandem repeats. Recombinant plasmids, including pUG35-Nbswp16 having full sequence of NbSWP16, pUG35-Nbswp16△1 having NbSWP16 sequence without three tandem repeats in the N-terminal and pUG35-Nbswpl6△2 having NbSWP16 sequence without three tandem repeats and the serine-rich region in the N-terminal, were constructed, respectively. Then, these three plasmids were transformed into yeast cell CEN PK2. PCR and Immunoblotting indicated that Nbswpl6 and its mutants Nbswpl6△1 and Nbswp16△2 had been transformed into yeast cells and expressed successfully. Yeast localization results showed that green fluorescence was fully filled the pUG35-Nbsp16 yeast strain, yet there is no remarkable difference when compare the mutant yeast strains with the pUG35-Nbswp16 yeast strain. It was speculated that the tandem repeats in NbSWP16 may not related to the protein location in yeast system. When exposing to the stress produced by 0.2% SDS、5% β-ME and 0.1M DTT, all the three yeast strains were not sensitive to these stress when comparing to the control. When exposed to the stress of 0.05M NaOH and 55℃, all the three strains ofpUG35-Nbswp]6, pUG35-Nbswp16△1 and pUG35-Nbswp16△2 were capable of resisting to these stress in a certain degree compared with the control. At 55 ℃ stress condition, there was no evident difference when compared the pUG35-Nbswpl6 strain with its mutant strains. However, the resistance had been decreased a little, comparing pUG35-Nbswp16△1 and pUG35-Nbswp16△2 strains with pUG35-Nbswp16 strain when they were exposed to the basic stimulus. And there was no remarkable difference between pUG35-Nbswp16△1 strain and pUG35-Nbswp16△2 strain to 0.05M NaOH stress. These results above demonstrated that NbSWP16 could resistant to the basic and heat stress in a certain degree and the tandem repeats of NbSWP16 may contribute to the resistant ability to the basic stress of this protein.2.3.3 Studies on the interaction proteins with NbSWP16 of N. bombycisTo survey the proteins interacting with NbSWP16, yeast two-hybrid system was used with the help of spore wall proteins yeast library of N. bombycis established in our laboratory. From the result of yeast two-hybrid system, it was found that NbSWP16 might interact with other four spore wall proteins including NbSWP1, NbSWP3, NbSWP5 and NbSWP6, which provided a new clue for the interaction of the spore wall proteins in N. bombycis.Ⅱ、Studies on the spore wall protein NbOTUl with otubains domain of N. bombycis1 Survey of the proteases associated with invasion process of N. bombycisIt had been described that protease played important function in invasion and host immune evasion process in pathogenic organisms and some of these protease were reported localizing on the sueface of certain species. The proteases in N. bombycis were surveyed using whole genome scanning and the results showed that a larger number of metalloproteases and little smaller of serine protease and cysteine protease exist in this parasite. Surprisingly, no aspartate proteases and threonine protease was predicted in N. bombycis. Among these proteases, there are 22 metalloproteases that can be divided into five protein families including M1, M16, M17, M24 and M48, there are also 3 serine proteases that belong to the Peptidase_S8 superfamily and 3 cysteine proteases possessing otubains domain which were annotated as deubiquitinating enzymes. It was described that the otubains could contribute to evasion of host ubiquin-dependent innate immune response against pathogenic organisms. To dissect whether the otubains in N. bombycis are related to this process, the studies about NbOTUl was performed.2 Sequence character of NbOTUl in N. bombycisNbOTUl consists of 383 amino acids and there is no signal peptide, transmembrance or GPI anchor sequence signals existing in this protein but a predicted otubains domain. Sequence alignment and phylogenetic analysis showed otubains are conserved in microsporidia. From the results of sequence alignment and the predicted 3D model, we found NbOTUl possesses the conserved amino acids Asp46, Cys49 and His 209 that form the specific catalytic triad of otubains. Moreover, it was noteworthy that the proteins homologous to NbOTU1 in Encephalitozoon genus are more relatively conserved than Nosema genus, suggesting that a certain degree of variation have been occurred in these parasites.3 Identification and functional analysis of NbOTUl in N. bombycisThe recombinant protein NbOTU1 was expressed and the soluble protein rNbOTUl was obtained. Then, the polyclonal antibody against rNbOTU1 was prepared and the characterization and functional analysis of NbOTUl were conducted by Western-blotting, IFA and IEM and deubiquitination analysis in vitro, respectively. The results of Western-blotting showed that native NbOTU1 could be detected in mature spores and it can be secreted when spores are germinating. The deubiquitination analysis in vitro suggested that rNbOTU1 possesses deubiquitination activity. Furthermore, IFA and IEM indicated that NbOTU1 localized at the regions around the endospore and plasma membrane on mature spores. To our best knowledge, this is the first description of a microsporidian otubain-like protease in N. bombycis.In conclusion, comparative genomics analysis towards the proteins with tandem repeats was conducted in N. bombycis. Moreover, a spore wall protein NbSWP16 with tandem repeats was characterized and the function of this protein was studied further. Besides, a protease named NbOTUl with deubiquitinating activity was also identified as a novel spore wall protein in N. bombycis. These studies not only enriched the components of spore wall proteins of N. bombycis but also provided some novel clues to studies on the molecular mechanism of microsporidian spore wall formation.