Studies On The Serpin Proteins From Microsporidia Nosema Bombycis

Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They can infect a wide variety of animals ranging from invertebrates to vertebrates, including human, and they are the common pathogens and a kind of important pests for sericulture, fishery or shrimp farms. Since the first microsporidia, Nosema bombycis, has been discovered in the middle of the nineteenth century, thus far, more than 1 200 microsporidia species belonging to 150 genera have been reported, from which 14 species belonging to 8 genera can infect humans, lead to several syndromes in immunocompetent hosts and cause opportunistic infections in acquired immunodeficiency syndrome (AIDS) patients. Microsporidia drew great interest of scientists because they are closely related to human health. N. bombycis is known as a pathogen of silkworm pebrine, which usually prevails in sericulture. The prevention and treatment of pebrine is still an important and difficult topic for sericulture. However, little is known about molecular biology of N. bombycis.Recently, the whole genome sequence project of N. bombycis was started in our laboratory in 2003. A whole genome shotgun library was constructed successfully, nearly 6×fold coverage of the N. bombycis haploid genome reads database and a draft sequences were obtained. It provided the basis for studies on N. bombycis. By using comparative genomics analysis, we identified some some serpin-like genes from the genome of N. bombycis. However, none of serpin homologs and partial sequences was found from all other sequenced microspore organisms, i.e., Encephalitozoon cuniculi, Antonospora locustae and Nosema ceranae. In the paper, the study was carried out in order to identified the microsporadia serpins, to study the funtions of these serpin and the target proteins. It will construct the foundation for clarify the mechanism of infection and interaction between N. bombycis and host.Serine proteinase inhibitors (serpin) genes comprise a large gene family, and their protein products regulate a wide variety of proteinase-dependent physiological functions, such as blood coagulation, fibrinolysis, activation of complement, and the inflammatory response. During the long co-evolutionary interaction with the host immune system, infectious organisms have evolved an array of specific adaptations to evade the host defence system for survival. Most of the Lepidopteras, including Bombyx. mori., contain abundant proteases. The major protease in the digestive tract is serine protease, which play an important role in the growth and development. It seems likely that N. bombycis secrete serpins to protect microsporidia from proteinase-rich environments during ingesta, interfere with host immunity for survival.In the present study, we fitrstly dentified microsporadia serpins from N. bombycis genomic data. Secondly, sequence alignments and phylogenetic relationships are presented. And we studied the origin, amplification and functional differentiation of the N. bombycis 11 serpins. Thirdly, RT-PCR, gene cloning, prokaryotic expression, monoclonal antibodies production, western blot were employed. Subsequently, immunoelectron microscopy analysis were employed. And we detected the proteinase inhibitory activity of the serpin protein NbSPN106 and NbSPN286 against trypsin and chymotrypsin. Finally, the GST-pull down combinding LC-MS/MS analysis was used to isolate and identify the target proteins that reacted specifically with the purified recombinant protein. The results are as follows:1. Identification of serpin genes in the N. bombycis genomeAll N. bombycis predicted proteins with an expect score less than 1×10^-6 were checked against serpin sequences which downloaded from NCBI, PFAM and MEROPS. By using BLASTP and Hidden Markov Models, 11 serpin-like genes were identified in the genome project of N. bombycis, and 6 sequences were predicted to contain signal peptides indicating that the microspore has adapted these genes to fulfill an extracellular function. And serpins appear to be absent from all other sequenced microspore organisms, i.e., Encephalitozoon cuniculi and Antonospora locustae. Although the amino acid sequence of serpins from N. bombycis has low overall homology to the other identified serpins, certain characteristic features of serpins are evident, including high conserved hinge region. We can't confirm the inhibitory activity of these serpins against protease through their identities due to high species-specificity of N. bombycis genome. The phylogenetic tree indicated that the the serpins from N. bombycis did not cluster with previously established clades, but maintained its independent phylogenetic relationship. They may be belonged to new clade. Moreover, they are distant with the celpin, serpin of Piromyces sp. strain E2, which is the only representative of serine proteinase inhibitor of the fungal kingdom has been reported until now. All N. bombycis serpin genes cluster in distinct groups related to the organism phylogeny, indicating that they have evolved from a single ancestor via gene duplication. It possibly reflect selection pressures to adapt to differential host immune-response challenges.2. Studied on the origin, amplification and functional differentiation of the serpin genes from N. bombycisBy the methods of phylogeny, statistics and evolutionary analysis, we studied the origin, amplification and functional differentiation of the TV. bombycis 11 serpins. Deduced from the data information to date, there is little possibility that N. bobycis serpins were produced by horizontal gene transfer, instead probably by de novo origin. Serpin were amplified and formed six families in TV. bombycis. Some serpins were distributed in clusters in TV. bombycis genome. The relative analysis between serpins and segmental duplications and transposable elements indicated that there are six out of eleven serpins have significant correlation to the TEs, and two pairs of serpins were produced by segmental duplication. The multialignments indicated that the serpins were functionally differentiated. Meanwhile, the substitution rate of amino acids showed that all TV. bombycis serpin experienced negative selection, which suggested that all serpins are in stable functional state, and furthermore, which demonstrated the differentiation of serpins was fixed.3. Molecular cloning and expression of the serpin genes from N. bombycisIn this study, we identified two microsporadia serpin gene, Nbspn106 and Nbspn286, from TV. bombycis genomic data. Reverse transcription PCR analysis isolated from different infected stages of the silkworm midguts showed the transcription of Nbspn106 and Nbspn286 begin with 24h post-infected and continued through to day 7 post-infected. The CDS of Nbspn106 and Nbspn286 without the signal peptide were subcloned into the pGEX4T-1 expression vector. The GST-NbSPN fusion proteins were expressed in Escherichia coli and soluble material was purified using GSTrapFF affinity column. Mouse polyclonal antibodies were raised against NbSPN106 and NbSPN286 recombinant proteins. In Western blot analysis, a specific band of approximately 45 kDa and 40 kDa was recognized by the anti-NbSPNs serum, respectively.4. Ultrastructural localization of the serpin proteinsIn order to determine the cellular location of NbSPN106 and NbSPN286, the specific mouse polyclonal antibodies against NbSPN 106 and NbSPN286 were used for immunoelectron microscopy (IEM). IEM of ultrathin sections of TV. bombycis-infected silk glands showed that the labelled gold particles are present in sporoplasm of spores and in tissue space of silk glands, while in the negative control no gold particles were found on the spores of TV. bombycis or the silkworm tissues. It indicated that the two serpin proteins are secretory protein expressed by TV. bombycis. This study will facilitate our understanding of the mechanisms underlying the host and parasite interactions.5. Inhibitory activity of the serpin proteinsBy using Native-Poly-acrylamide gel electrophoresis followed by active staining, we investigated the inhibitory activities NbSPN106 and NbSPN286. To eliminate the influence of GST tag, the purified recombinant serpin proteins were digested with thrombin cleavage capture kit. After digestion, the serpin proteins were loaded onto Native-PAGE and stained. The results indicated that the digested recombinant serpin proteins of NbSPN106 and NbSPN286 showed no inhibitory activity against bovine trypsin and bovine chymotrypsin. It may attributed to three factors. First, the serpin proteins secreted by N. bombycis perform a noninhibitory function, not to inhibit proteases, but play other roles. Second, the proteins expressed in Escherichia coli is not active or low active, because some functional proteins with the formation of protein folding, such as molecular chaperones and some with glycosylation, acetylation, and disulfide formation, lack in prokaryotic cells. Third, NbSPN106 and NbSPN286 show a high substrate specificity, and they can not inhibit the commercial bovine trypsin and bovine chymotrypsin. Their possible role in pathogenicity deserve further studies.6. Isolation and identification of the target proteins of the serpin proteinsIn the study, we used the recombinant GST-NbSPNs fusion proteins to pull down the target proteins interact with NbSPN106 and NbSPN286. The GST-NbSPNs fusion proteins were expressed in Escherichia coli and purified using using GSTrapFF affinity column and bounded to gultathione agarose beads. The gultathione agarose bead bounded with the GST-fusion serpin proteins was added to the supernantant produced form N. bombycis-infected midguts of silkworm. We detected three differential bands by SDS-PAGE and obtained four hypothetical interacting proteins by analysis of LC-MS/MS. However the results are not agree with our hypothesis, which the target proteins of NbSPNs may be the host proteases. Thus, we know that NbSPN106 and NbSPN286 can recognize and interact with the host cell proteins, and the interaction mechanism is quite complex. But, the interaction mechanism and the particular biological function of NbSPNs will be studied subsequently. Taken as a whole, this research will have laid a foundation for further identification of the interacting proteins.