Analysis Of Differentially Expressed Genes Among The Schistosomula Of Schistosoma Japonicum From Different Hosts

Schistosomiasis is one of the wide spread and prevalent parasitic diseases worldwide. Nearly 40 kinds of animals can be infected by Schistosoma japonicum (Chinese mainland strain). The previous research had demonstrated that BALB/c mouse as a susceptible host of schistosome, Wistar rat as a unsusceptible host and Microtus fortis as resistant host of Schistosoma japonicum infection. Microtus fortis is the resistant host of Schistosoma japonicum. Animals have different symptoms after infected by cercaria: 60-70% of the adult Schistosoma japonicum can be perfused post-cercarial challenge from the host in BALB/c mouse, but 10-20% from Wistar Rat. Most of the schistosolums have incomplete development and fail to accomplish their life cycles in Microtus fortis. Till now, Microtus fortis is the only unmorbific host after infected with schistosoma japonicum. The microarray analysis of the gene expression difference between mouse, Wistar rat and Microtus fortis demonstrated that the genes related with immune mechanism, apoptosis, growth and development were differential expressed. Our research work focused on the different expression genes in 10 days Schistosolum from the hosts of Microtus fortis, BALB/c mouse and Wistar rat with the analysis of oligonucleotide microarray and bioinformatics techniques. A great number of genes related with signal transduction, immune escape, development and metabolism had been screen out with the analysis. Several genes of Schistosoma japonicum were selected for further research: genes referred with apoptosis such as Sj IAP,caspase3 and caspase 7, genes related with development and growth such as Sj MAP 2 and Sj mago nashi, in signal transduction such as Cyclophilin A,B and C genes.Our data presented here suggest that such genes may play important role in the development; reproduction and host-parasite interplay in schistosome living, and may be a potential new drugs or vaccine target for controlling schistosomiasis.1,Scanning electron microscope(SEM) and transmission electron microscopy (TEM) observation of Schistosolum from the three different hostsThere are obvious changes in the development and growth of Schistosoma japonicum in different hosts. After two weeks post infected with cercaria, Schistosolum gradually withered and stopped growth, then died. The width and length of schistosomula were measured with optical microscope.The length of schistosomula from Microtus fortis, Wistar rat and BALB/c mouse was 402.2±102.2, 828.3±127.4 and 878.5±137.4. The width of schistosomula from Microtus fortis, Wistar rat and BALB/c mouse was 159.1±47.3,103.4±22.8 and 88.3±20.0.The morphologic comparion of the schistosolums from three different hosts with SEM and TEM showed that microscopic and ultrastructural structure of the schistosolums have significant difference. The surfaces of the schistosomula from Microtus fortis were stunted, shrinking and vacuole.The nucleus of the cells in schistosomula from Microtus fortis were agglutinated. All these revealed that the cells of Schistosolum may have the phenomenon of apoptosis. Schistosolum were divided into monoplast with trypsinization, and then apoptosis were determined by flow cytometry (FCM) with the analysis of Annexin V-FITC/propidium iodide (PI).2,Analyze differentially expressed genes of 10 days schistosolum from the three different hostsBased on the data of oligonucleotide microarray and bioinformatics analysis of the differentially expressed genes from the three different hosts: in the schistosolums from Microtus fortis,3293 genes were down-regulated (fold <0.5)and 71 genes were up-regulated (fold >2) in comparison with the schistosolums in BALB/c mouse; in the schistosolums in from Wistar rat , 3335 geneswere down-regulated and 133 genes were up-regulated in comparison with the schistosolums in BALB/c mouse.Preliminary function analysis with Gene Ontology GO classification and KEGG pathways analysis indicate that the up-regulated expressed genes in the schistosolums from Microtus fortis focused on the function as cell, extracellular region part, cell part, enzyme regulator activity, translation regulator activity, the down-regulated expressed genes in the schistosolums from Microtus fortis focused on the function as metabolic process, catalytic activity, developmental process, transporter activity, cellular process, localization and binding. The significant down-regulated expressed genes (fold <0.2) in the schistosolums from both Microtus fortis and Wistar rat included metabolic process, localization, anatomical structure formation and catalytic activity. Also another some genes not related with the above process are also co-down regulated, such as serine/threonine-protein kinase, NAD(P)H-quinone oxidoreductase, Pericentriolar material, Cell surface receptor daf-4. The KEGG pathway analysis revealed that the co-down regulated expressed genes in the schistosolums from both Microtus fortis and Wistar rat consist of the pathway of Glycine, serine and threonine metabolism, DNA replication, MAPK signaling pathway, Protein export, Aminoacyl-tRNA biosythesis. Because the genomic research works of S. japonicum and S. mansoni were fall behind the Model organism Caenorhabditis elegans, we do the KEGG pathway analysis of the co-down regulated expressed genes with the C. elegans pathway model. We found that some genes are involved in the pathway as Apoptosis, Oxidative phosphorylation, Insulin signaling pathway and Fatty acid metabolism and so on.The 10 days schistosolum specific expression genes were selected and analyzed with the gene expression of NADH dehydrogenase [ubiquinone] flavoprotein 2 as the internal control. The result showed that 1178 genes were down-regulated (fold <0.5) and 524 genes were up-regulated (fold >2), also 124 significant down-regulated genes (fold <0.2) and 578 significant up-regulated genes (fold >5). The result of the Gene Ontology GO classification analysis of the significant up-regulated/high genes demonstrated that the different function genes between these two trend genes focused on metabolic process, catalytic activity, enzyme regulator activity, transcription regulator activity, transporter activity, antioxidant activity, electron carrier activity, structural molecule activity. In the other part, the KEGG pathways of the different function genes are involved in Protein export, MAPK signaling pathway, Oxidative phosphorylation, Inositol phosphate metabolism, Calcium signaling pathway, Progesterone-mediated oocyte maturation), Ubiquitin mediated proteolysis, Notch signaling pathway and estrogen-related receptor beta like 2.3,Analyzed of apoptosis in Schistosoma japonicum from different hosts and molecular characterizations of an inhibitor of apoptosisApoptosis is a normal process for regulating cellular death of many organisms. We molecularly characterized an inhibitor of apoptosis from Schistosoma japonicum (SjIAP). The transcription of the SjIAP predominantly occurred at the developmental stages in a final host. The immunohistochemistry analysis revealed that Sj IAP protein was located in the tegument of the adult male worm of schistosoma japonicum. Functional assay indicated that the SjIAP could inhibit caspase activity either in 293T cell or in schistosome lysates. Additionally, there were differently expressed profiles of the SjIAP in S. japonicum living in different hosts. The gene expression of two important molecules caspase 3 caspase 7 and IAP were certified with Real Time RT-PCR, and then the caspase activity in schistosome lysates was determined by using Glo? 3/7 Assay kit. Our preliminary results suggest that the SjIAP may play important roles in parasitic living and development as well as in the host–parasite interactions, and drug target of SjIAP might be a potential for controlling schistosomiasis.4,Molecular characterizations and identification of two important genes Sj Methionine aminopeptidase 2 and Sj mago nashi those those may play important roles in growth and development of Schistosoma japonicumBased on the result of the differentially expressed genes from the three different hosts, we selected two important functional genes Sj Methionine aminopeptidase 2 and Sj mago nashi those may play important role in growth and development of schistosoma japonicum for further research.Genes encoding methionine aminopeptidase 2 and mago nashi of Schistosoma japonicum (Sj MAP2, Sj mago nashi) were cloned and characterized. Real time RT-PCR(reverse-transcription polymerase chain reaction) and(or) Western blot analysis revealed that SjMAP2 and Sj mago nashi were expressed by each developmental stage of the schistosome tested, but was expressed at a higher level in the schistosomulum stage and the adult male worm at 42 days. The results also showed that the two genes were differentially expressed in worms from three different host species. Sj MAP 2 and Sj mago nashi were both highly expressed in worms from the schistosome susceptible mouse, expressed at a lower level in worms from the less susceptible rat, and at an even lower level in worms from the non-permissive host Microtus fortis. The expression of Sj MAP 2 was affected significantly when the hosts were treated with praziquantel: expression was down-regulated by 92.17% and 49.01%, respectively, in treated male and female adult worms in comparison with untreated worms. An immuno-experiment in mice indicated that recombinant Sj MAP 2 and Sj mago nashi could induce partial protective efficacy against schistosome infection. The data presented here suggest that SjMAP2 and Sj mago nashi were important molecules in the growth and development of the schistosome, and that it may be a potential new drug target or vaccine candidate for schistosomiasis.4,Molecular characterizations and identification of genes Cyclophilin A,B and C those may play important roles in signal transduction of Schistosoma japonicumThe result of the differentially expressed genes from the three different hosts revealed that the molecules as cell signaling and modification of protein translation may play important roles in the development of schistosoma japonicum. We selected Cyclophilin family genes those function may be chaperones and cell signaling as our research objects. The Cyclophilin family genes included Cyclophilin A, B and C.The full-length cDNA encoding Cyclophilin A, B and C in Schistosoma japonicum of schistosomula were amplified by RT-PCR technique. As determined by Real-time RT-PCR, the highest expression of Sj CyPA, B and C was observed at the transcript level at schistosomula stage, indicating that Sj CyPA, B and C was a abundant expression gene in schistosomula stage.The expressions of Sj CyPA both at transprict level and at protein level were not significantly alternated upon the treatment by Cyclosporin A as determined by Real time RT-PCR and western blot. Expertly, Chymotrypsin- coupled chromogenic assay confirmed that Sj CyPA had PPIase activity. Western blot indicated that Sj CyPA, B and C were able to induce specific antibodies. Additionally, animal experiment showed that partial worm reduction and egg reduction were achieved in mice vaccinated with recombinant Sj CyPA, B and C protein, respectively.In total, the differentially expressed genes from the three different hosts were focused on many signal pathway and function. Our research work present here demonstrated that the independentof development and growth in schistosoma japonicum from Microtus fortis may due to the nutrition metabolism, signal transduction, immune escape and apoptosis and so on. With the further research work on the differentially expressed genes, many important function molecules in Schistosolum will be characterized and its biological function will be identified. All the research work will help us to explain the host and parasite inter play, growth and development of the schistosome, and the identification of different expression genes may potentially represent new drugs or vaccine targets, applicable for the future controlling of schistosomiasis.