Proteomic Analysis Of Proteins Expressed By Schistosomula Of Schistosoma Japonicum In Hosts With Different Susceptibilities To The Infection

Schistosomiasis is the second most prevalent tropical disease caused by parasitic blood flukes. It causes symptomatic infection in approximately 200 million individuals and more than 200 thousand deaths per year in 74 endemic countries, with more than 600 million people at risk of infection. The current strategy for control of schistosomiasis aims at the reduction of morbidity and involves treatment with praziquantel. However, chemotherapy does not prevent re-infection, and some isolates of Schistosoma mansoni (S. mansoni) that are resistant to high doses of praziquantel have been found in Egypt. Thus it has been argued that the identification of target proteins for use in the development of vaccines or new drugs would contribute enormously to the control of this disease.It was reported that in China S. japonicum can infect more than 40 kinds of mammals including goat, cattle, sheep, rabbit and mice, which are susceptible to the infection, and some others such as rat, which are less susceptible as indicated by the observations that worms exhibit a low developmental rate and a smaller worm size in these hosts. The reed vole or Microtus fortis, is the only mammal reported so far in which the schistosome cannot develop and thus do not have any significant pathological effects. As reported earlier, growth and survival of the worms was extremely poor in M. fortis 15 days after a challenge with cercariae. This may be that M. fortis developed a stronger immune response and more severe pathological lesion in response to the schistosomes during the early phase of the infection. Different hosts provide a distinctive environment for the schistosome, which impacts the survival and development of the parasite. Protein expression variation in schistosome would be influenced directly by the development of worms in different hosts, and some of the proteins that are differentially expressed in worms from different hosts may be key molecules required for the survival of the worms, and the establishment of the parasite.1. Proteomic analysis of proteins expressed by schistosomula of Schistosoma japonicum in hosts with different susceptibilities to the infectionThe aim of this study was to identify proteins involved in the growth and survival of the parasitic forms inside a host. Schistosomula of Schistosoma japonicum were isolated from three different hosts:the susceptible BALB/c mice; the Wistar rats, which have a considerably lower susceptibility; and the resistant reed vole, Microtus fortis. Soluble proteins of the schistosomula collected from the above three hosts 10 days post-infection were subjected to two-dimensional difference gel electrophoresis (2-D DIGE). Soluble proteins extracted from the schistosomula were compared by the DeCyder 6.5 software. A total of 1721 spots were matched across four analytical gels and one preparative gel. Thirty-nine protein spots were found to be differentially expressed between the 10 d schistosomula from mice or rats, of which 27 were up-regulated and 12 were down-regulated in schistosomula from mice. Twenty-one protein spots were found to be differentially expressed between 10 d schistosomula from mice or M. fortis, of which 13 were up-regulated and 8 were down-regulated in schistosomula from mice.The proteins selected as differentially expressed were separated on a preparative gel and visualized by staining with Coomassie blue. Gel pieces containing the proteins of interest were excised and subjected to digestion with trypsin. Thus,44 protein spots were considered suitable for downstream analysis by MALDI MS/MS, based on the amount of protein, matching across gels and the presence of the protein spot on the preparative gels. Out of these,41 protein spots which represented 33 proteins were successfully identified by MS.Six genes corresponding to the protein spots were chosen for quantitative real-time PCR (qPCR) analysis to quantify their transcript levels. The qPCR results were consistent with those of the DIGE studies, and suggested that these proteins identified as differentially expressed were regulated at transcriptional level.In order to better understand the biological functions of the 33 differentially expressed proteins, Gene Ontology Annotation was performed. The annotated S. japonicum proteins from the NCBI non-redundant database were analyzed by the Blast2GO and AmiGO servers initially. Each identified known protein was classified according to its GO functional annotation. These differentially expressed proteins were essentially those involved in metabolism of proteins, ribonucleotides or carbohydrates, or in stress response or cytoskeleton/movement. Mitochondrial import receptor subunit TOM34 protein, proteasome subunits, heterogeneous nuclear ribonucleoprotein K and actin 5C protein were highly expressed in the susceptible host, mice compared to the unsusceptible host, rats or the resistant host, M. fortis. In contrast, cysteine protease inhibitor, aldolase and heat shock 70kDa protein were relatively highly expressed in rats or M. fortis compared to mice. Most of the differentially expressed proteins were involved in important biological functions. The analysis also revealed that the proteins of 10 d schistosomula that were differentially expressed between rats and mice were mainly related to protein (40%) or carbohydrate metabolism (26%), while those differentially expressed between M. fortis and mice were mainly related to protein (53%) or ribonucleotide metabolism (23%).This study represents the first attempt at profiling S. japonicum living in different states and provides a basis for a better understanding of the molecular mechanisms in the development and survival of S. japonicum in different host environments.2. Cloning, expression and immunoprotection of<alpha>5-subunit and <alpha>2-subunit of the proteasome in Schistosoma japonicumA full-length cDNA encoding the Schistosoma japonicum proteasome subunit alpha type 5 protein (SjPSMA5) was first isolated from 18-day-schistosomulum cDNAs. The cDNA had an open reading frame (ORF) of 747 bp and encoded 248 amino acids. Real-time quantitative RT-PCR analysis revealed that SjPSMA5 is up-regulated in 18-day and 32-day schistosomes, and the level of expression in male is around 4-fold higher than that in female worms at 42 days. Real-time quantitative RT-PCR analysis also showed that SjPSMA5 is up-regulated in 10d schistosomulum from BALB/c mice than that from rats and M. fortis. The SjPSMA5 was subcloned into pET28a(+) and expressed as inclusion bodies in E. coli BL21 (DE3) cells. Western blotting showed that the recombinant SjPSMA5 (rSjPSMA5) was immunogenic. Immunolocalization studies revealed ubiquitous expression of SjPSMA5 in S. japonicum organs and tissues. After immunization of BALB/c mice with rSjPSMA5, reductions of 23.29% and 35.23% were obtained in the numbers of worms and eggs in the liver, respectively. The levels of specific IgG antibodies and CD4+ cells were significantly higher (P<0.01) in the group vaccinated with rSjPSMA5 combined with Seppic 206 adjuvant than in the other groups, as detected by enzyme linked immunosorbent assay (ELISA) and flow cytometry. The study suggested that rSjPSMA5 induced partial immunoprotection against Schistosoma japonicum in BALB/c mice.A full-length cDNA encoding the Schistosoma japonicum proteasome subunit alpha type 2 protein (SjPSMA2) was first isolated from 18-day-schistosomulum cDNAs. The cDNA had an open reading frame (ORF) of 708 bp and encoded 235 amino acids. Real-time quantitative RT-PCR analysis revealed that SjPSMA2 is down-regulated in 7-day and 23-day schistosomes. Real-time quantitative RT-PCR analysis also showed that SjPSMA2 is a little up-regulated in 10d schistosomulum from BALB/c mice than that from rats and M. fortis. The SjPSMA2 was subcloned into pET28a(+) and expressed as inclusion bodies in E. coli BL21 (DE3) cells. Western blotting showed that the recombinant SjPSMA2 (rSjPSMA2) was immunogenic. Immunolocalization studies revealed ubiquitous expression of SjPSMA2 in S. japonicum organs and tissues. After immunization of BALB/c mice with rSjPSMA2, reductions of 12.33% and 35.23% were obtained in the numbers of worms and eggs in the liver, respectively. The levels of specific IgG antibodies was significantly higher (P< 0.01) in the group vaccinated with rSjPSMA2 combined with Seppic 206 adjuvant than in the other groups, as detected by enzyme linked immunosorbent assay (ELISA). The study suggested that rSjPSMA2 induced partial immunoprotection against Schistosoma japonicum in BALB/c mice.All the above suggested that SjPSMA5 and SjPSMA2 could be potential vaccine candidates against schistosomiasis. This study provided an important basis for investigating the role of the proteasome during the development of Schistosoma japonnicum.3. Cloning, expression and characterization of thioredoxin peroxidase gene of Schistosoma japonicumBased on the results of the differentially expressed proteins of 10d schistosomulum from three different hosts, we selected a functional gene thioredoxin peroxidase which plays an important role in anti-oxidant in Schistosoma japonicum for further research. A full-length cDNA encoding the Schistosoma japonicum thioredoxin peroxidase (SjTPx) was analyzed by some bioinformatics softwares. The cDNA had an open reading frame (ORF) of 681 bp and encoded 227 amino acids. The ORF contains a part of signal peptides of 24 amino acids. The cDNA encoding SjTPx without the signal peptides sequence was isolated from 42-day-schistosome cDNAs. Real-time quantitative RT-PCR analysis revealed that SjTPx is up-regulated in 7-day and 13-day schistosomes, and the level of expression in female is around 2-fold higher than that in male worms at 42 days. The SjTPx was subcloned into pET28a(+) and expressed as both inclusion bodies and supernatant in E. coli BL21 (DE3) cells. Western blotting showed that the recombinant SjTPx (rSjTPx) was immunogenic. Immunolocalization studies revealed that SjTPx was predominantly localized in the sub-tegumental layer and it was also expressed in other organs and tissues. The result of mass spectrum revealed that purified protein could inform disulfide-bonded dimmers in vitro. H2O2 peroxidase assay and protection of super-coiled DNA assay demonstrated that SjTPx owned the peroxidase activity in vitro.