Jianmei Yang

Proteomic Analysis of Tegument-Exposed Proteins of Female and Male Schistosoma japonicum Worms

The interplay between sexes is a prerequisite for female growth, reproductive maturation, and egg production, and the basis of schistosome pathopoiesis and propagation. The tegument is in direct contact with the host environment and its surface membranes are particularly crucial for schistosome survival in the definitive host. In this study, a streptavidin-biotin affinity purification technique combined with LC-MS/MS was used to analyze putative tegument-exposed proteins in female and male adult Schistosoma japonicum worms. In total, 179 proteins were identified in females and 300 in males, including 119 proteins common to both sexes, and 60 female biased and 181 male biased proteins. Some (e.g., serpin and CD36-like class B scavenger receptor) were involved in host-schistosome interactions, while some (e.g., gynecophoral canal protein) were important in the interplay between sexes. Gene Ontology analysis revealed that proteins involved in protein glycosylation and lysosome were highly expressed in females, while proteins involved in intracellular signal transduction, regulation of actin filament polymerization, and proteasome core complex were highly expressed in males. These results might elucidate physiological differences between the sexes. Our study provides new insights into schistosome growth and sexual maturity in the final host and permits the screening of vaccine candidates or drug targets for schistosomiasis.

Comparison of worm development and host immune responses in natural hosts of schistosoma japonicum, yellow cattle and water buffalo

BackgroundYellow cattle and water buffalo are two of the most important natural hosts for Schistosoma japonicum in China. Previous observation has revealed that yellow cattle are more suited to the development of S. japonicum than water buffalo. Understanding more about the molecular mechanisms involved in worm development, as well as the pathological and immunological differences between yellow cattle and water buffalo post infection with S japonicum will provide useful information for the vaccine design and its delivery procedure.ResultsThe worm length (p < 0.01), worm recovery rate (p < 0.01) and the percentage of paired worms (p < 0.01) were significantly greater in yellow cattle than those in water buffalo. There were many white egg granulomas in the livers of yellow cattle, but fewer were observed in water buffalo at 7 weeks post infection. The livers of infected yellow cattle contained significantly increased accumulation of inflammatory cells, and the schistosome eggs were surrounded with large amounts of eosinophil infiltration. In contrast, no hepatocyte swelling or lymphocyte infiltration, and fewer white blood cells, was observed in water buffalo. The percentage of CD4+ T cells was higher in yellow cattle, while the percentage of CD8+ T cells was higher in water buffalo from pre-infection to 7 w post infection. The CD4/CD8 ratios were decreased in both species after challenge with schistosomes. Comparing with water buffalo, the IFN-γ level was higher and decreased significantly, while the IL-4 level was lower and increased gradually in yellow cattle from pre-infection to 7 w post infection.ConclusionsIn this study, we confirmed that yellow cattle were more suited to the development of S. japonicum than water buffalo, and more serious pathological damage was observed in infected yellow cattle. Immunological analysis suggested that CD4+ T cells might be an integral component of the immune response and might associate with worm development in yellow cattle. A shift from Th1 to Th2 type polarized immunity was only shown clearly in schistosome-infected yellow cattle, but no shift in water buffalo. The results provide valuable information for increased understanding of host-schistosome interactions, and for control of schistosomiasis.

Wnt4, the first member of the Wnt family identified in Schistosoma japonicum, regulates worm development by the canonical pathway

The Wnt signaling pathway is an evolutionarily conserved signal transduction pathway used extensively during animal development. We aim, by increasing our understanding of the Wnt signaling pathway, to find a key gene or protein present in schistosomes that can be developed into vaccine candidate or drug target. We therefore isolated the Wnt4 gene from Schistosoma japonicum. Wnt4 encodes a putative protein of 558 amino acids which contains the conserved functional domain of the Wnt gene family. We suppressed the expression of Wnt4 mRNA in 10-day schistosomulae by RNA interference. Quantitative PCR analysis showed that Wnt4 displayed a 73% reduction in the transcript level. And GSK-3β and β-catenin, which are involved in Wnt canonical pathway, showed a 45% and 39% reduction in mRNA levels, respectively. PLC, CaMKII, DVL, and JNK, which are involved in Wnt non-canonical pathway, showed no reduction. These results suggest that the Wnt4 signal protein in S. japonicum regulates downstream genes by a canonical pathway. Wnt4 is the first member of the Wnt family to be identified in S. japonicum. An increased understanding of the Wnt signal transduction pathway will allow us to elucidate further the molecular mechanism of development in schistosomes.

Characterization and Expression of the Schistosoma japonicum Thioredoxin Peroxidase-2 Gene

Abstract:  We analyzed proteins that were differentially expressed by 10-day-old schistosomula from 3 different hosts and determined that a functional thioredoxin peroxidase-2 gene has an important antioxidant role in Schistosoma japonicum, which we investigated further. A full-length cDNA encoding the S. japonicum thioredoxin peroxidase-2 (SjTPx-2) had an open reading frame of 681 bp that encoded 226 amino acids with a signal peptide of 24 amino acids. A cDNA encoding SjTPx-2 without the signal peptide sequence was isolated from 42-day-old schistosome cDNAs. Real-time quantitative RT-PCR analysis revealed that SjTPx-2 was upregulated in 7- and 13-day-old schistosomes, while the expression level in females was around 2-fold higher than that in male worms at 42 days. SjTPx was subcloned into pET28a(+) and expressed as both inclusion bodies and supernatant in Escherichia coli BL21 (DE3) cells. Western blotting showed that the recombinant SjTPx-2 (rSjTPx-2) was immunogenic. The purified recombinant protein could form disulfide-bonded dimers and it had peroxidase activity in vitro. An immunoprotection experiment in BALB/c mice showed that vaccination with recombinant SjTPx-2 could induce 31.2% and 34.0% reductions in the numbers of worms and eggs in the liver, respectively. This study suggests that SjTPx-2 may be an important antioxidative enzyme in scavenging ROS, and it may be a potential vaccine candidate or new drug target for schistosomiasis.

Ultrastructural Observation and Gene Expression Profiling of Schistosoma japonicum Derived from Two Natural Reservoir Hosts, Water Buffalo and Yellow Cattle

Water buffalo and yellow cattle are the two of the most important natural reservoir hosts for Schistosoma japonicum in endemic areas of China, although their susceptibility differs, with water buffalo being less conducive to the growth and development of S. japonicum. Results from the current study show that the general morphology and ultrastructure of adult schistosomes derived from the two hosts also differed. Using high-throughput microarray technology, we also compared the gene expression profiles of adult schistosomes derived from the two hosts. We identified genes that were differentially expressed in worms from the two natural hosts. Further analysis revealed that genes associated with protein kinase and phosphatase, the stimulus response, and lipid and nucleotide metabolism were overexpressed, whereas genes associated with reproduction, anatomical structure morphogenesis and multifunctional motif were underexpressed in schistosomes from water buffalo. These differentially expressed genes were mainly involved in nucleotide, energy, lipid metabolism, energy metabolism, transcription, transport and signaling pathway. This suggests that they are key molecules affecting the survival and development of schistosomes in different natural host species. The results of this study add to current understanding of the interplay between parasites and their natural hosts, and provide valuable information for the screening of vaccine candidates or new drug targets against schistosomiasis in the natural reservoir hosts in endemic areas.

Characterization and expression of a novel Frizzled 9 gene in Schistosoma japonicum.

Wnt signaling regulates a diverse array of eukaryotic development processes, which are mediated by the Frizzled family receptors. However, the role of this signaling pathway in the development of Schistosoma japonicum remains poorly understood. We isolated a novel S. japonicum Frizzled member (SjFz9), which encodes a 923 amino acid protein, sharing the general feature of Frizzled proteins. We investigated its mRNA and protein expression patterns during different life stages in definitive hosts. Quantitative real-time PCR analysis revealed that SjFz9 transcripts were highly expressed in the schistosomulum. In adult stages, SjFz9 expression exhibited high level at day 23 and day 42 in both male and female, compared to other adult stages. The immunohistochemical localization pattern of the SjFz9 protein showed a broad tissue distribution in the subtegumental musculature and acetabulum musculature of schistosomulum and adult worms. Furthermore, SjFz9 was found prominently expressed in the testes of the male and the ovary as well as the vitellarium of the female. Our data suggest that SjFz9 may be an important Wnt receptor with potential functions in regulating the cell differentiation and proliferation within the musculature, as well as the development of the reproductive organs of both sexes.

Microarray analysis of gene expression profiles of Schistosoma japonicum derived from less-susceptible host water buffalo and susceptible host goat.

Background Water buffalo and goats are natural hosts for S. japonicum in endemic areas of China. The susceptibility of these two hosts to schistosome infection is different, as water buffalo are less conducive to S. japonicum growth and development. To identify genes that may affect schistosome development and survival, we compared gene expression profiles of schistosomes derived from these two natural hosts using high-throughput microarray technology. Results The worm recovery rate was lower and the length and width of worms from water buffalo were smaller compared to those from goats following S. japonicum infection for 7 weeks. Besides obvious morphological difference between the schistosomes derived from the two hosts, differences were also observed by scanning and transmission electron microscopy. Microarray analysis showed differentially expressed gene patterns for parasites from the two hosts, which revealed that genes related to lipid and nucleotide metabolism, as well as protein folding, sorting, and degradation were upregulated, while others associated with signal transduction, endocrine function, development, immune function, endocytosis, and amino acid/carbohydrate/glycan metabolism were downregulated in schistosomes from water buffalo. KEGG pathway analysis deduced that the differentially expressed genes mainly involved lipid metabolism, the MAPK and ErbB signaling pathways, progesterone-mediated oocyte maturation, dorso-ventral axis formation, reproduction, and endocytosis, etc. Conclusion The microarray gene analysis in schistosomes derived from water buffalo and goats provide a useful platform to disclose differences determining S. japonicum host compatibility to better understand the interplay between natural hosts and parasites, and identify schistosome target genes associated with susceptibility to screen vaccine candidates.

Differential expression of microRNAs in the non-permissive schistosome host Microtus fortis under schistosome infection.

The reed vole Microtus fortis is the only mammal known in China in which the growth, development and maturation of schistosomes (Schistosoma japonicum) is prevented. It might be that the anti-schistosomiasis mechanisms of M. fortis associate with microRNA-mediated gene expression, given that the latter has been found to be involved in gene regulation in eukaryotes. In the present study, the difference between pathological changes in tissues of M. fortis and of mice (Mus musculus) post-schistosome infection were observed by using hematoxylin-eosin staining. In addition, microarray technique was applied to identify differentially expressed miRNAs in the same tissues before and post-infection to analyze the potential roles of miRNAs in schistosome infection in these two different types of host. Histological analyses showed that S. japonicum infection in M. fortis resulted in a more intensive inflammatory response and pathological change than in mice. The microarray analysis revealed that 162 miRNAs were expressed in both species, with 12 in liver, 32 in spleen and 34 in lung being differentially expressed in M. fortis. The functions of the differentially expressed miRNAs were mainly revolved in nutrient metabolism, immune regulation, etc. Further analysis revealed that important signaling pathways were triggered after infection by S. japonicum in M. fortis but not in the mice. These results provide new insights into the general mechanisms of regulation in the non-permissive schistosome host M. fortis that exploits potential miRNA regulatory networks. Such information will help improve current understanding of schistosome development and host–parasite interactions.

Characterization Analysis of Schistosoma japonicum Plasma Membrane Repair Relative Gene Myoferlin.

Myoferlin is a member of the ferlin family of proteins, which are involved in plasma membrane repair, and has been identified as one of the tegument proteins of Schistosoma japonicum. The tegument proteins are potential candidates for vaccines and new drug targets. In this study, myoferlin of S. japonicum (SjMF) was cloned, expressed and characterized, the potential of SjMF recombinant protein (rSjMF) as a vaccine candidate was evaluated, and the effect of praziquantel on SjMF was detected by Real-time PCR. Immunofluorescence showed that this protein was mainly distributed on the surface of worms at different stages. Sequence analysis revealed that the SjMF open reading frame was conserved at all stages of the S. japonicum life cycle. And SjMF transcription was upregulated in 42-day-old worms, and was significantly higher in female worms. Western blotting revealed that rSjMF showed strong immunogenicity. The cytokine profile and IgG isotype analysis demonstrated that rSjMF plus ISA206 immunization induced a mixed T helper (Th)1/Th2 response. Purified rSjMF emulsified with ISA206 adjuvant significantly reduced worm burden from 21.8% to 23.21% and liver egg number from42.58% to 28.35%. Besides, SjMF transcription was downregulated when worms were exposed to low-dose praziquantel (PZQ) and upregulated when PZQ was degraded, accompanied by recovery of damaged tegument. When worms were exposed to high-dose PZQ, SjMF transcription was downregulated all the time and the damaged tegument did not recover. These findings indicated that SjMF is a potential vaccine against S. japonicum and provides the basis for further investigations into the biological function of SjMF.

The Differential Expression of Immune Genes between Water Buffalo and Yellow Cattle Determines Species-Specific Susceptibility to Schistosoma japonicum Infection.

Water buffalo are less susceptible to Schistosoma japonicum infection than yellow cattle. The factors that affect such differences in susceptibility remain unknown. A Bos taurus genome-wide gene chip was used to analyze gene expression profiles in the peripheral blood of water buffalo and yellow cattle pre- and post-infection with S. japonicum. This study showed that most of the identified differentially expressed genes(DEGs) between water buffalo and yellow cattle pre- and post-infection were involved in immune-related processes, and the expression level of immune genes was lower in water buffalo. The unique DEGs (390) in yellow cattle were mainly associated with inflammation pathways, while the unique DEGs (2,114) in water buffalo were mainly associated with immune-related factors. The 83 common DEGs may be the essential response genes during S. japonicum infection, the highest two gene ontology (GO) functions were associated with the regulation of fibrinolysis. The pathway enrichment analysis showed that the DEGs constituted similar immune-related pathways pre- and post-infection between the two hosts. This first analysis of the transcriptional profiles of natural hosts has enabled us to gain new insights into the mechanisms that govern their susceptibility or resistance to S. japonicum infections.