Yanhui Han

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.

Apoptosis phenomenon in the schistosomulum and adult worm life cycle stages of Schistosoma japonicum

Apoptosis is an important aspect of a number of biological processes, from embryogenesis to the stress-injury response. It plays a central role in balancing cell proliferation and tissue remodeling activity in many organisms. In the present study, apoptosis in 14 days post infection schistosomula was evaluated using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assays and DAPI staining. Additionally, flow cytometry using the Annexin V-FITC/propidium iodide (PI) (Annexin V/PI) assay confirmed the percentage of early apoptotic, late apoptotic, and necrotic cells in 14 and 23 days post infection worms. Conserved Domain Database (CDD) BLAST analysis and alignment analysis of known schistosome proteins demonstrated the feasibility of detecting the activity of caspase-3 and -7 using the caspase-3/7 Glo analysis assay. Analysis of caspase-3 and -7 activities in schistosome demonstrated that both caspases were active in each developmental stage of Schistosoma japonicum, but was highest in the 14 days post infection schistosomula. Additionally, the caspase peptide inhibitor (Z-VAD-FMK) inhibited the caspase-3/7 activity at all developmental stages examined. Therefore, we hypothesized that two main signaling pathways are involved in apoptosis in S. japonicum, the caspase cascade and the mitochondrial-initiated pathway. We have constructed a model of these two pathways, including how they may interact and their biological outcomes. qRT-PCR analyses of the gene expression profiles of apoptosis-related genes supported our hypothesis of the relationship between the apoptotic pathway and parasite development. The data presented here demonstrates that apoptosis is an important biological process for the survival and development of the schistosome, and identifies potential novel therapeutic targets.

Proteomics analysis of differentially expressed proteins in schistosomula and adult worms of Schistosoma japonicum

Schistosoma japonicum has a complex lifecycle and exhibits dramatic changes in its biology and morphology at different developmental stages. The schistosomulum and adult worm are two stages of this complex lifecycle and differentially expressed proteins in these two stages should be important for survival, development, and reproduction of the parasites. In this study, soluble and hydrophobic proteins were extracted from eggs, cercariae, schistosomula (8d and 19d), and male and female adult worms (42d) of Schistosoma japonicum, and separated by two-dimensional (2D) gel electrophoresis. A total of 1376±52, 928±61, 1465±41, 1230±30, 904±34, and 1080±26 soluble proteins and 1437±44, 845±53, 986±22, 1145±35, 1066±39, and 1123±45 hydrophobic proteins were separated from eggs, cercariae, schistosomula (8d and 19d), and male and female adult worms (42d), respectively. There were 65±14, 27±7, 37±17 and 48±9 soluble protein spots only present in schistosomula (8d and/or 19d) and adult schistosomes (male and/or female). We successfully identified 22 spots from schistosomula and 11 spots from adult schistosomes by mass spectrometry. Quantitative real-time RT-PCR was used to examine six differentially expressed proteins at the transcription level. These proteins only found in schistosomula or adults stage by the proteomics analysis were highly expressed in the corresponding stage at mRNA level. Bioinformatics analysis showed that the differentially expressed proteins from schistosomula were mainly involved in cellular metabolic processes, stress response and developmental process. Differentially expressed proteins from adult schistosomes were involved with gene expression and protein metabolism processes. The results of this study might provide new insights to stimulate further exploration of the mechanism of growth and development in schistosomes and help identify candidate molecules for developing new vaccines or drugs.

Characterization of thioredoxin glutathione reductase in Schiotosoma japonicum.

Schistosomiasis is one of the most prevalent and serious parasitic diseases in the world and remains an important public health problem in China. Screening and discovery of an effective vaccine candidate or new drug target is crucial for the control of this disease. In this study, we cloned a cDNA encoding Schistosoma japonicum (S. japonicum) thioredoxin glutathione reductase (SjTGR) from the cDNA of 42-day-old adult worms. The open reading frame (ORF) of the gene was 1791 base pairs (bp) encoding a protein of 596 amino acids. SjTGR was subcloned into pET-32a (+) and expressed in Escherichia coli (E. coli) BL21 (DE3). The recombinant protein rSjTGR exhibited enzymatic activity of 5.13U/mg with DTNB as the substrate, and showed strong immunogenecity. Real-time PCR results indicated that SjTGR was expressed at a higher level in 35-day-old schistosome worms in transcript. We vaccinated BALB/c mice with rSjTGR in combination with MONTANIDE™ ISA 206 VG (ISA 206) and observed a 33.50% to 36.51% (P<0.01) decrease in the adult worm burden and a 33.73%to 43.44% (P<0.01) decrease in the number of eggs counted compared to the ISA 206 or blank control groups in two independent vaccination tests. ELISA analysis demonstrated that rSjTGR induced a high level of SjTGR-specific IgG, IgG1, and IgG 2a antibodies and induced elevated production of IFN-γ. This study provides the basis for further investigations into the biological function of SjTGR and further evaluation of the potential use of this molecule as a vaccine candidate or new drug target is warranted.

MicroRNA expression profile in different tissues of BALB/c mice in the early phase of Schistosoma japonicum infection.

Schistosomiasis remains an important global public health problem that affects 200 million people in 76 countries. The molecular mechanisms of host-parasite interaction are complex, and in schistosome infection regulation of microRNA (miRNA) and the host micro-environment may be involved. In this study, an miRNA microarray was applied to investigate differences in miRNA expression in different tissues of mice before and 10 days post infection. In total, 220 miRNAs were detected in different tissues of the BALB/c mice before and after infection, including 8 miRNAs in liver, 8 in spleen and 28 in the lungs with up-regulated expression, and 3 miRNAs in liver, 5 in spleen and 28 in the lungs with down-regulated expression in mice 10 days post infection with schistosomes. The functions of these differentially expressed miRNAs are related mainly to the immune response, nutrient metabolism, cell differentiation, apoptosis, and signal pathways. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the differentially expressed miRNAs revealed that many important biological pathways are triggered by schistosome infection in BALB/c mice, such as the MAPK signaling pathway, insulin signaling pathway, Toll-like receptor signaling pathway and TGF-β signaling pathway.The results reveal that miRNAs may be an important regulator of schistosome-host interaction in the early phase of Schistosoma japonicum infection. The data presented here provide valuable information to increase understanding of the regulatory function of the miRNAs in the host micro-environment, as well as the mechanism of host-parasite interactions. This may be helpful in the search for potential new drugs, and for biomarkers of early S. japonicum infection applicable in the future control of schistosomiasis.

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.

iTRAQ-based comparative proteomic analysis of excretory–secretory proteins of schistosomula and adult worms of Schistosoma japonicum

Schistosomiasis remains a serious public health problem with 200 million people infected and 779 million people at risk worldwide. The schistosomulum and adult worm are two stages of the complex lifecycle of Schistosoma japonicum and excretory/secretory proteins (ESPs) play a major role in host-parasite interactions. In this study, iTRAQ-coupled LC-MS/MS was used to investigate the proteome of ESPs obtained from schistosomula and adult worms of S. japonicum, and 298 differential ESPs were identified. Bioinformatics analysis of differential ESPs in the two developmental stages showed that 161 ESPs upregulated in schistosomula were associated with stress responses, carbohydrate metabolism and protein degradation, whereas ESPs upregulated in adult worms were mainly related to immunoregulation and purine metabolism. Recombinant heat shock protein 70 (HSP70) and thioredoxin peroxidase (TPx), two differential proteins identified in this study, were expressed. Further studies showed that rSjHSP70 and rSjTPx stimulated macrophages expressing high levels of the anti-inflammatory factors TGF-β, IL-10 and Arg-1, and suppressed the expression of the pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and iNOS in LPS-induced macrophages. This study provides new insights into the survival and development of schistosomes in the final host and helps identify vaccine candidates or new diagnostic reagents for schistosomiasis.

Screening diagnostic candidates for schistosomiasis from tegument proteins of adult Schistosoma japonicum using an immunoproteomic approach.

Background Schistosomiasis is one of the world’s most prevalent zoonotic diseases and a serious worldwide public health problem. Since the tegument (TG) of Schistosoma japonicum is in direct contact with the host and induces a host immune response against infection, the identification of immune response target molecules in the schistosome TG is crucial for screening diagnostic antigens for this disease. Methodology/Principal Findings In this study, an immunoproteomics approach used TG proteins as screening antigens to identify potential diagnostic molecules of S. japonicum. Ten spots corresponding to six proteins were identified that immunoreacted with sera from S. japonicum-infected rabbits but not sera from uninfected rabbits and their specific IgG antibody levels declined quickly after praziquantel treatment. Recombinant phosphoglycerate mutase (PGM) and UV excision repair protein RAD23 homolog B (RAD23) proteins were expressed and their diagnostic potential for schistosomiasis was evaluated and compared with schistosome soluble egg antigen (SEA) using ELISA. The results showed high sensitivity and specificity and low crossreactivity when rSjPGM-ELISA and rSjRAD23-ELISA were used to detect water buffalo schistosomiasis. Moreover, antibodies to rSjPGM and rSjRAD23 might be short-lived since they declined quickly after chemotherapy. Conclusion/Significance Therefore, the two schistosome TG proteins SjPGM and SjRAD23 were identified as potential diagnostic markers for the disease. The two recombinant proteins might have the potential to evaluate the effectiveness of drug treatments and for distinguishing between current and past infection.

Comparison of the differential expression miRNAs in Wistar rats before and 10 days after S.japonicum infection

BackgroundWhen compared to the murine permissive host of Schistosoma japonicum, Wistar rats are less susceptible to Schistosoma japonicum infection, and are considered to provide a less suitable microenvironment for parasite growth and development. MicroRNAs (miRNAs), are a class of endogenous, non-coding small RNAs, that impose an additional, highly significant, level of gene regulation within eukaryotes.MethodsTo investigate the regulatory mechanisms provided by miRNA in the schistosome-infected rat model, we utilized a miRNA microarray to compare the progression of miRNA expression within different host tissues both before and 10 days after cercarial infection, in order to identify potential miRNAs with roles in responding to a schistosome infection.ResultsAmong the analysed miRNAs, 16 within the liver, 61 within the spleen and 10 within the lung, were differentially expressed in infected Wistar rats. Further analysis of the differentially expressed miRNAs revealed that many important signal pathways are triggered after infection with S. japonicum in Wistar rats. These include the signal transduction mechanisms associated with the Wnt and MAPK signaling pathways, cellular differentiation, with a particular emphasis on adipocyte and erythroid differentiation.ConclusionsThe results presented here include the identification of specific differentially expressed miRNAs within the liver, lungs and spleen of Wistar rats. These results highlighted the function of host miRNA regulation during an active schistosome infection. Our study provides a better understanding of the regulatory role of miRNA in schistosome infection, and host–parasite interactions in a non-permissive host environment.

Gene gun bombardment with DNA-coated golden particles enhanced the protective effect of a DNA vaccine based on thioredoxin glutathione reductase of Schistosoma japonicum.

Schistosomiasis, caused by infection with Schistosoma species, remains an important parasitic zoonosis. Thioredoxin glutathione reductase of Schistosoma japonicum (SjTGR) plays an important role in the development of the parasite and for its survival. Here we present a recombinant plasmid DNA vaccine, pVAX1/SjTGR, to estimate its protection against S. japonicum in BALB/c mice. The DNA vaccine administrated by particle bombardment induced higher protection than by intramuscular injection. All animals vaccinated with pVAX1/SjTGR developed significant specific anti-SjTGR antibodies than control groups. Moreover, animals immunized by gene gun exhibited a splenocyte proliferative response, with an increase in IFN-γ and IL-4. The recombinant plasmid administrated by gene gun achieved a medium protective efficacy of 27.83–38.83% (P < 0.01) of worm reduction and 40.38–44.51% (P < 0.01) of liver egg count reduction. It suggests that different modes of administering a DNA vaccine can influence the protective efficacy induced by the vaccine. Interestingly, from the enzymatic activity results, we found that worms obtained from pVAX1/SjTGR-vaccinated animals expressed lower enzymatic activity than the control group and the antibodies weakened the enzymatic activity of SjTGR in vitro, too. It implies that the high-level antibodies may contribute to the protective effects.