Xiaodan Cao

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.

Proteome-wide analysis of lysine acetylation in adult Schistosoma japonicum worm.

UNLABELLED Lysine acetylation, a ubiquitous and conserved posttranslational modification, has recently been shown to participate in many diverse non-chromatin-associated biological processes in prokaryotes and eukaryotes. However, the full extent and functional significance of acetylation in Schistosoma japonicum is still unknown. To investigate the nature, extent, and biological functions of lysine acetylation in schistosomes, immunoaffinity-based acetyl-lysine peptide enrichment, integrated with mass spectrometry, was used to comprehensively characterize the lysine-acetylated proteins in this parasite. In total, 1109 acetylated proteins and 2393 acetylation sites in S. japonicum were identified, representing the largest acetylome yet reported in a parasite. In a bioinformatic analysis showed that these acetylated proteins were mainly enriched in the biological process categories of metabolism, gene expression, translation, and transport. The classification according to molecular function revealed that the largest class involved the catalytic activity of different enzymes, including oxidoreductase, transferase, and pyrophosphatase activities. Most of the acetylated proteins in the cellular component category occurred in the cytoplasm, membrane, cytoskeleton, and nucleus. These data demonstrate the generality of lysine acetylation and provide the first global survey of acetylation in schistosomes. Our findings are an exciting starting point for the further exploration of the functions of acetylation in the biology of this parasite. SIGNIFICANCE Schistosomiasis is one of the worlds most prevalent and neglected tropical parasitic zoonotic diseases, and it causes almost 200,000 deaths annually. To control and eradicate schistosomiasis, effective vaccines are urgently required, and drug targets that are essential for schistosome survival must be identified in fundamental studies of schistosome biology. Posttranslational modifications are complex, fundamental, and important mechanisms that regulate the physiological functions of organisms. Lysine acetylation, a ubiquitous and conserved posttranslational modification, has recently been shown to participate in many diverse non-chromatin-associated biological processes in prokaryotes and eukaryotes. However, the full extent and functional significance of acetylation in Schistosoma japonicum is still unknown. To investigate the nature, extent, and biological functions of lysine acetylation in S. japonicum, we employ immunoaffinity-based acetyl-lysine peptide enrichment, integrated with mass spectrometry to comprehensively characterize the lysine-acetylated proteins in this parasite. The results of our data demonstrate the generality of lysine acetylation and provide the first global survey of acetylation in schistosomes. Our findings are an exciting starting point for the further exploration of the functions of acetylation in the biology of this parasite. Meanwhile, identifying the mechanisms and proteins targeted by acetylation may also provide a promising avenue for specific drug design and the development of sophisticated therapeutic strategies.

Excretory/secretory proteome of 14-day schistosomula, Schistosoma japonicum

Schistosomiasis remains a serious public health problem, with 200 million people infected and 779 million people at risk worldwide. The schistosomulum is the early stage of the complex lifecycle of Schistosoma japonicum in their vertebrate hosts, and is the main target of vaccine-induced protective immunity. Excretory/secretory (ES) proteins play a major role in host-parasite interactions and ES protein compositions of schistosomula of S. japonicum have not been characterized to date. In the present study, the proteome of ES proteins from 14 day schistosomula of S. japonicum was analyzed by liquid chromatography/tandem mass spectrometry and 713 unique proteins were finally identified. Gene ontology and pathway analysis revealed that identified proteins were mainly involved in carbohydrate metabolism, degradation, response to stimulus, oxidation-reduction, biological regulation and binding. Flow cytometry analysis demonstrated that thioredoxin peroxidase identified in this study had the effect on inhibiting MHCII and CD86 expression on LPS-activated macrophages. The present study provides insight into the growth and development of the schistosome in the final host and valuable information for screening vaccine candidates for schistosomiasis.

Changes in microRNA expression in response to Schistosoma japonicum infection

Schistosomiasis japonicum is one of the most serious zoonotic diseases in the world. There is increasing evidence to show that host miRNAs are modulated following Schistosoma japonicum infection, and some of these miRNAs may play important regulatory roles in response to schistosome infection. Several host miRNAs have been identified and shown to be potential diagnostic biomarkers or novel therapeutic targets for schistosomiasis. These studies have paved the way to a better understanding of the mechanisms of schistosome‐host interaction and may facilitate the development of novel approaches to the control of the disease.

Comparison of apoptosis between adult worms of Schistosoma japonicum from susceptible (BALB/c mice) and less-susceptible (Wistar rats) hosts

Schistosomiasis remains a serious public health concern in China. BALB/c mice are susceptible to Schistosoma japonicum infection, whereas the Wistar rats are less susceptible. Apoptosis phenomenon was observed in 42d adult worms of S. japonicum from both rats and mice at the morphologic, DNA, cellular, and gene levels by transmission electron microscopy (TEM), fluorometric terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis, fluorescein isothiocyanate-annexin-V/propidium iodide staining flow cytometry (FCM) analysis, and real-time PCR. The results showed that the apoptotic state in worms from two different susceptible hosts was diverse. Several classical hallmarks of apoptosis, including cell shrinkage, chromatin condensation and lunate marginalization, splitting of the nucleoli, nuclear shrinkage and apoptotic body formation were observed by TEM. TUNEL analysis showed that there were much more apoptosis spots in adult worms from rats than those from mice. Statistical analysis revealed that the degree of apoptosis and percentage of necrotic cells in adult worms from Wistar rats were significantly greater (P<0.01) than those from BALB/c mice by flow cytometry. A total of 15 apoptosis-associated genes including the major components of an intrinsic cell-death pathway were identified from S. japonicum in this study, suggested that a similar apoptosis pathway might occur in S. japonicum. Real-time PCR analyses revealed that the expression levels of most of the tested apoptosis-associated genes, except CASP7, were significantly higher or at the similar level in adult worms from Wistar rats, as compared to those from BALB/c mice. The results obtained in this study collectively demonstrated that differential development of adult S. japonicum in less-susceptible rats and susceptible mice was significantly associated with apoptosis in the worm, and provided valuable information to guide further investigations of the mechanisms governing apoptosis and host interactions in schistosome infection.

Characterization of VAMP2 in Schistosoma japonicum and the Evaluation of Protective Efficacy Induced by Recombinant SjVAMP2 in Mice.

Background The outer-tegument membrane covering the schistosome is believed to maintain via the fusion of membranous vesicles. Fusion of biological membranes is a fundamental process in all eukaryotic cells driven by formation of trans-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes through pairing of vesicle associated v-SNAREs (VAMP) with complementary t-SNAREs on target membranes. The purpose of this study was to characterize Schistosoma japonicum vesicle-associated membrane protein 2 (SjVAMP2) and to investigate its potential as a candidate vaccine against schistosomiasis. Methodology/Principal Findings The sequence of SjVAMP2 was analyzed, cloned, expressed and characterized. SjVAMP2 is a member of the synaptobrevin superfamily harboring the v-SNARE coiled-coil homology domain. RT–PCR analysis revealed that significantly higher SjVAMP2 levels were observed in 14-, 28- and 42-day-old worms, and SjVAMP2 expression was much higher in 42-day-old female worms than in those male worms. Additionally, the expression of SjVAMP2 was associated with membrane recovery in PZQ-treated worms. Immunostaining assay showed that SjVAMP2 was mainly distributed in the sub-tegument of the worms. Western blotting revealed that rSjVAMP2 showed strong immunogenicity. Purified rSjVAMP2 emulsified with ISA206 adjuvant induced 41.5% and 27.3% reductions in worm burden, and 36.8% and 23.3% reductions in hepatic eggs in two independent trials. Besides, significantly higher rSjVAMP2-specific IgG, IgG1, IgG2a levels were detected in rSjVAMP2-vaccinated mice. Conclusion Our study indicated that SjVAMP2 is a potential vaccine candidate against S. japonicum and provided the basis for further investigations into the biological function of SjVAMP2.

Suppression of VAMP2 Alters Morphology of the Tegument and Affects Glucose uptake, Development and Reproduction of Schistosoma japonicum

Schistosomiasis caused by schsitosomes is a serious global public health concern. The tegument that surrounds the worm is critical to the schistosomes survival. The tegument apical membrane undergoes a continuous process of rupture and repair owing to membranous vacuoles fusing with the plasma membrane. Vesicle-associated membrane protein 2 (VAMP2), a member of soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNAREs) is required for membrane fusion. Here, we used RNA interference (RNAi) to knock down the expression of VAMP2 of Schistosoma japonicum (SjVAMP2), and both real-time PCR and western blot analysis confirmed the suppression of this molecule, as well as the suppression of the transcript levels of schistosome glucose transporters (SGTP1 and SGTP4), and insulin receptors (SjIR1 and SjIR2). SjVAMP2-suppressed worms exhibited a lower viability, and phenotypic alterations were also observed in the tegument. Moreover, the glucose consumption of SjVAMP2-suppressed worms decreased significantly in 4 and 6 days, respectively, as well as a significant reduction in egg production. We also observed a significant reduction in worm burden and hepatic eggs burden in two independent RNAi experiment in vivo, and minor pathological changes in mice treated with SjVAMP2 specific small interfering (si)RNA. These findings reveal that SjVAMP2 may play important roles in the maintenance of tegument, glucose uptake, worm development and egg production in schistosomes.

Characterization of Schistosoma japonicum tetraspanning orphan receptor and its role in binding to complement C2 and immunoprotection against murine schistosomiasis

BackgroundSchistosomiasis remains an important global public health problem, as millions of people are at risk of acquiring infection. An ideal method for sustainable control of schistosomiasis would be to develop an efficient vaccine. Schistosomes can survive in the host vascular system by immune evasion, regulating the host complement cascade. Schistosoma japonicum tetraspanning orphan receptor (SjTOR) is a complement regulator, which is a tegument membrane protein. To date there is no experimental evidence to explain the function of SjTOR.ResultsWe cloned the first extracellular domain of the SjTOR (SjTOR-ed1) gene and expressed the gene in Escherichia coli. The expression level of SjTOR in different developmental stages of S. japonicum was assessed by quantitative real-time RT-PCR. Western blotting showed that recombinant SjTOR-ed1 (rSjTOR-ed1) could be recognised by schistosome-infected mouse serum. Immunolocalization indicated that the protein was mainly distributed on the tegument of the parasite. Haemolytic assays and ELISA revealed that rSjTOR-ed1 could inhibit complement hemolysis and bind to complement C2. Purified rSjTOR-ed1 emulsified with ISA206 adjuvant could induce a significant reduction of worm burden from 24.51 to 26.51%, and liver egg numbers from 32.92 to 39.62% in two independent trials in mice.ConclusionsThe results of this study indicated that rSjTOR-ed1 could inhibit complement hemolysis and bind to complement C2, and it is a potential vaccine candidate that protects against S. japonicum infection.

日本血吸虫凋亡基因Sjcaspase3的克隆、真核表达 及其功能分析

For further research of the apoptosis mechanism of Schistosoma japonicum (S. japonicum). The cDNA encoding Sjcaspase3 of Schistosoma japonicum was amplified by polymerase chain reaction (PCR) technique, which contained 900 nucleotides and encoded 299 amino acids. The theory molecular weight and isoelectric point (PI) of the deduced protein is 33.5 kDa and 6.39, respectively. Real-time PCR was used to analyze the transcription profiles of Sjcaspase3 at different development stages of S. japonicum. The results showed that this gene was expressed in all stages of S. japonicum with the highest expression in 21d worms, and the level of gene transcription in 42 d female worms was higher than that of male worms. The recombinant plasmid pXJ40-FLAG-Sjcaspase3 was constructed and transfection into Hela cells successfully. Real-time PCR and Western blotting analysis showed Sjcaspase3 was successfully expressed in Hela cells. Enzyme activity analysis revealed that recombinant Sjcaspase3 possessed the activity to cut substrate DEVD. Flow cytometry proved that Sjcaspase3 could induce early apoptosis of Hela cells. The results provide the basis for proceeding further study on the biological function of Sjcaspase3 and better understand the apoptosis mechanism of S. japonicum.