Analysis Of Excretory-Secretory Proteins Of Schistosoma Japonicum

Schistosomiasis is a chronic and seriously harmful parasitic zoonosis caused by Schistosoma japonicum(S. japonicum). Schistosome release or secrete some antigens directly exposed to their host, inducing and regulating host immune responses. Some excretory/secretory proteins(ESPs), especially those from early stage worms and those differential expressed between stages, should be important for the establishment and maintenance of the parasite infection in their definitive hosts. Proteomic analysis on the composition and alteration of ESPs of schistosomulum and adult worm of S. japonicum and the identification of crucial molecules associated with the growth, development and reproduction of worms, will increase our better understanding of the mechanism about host–pathogen interplay, and provide valuable information for the screening of immunomodulatory molecules and vaccine candidates for schistosomiasis.1. Excretory/secretory proteome of 14-day schistosomula, Schistosoma japonicumThe schistosomulum is the early stage of the complex lifecycle of S. japonicum in their vertebrate hosts, and is the main target of vaccine-induced protective immunity. ESPs play a major role in host–parasite interactions and ESPs compositions of schistosomula of S. japonicum have not been characterized to date. The proteome of ESPs from 14 day schistosomula of S. japonicum was firstly analyzed by high performance liquid chromatography/tandem mass spectrometry in the present study and 713 unique proteins were finally identified. The molecular mass was mainly distributed between 10 kDa and 70 kDa and the pI of identified proteins ranged from 3 to 13. Bioinformatics analysis revealed that the identified ESPs of schistosomula were mainly involved in response to stimulus(e.g., heat shock protein), carbohydrate metabolism(e.g., glyceraldehyde-3-phosphate dehydrogenase), and protein degradation(e.g., protein disulfide isomerase) et al. Some ESPs may be crucial molecules associated with the adapting to parasitism, the survival and development of schistosomulum in the final host. The present study provides insight into the interaction between host and schistosome, and the screening of vaccine candidates for schistosomiasis.2. Comparative proteomic analysis of excretory–secretory proteins of schistosomula and adult worms of Schistosoma japonicumThe schistosomulum and adult worm are two developmental stages of S. japonicum in the final host and schistosome exhibit dramatic changes in their morphology and biology at different developmental stages. Differential ESPs between schistosomulum and adult worm perform different biological functions for the establishment and maintenance of infection, the growth and reproduction of schistosome. In this study, iTRAQ-coupled LC-MS/MS was firstly used to investigate the proteome of ESPs obtained from 14 d schistosomula and 42 d 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(e.g., hot shock proteins, glucose-6-phosphate isomerase, protein disulfide isomerase), whereas 137 ESPs upregulated in adult worms were mainly related to immunoregulation and purine metabolism(e.g., thioredoxin peroxidase, adenine phosphoribosyltransferase). 31 ESPs with no significant difference between schistosomula and adult worms were identified in the present work(e.g., phosphoglycerate mutase). This study might elucidate physiological differences between schistosomulum and adult worm, and provides new insights into the roles of differential ESPs in the growth and development of shistosomes and helps identify vaccine candidates for schistosomiasis.3. Cloning, expression and immunoprotection of protein disulfide isomerase of Schistosoma japonicumProtein disulfide isomerase(PDI)was identified as an ESP of S. japonicum in the proteome analysis of the present study. The gene of the S. japonicum PDI(SjPDI)was cloned by PCR and SjPDI had an open reading frame(ORF)of 1410 bp encoding 469 amino acids. The recombinant expression plasmid pET-28a-SjPDI was successfully expressed as soluble recombinant protein(rSjPDI) of 55 kDa in E. coli/BL21 cells. Western blot analysis showed that rSjPDI was recognized by serum from rabbits vaccinated with schistosome worm antigen, and protein extracts from S. japonicum or ESPs extracts from S. japonicum reacted with anti-rSjPDI mouse serum, which indicating rSjPDI had good immunogenicity and antigenicity, and SjPDI indeed existed in ESPs of S. japonicum. Real-time PCR analysis indicated that SjPDI was expressed at all tested developmental stages, and a high expression level was detected in 42-day-old worms and early schistosomula. Immunofluorescence analysis revealed that SjPDI was mainly distributed on the tegument and parenchyma of S. japonicum worms. The biological activity of purified rSjPDI was confirmed by isomerization and antioxidative activity assays. An enzyme-linked immunosorbent assay(ELISA) demonstrated that rSjPDI could induce a high level of rSjPDI-specific IgG antibodies in vaccinated mice. The 35.32%, 26.19% reduction in the worm burden and 33.17%, 31.7% lower liver egg count were obtained in mice vaccinated with rSjPDI compared with the blank control group in two independent animal experiments. Our preliminary results suggest that SjPDI plays an important role in the development of the schistosome and is a potential vaccine candidate for schistosomiasis.4. The effect and potential regulatory mechanism of glutathione dehydrogenase of Schistosoma japonicum on the activity of LPS-induced RAW264.7 macrophage.Macrophages, a target for immunomodulation by the helminth parasite, play a crucial role in both pro- and anti-inflammatory response to parasite infection. Glutathione dehydrogenase was identified as another ESP of S. japonicum in the proteome analysis of the present study. The protein has a domain characteristic of GSTs which is the same as an immunomodulatory molecule Tc52 of Trypanosoma cruzi. In this study, the gene encoding S. japonicum glutathione dehydrogenase was firstly cloned and expressed; the effect of this recombinant protein on modulating RAW264.7 macrophage activities and the potential regulatory mechanism were investigated. The result showed that stimulation of recombinant S. japonicum glutathione dehydrogenase could suppress the expression of pro-inflammatory cytokine TNF-α, IL-1β and iNOS and increase the expression of microRNA-146 a in LPS-activated RAW264.7 macrophages. Further study revealed that miR-146 a negatively regulated LPS-induced IL-1β expression in RAW264.7 macrophage and might be involved in the pro-inflammatory response. In conclusion, recombinant S. japonicum glutathione dehydrogenase regulates the host immune response through altering macrophage activity and inflammatory responses which may be important for worm survival and development.In summary, the high performance liquid chromatography/tandem mass spectrometry was used to analyze the composition of ESPs from 14 day schistosomula of S. japonicum and iTRAQ-coupled LC-MS/MS was used to investigate the proteome of ESPs obtained from 14 d schistosomula and 42 d adult worms of S. japonicum. PDI, identified as an ESP of S. japonicum, was cloned, expressed and charactered. Immune protection induced by rSjPDI was assessed in mouse model. Glutathione dehydrogenase, identified as another ESP of S. japonicum, was cloned and expressed. The effect of recombinant glutathione dehydrogenase on the activity of RAW264.7 macrophages and potential immunoregulation mechanism were investigated. The present study increases our understanding of the mechanism about growth and development of schistosome in their host and the interplay between host and schistosome, and provides valuable information for the screening of vaccine candidates for schistosomiasis.