Proteomic Analysis Of Hydatid Cyst Fluid And Effect Of Mainly Secreted Protein On Host Immune Regulation

Objective: Hydatidosis, also known as echinococcosis, is a cosmopolitan zoonosis caused by larval stages of cestodes belonging to the genus Echinococcus(family Taeniidae). The two major species in china are Echinococcus granulosus(E. granulosus)and Echinococcus multilocularis(E. multilocularis), which cause cystic echinococcosis(CE) and alveolar echinococcosis(AE), respectively. As the serious harmful of the two types of hydatid disease, the prevention and treatment research work is urgently needed. There are four study aims:(1) Establish a more stable in vitro culture system culturing protoscoleces to producing hydatid cysts of E. granulosus and E. multilocularis respectively. I used the method to produce hydatid cysts of E. granulosus and E. multilocularis in equal condition, which allow me to compare the cyst fluid proteins of the two parasites.(2) Use the liquid chromatography-tandem mass spectrometry(LC/MS/MS) proteomic analysis method to define the secreted proteins in hydatid cyst fluid from cysts cultured in vitro.(3) Identify differentiation and polarization of macrophages in mice after Echinococcus infection and stimulated by antioxidant protein Eg TPx, to explore the early immune escape mechanisms of larval stages of echinococcus infection.(4) Explore the possible mechanisms of E. granulosus infection for reducing or inhibiting the allergic asthma induced by OVA, which will provide important knowledge for some new prevention strategies for autoimmune disease and allergic disease use the immune regulation mechanisms of the worm.Methods:(1) Protoscoleces of E. granulosus were aspirated and pooled from sheep liver hydatid cysts collected from a slaughterhouse in Urumqi. Protoscoleces of E. multilocularis were aspirated and pooled from hamster rats infected with E. multilocularis feeding in our laboratory. After 1%(w/v) pepsin digestion to remove immatureprotoscoleces, the activity and number of protoscoleces were tested and then cultured in a carbon dioxide incubator. Collected adequate amount of protoscoleces at different time points randomly and then observed using an inverted microscope and took photos and recorded the growth situation. The ultrastructure of cysts were observed by transmission electron microscopy(TEM). The BALB/c mice were each intraperitoneally(i.p) transplanted with 50 small E.granulosus or E. multilocularis hydatid cysts, respectively. Hydatid cysts collected from mice were taken and processed for histopathological examination by HE staining and counted for infection and recovery rate.(2) Collected hydatid cyst fluid from E.granulosus and E. multilocularis cysts cultured in vitro, respectively. Then went through the vacuum freeze-drying, quantitative, enzymolysis and SCX separation, using the LC-ESI-MSMS analysis system based on the Triple TOF 5600 and protein databases of E. granulosus, E. multilocularis and bovine serum respectively(ie, Eg_proteins. fa and Em_proteins. fa and uniprot_Bovine_serum. fa) to identify the protein expression spectrum and useed Signa IP, TMHMM, Targetp software for predicting secretory protein.(3) Used RNAi technology silence the expression of Eg TPx gene to determine the necessity of Eg TPx for normal growth and survival of PSC; Cloneed the Eg TPx gene by RT-PCR and constructed the Eg TPx mutants and then expressed Eg TPx and mutated proteins using E. coli expression system. To determine antioxidant activity of Eg TPx and Eg TPx mutants, a DNA nicking assay was carried out to assess the protection of DNA damage from metal-catalyzed oxidation(MCO) and also by assessing their migration in SDS-PAGE under reducing and nonreducing conditions. Peritoneal exudate cells(PEC) of E.granulosus and E. multilocularis infection mice were harvested by adherence method and then identified macrophage polarization types by PCR using specific primers of macrophage(i NOS, Ym1, Fizz1 and Arginase1); Macrophage induced by M-CSF from bone marrow of mice was stimulated by adding LPS, IL-4, HCF, recombinant protein Eg TPx and mutant protein for 16 h, extracted the cells total RNA and then identified macrophage polarization types by PCR using specific primers.(4) To establish E. granulosus infection complicated with experimental animal model of allergic asthma, BALB/c mice were first intraperitoneally transplanted with 50 small E. granulosus cysts cultured in vitro and then sensitized and challenged with ovalbumin(OVA). Airway hyperresponsiveness was assessed in response to increasing doses of methacholine. For histopathological studies, hematoxylin eosin(HE) and periodic acid schiff(PAS)staining was used to examine the inflammatory cells infiltration and goblet cells hyperplasia, respectively. Cytokine levels were measured by mouse cytometric beadarray(CBA) Kit and quantitative RT-PCR and other molecular biological approaches. To explore whether E. granulosus infection can reduce or inhibit allergic asthma airway inflammation, and to analyze its possible mechanism.Results:(1) At the time of 15 days cultured in vitro, we observed E.granulosus and E. multilocularis small cysts begin to gradually formed; at 60 days, the cysts with transparent cuticle structure were formed;after 100 days,the cysts were grown even more larger and visible. The ultrastructure results showed that E. granulosus cysts have a thicker cell free laminated layer, and the laminated layer of E. multilocularis cysts was thinner, but the germinal layer was relatively abundant in cells, and as the first time for in vitro culture, protoscoleces were observed in the small brood capsules. All the mice were infected successfully, the infection rates were 100%. E. granulosus hydatid cysts formed many single capsules in the peritoneal cavity of mice and we obtained a recovery rate(viability) of 70%. However, E. multilocularis hydatid cysts formed many big crumb structures, seem like tumor tissues and brood capsule were formed and protoscolexs was observed in the small brood capsules.(2) SDS-PAGE results showed that approximately 90% of E. granulosus and E. multilocularis hydatid cyst fluid proteins were distributing in a range of MW 1-100 KDa. We identified 774 E. granulosus proteins, 2305 E. multilocularis proteins and 39 bovine serum proteins by LC-MS/MS. Among them, the classical secretory proteins of E. granulosus and E. multilocularis was 52 and 122 respectively. Which include different subtypes of antigen B. Gene Ontology analysis revealed that the proteins involved in cellular process, metabolic process, response to stimulus, biological regulation, binding catalytic activity and other GO terms were highly expressed in E. granulosus and E. multilocularis.(3) The maximum tolerance concentration of PSC against H2O2 is 0.6 m M for 1 hour. Using RNAi technology to silence Eg TPx gene expression, I screened out the most effective interference sequence, which showed TPx si RNA-3 was the target silence sequence Eg TPx gene and its three mutants were successfully amplified by PCR and expressed in E. coli expression system. The biological characteristics of Eg TPx and its three mutants were confirmed by MCO system. E.granulosus and E. multilocularis infection, recombinant Eg TPx and its mutant proteins had the ability of activating macrophages to M2 type.(4) E. granulosus infection ameliorated OVA induced airway hyperresponsiveness. HE and PAS staining of lung tissues showed that E. granulosus infection significantly reduced the severity of OVA-induced airway inflammation including reduction of eosinophil cell infiltration and mucus production. E. granulosus infection significantly increased Th2 and Treg cytokinelevels in serum and lung tissues, but down-regulated the expression of IL-5 in the lungs and IL-17 A in serum and lung tissues of asthmatic mice sensitized and challenged with OVA, speculated that E. granulosus infection remarkably reduces the severity of OVA-induced airway inflammation likely through enhancing IL-10 and down-regulation of IL-17 A leads to the imbalance of Th17/Treg.Conclusion:(1) A stable in vitro culture system of E.granulosus and E. multilocularis from PSC to cyst was successfully established, which can completely eliminate the interference of host factors to study the specific factors on the role of the parasite growth and development. I used this method to produce experimental materials for sequencing cystic fluid protein sequences using proteomic analysis.(2) A secondary infection mouse model of E. granulosus and E. multilocularis was successfully established. I used the procedure to transplante 50 small cysts of E.granulosus and E. multilocularis respectivley into intraperitoneal cavity. This animal model will be helpful for the research and development of vaccine, drug screening and prevention and control of echinococcosis.(3) It is the first time to do the proteomic analysis of HCF collected from hydatid cysts cultured in vitro. Some I identified highly secreted proteins are likely associated to the interaction between parasite and host, energy metabolism, oxidation damage which play an important role in maintaining the parasite’s development and survival in the host.. Further study of the function of these proteins will provided an important theoretical basis for the molecular mechanism of immune escape of echinococcus.(4) Using RNAi technology, I effectively silenced Eg TPx gene expression and confirmed that Eg TPx plays an important role in protecting the parasite from oxidative damage. The results will provide technical reference for gene function study of echinococcus and other parasite.(5) Successfully constructed Eg TPx gene and its three mutants and illustrated echinococcus infection and antioxidant protein Eg TPx can induce macrophages to M2 polarization. M2 macrophage inhibits Th1 inflammation and promotes the Th2 immune response benefit for the parasitism in the host.(6) E. granulosus infection ameliorated OVA induced airway hyperresponsivenes. E. granulosus infection significantly increased Treg cytokine(IL-10)level but down-regulated Th17 cytokine(IL-17A)level. It is speculated that IL-10 inhibits the differentiation of Th17 cells lead to imbalance of Th17 /Treg and this imbalance may be the possible mechanism reduced or inhibit allergic airway inflammation of asthma. These results will provide important basis for use the immunoregulation mechanism of worm and its major secretory protein to develop new prevention strategies for autoimmune disease and allergic disease.