Molecular Characterization And Function Of Infertile Cysts Formation Associated Genes From Echinococcus Granulosus

Cystic echinococcosis（CE）,a serious parasitic zoonosis that causes significant economic losses and poses a threat to public health,is caused by infection with the larval stages（metacestodes）of the tapeworm Echinococcus granulosus.This disease is distributed worldwide.Infection causes infertile cysts in intermediate hosts that are unable to produce protoscoleces and hence incapable of completing the life cycle of this tapeworm,but they still cause mechanical oppression in host organs,leading to atrophy and dysfunction.Previous studies indicate that apoptosis participates in infertility in hydatid cysts,but the genes regulating this process remain unknown.Herein,we clone,express and characterise the Bcl2-associated athanogene 3（Eg-BAG3）,endophilin B1（Eg-EB1）,mitochondrial fission protein 1（Eg-Fis1）,Programmed cell death protein 6（Eg-PDCD6）and citrate synthase（Eg-CS）of E.granulosus.The function of these proteins in regulating the formation of infertile cysts were also explored which provided the foundational infromation for the studies of functional genes in E.granulosus.The results obtained in this study are summarized as follow:1.Identification and characterisation of Echinococcus granulosus BAG3 and EB1 in oxidative stress-induced apoptosis and infertility of cystsIn our study,the anti-apoptosis protein BAG3 and pro-apoptosis protein EB1 of E.granulosus were successfully cloned and expressed.The Eg-BAG3 gene encodes a putative protein of 232 amino acids,while the Eg-EB1 gene encodes a 252 residue polypeptide.Immunohistochemical localisation showed that Eg-BAG3 was ubiquitously distributed in adult worms,protoscoleces and the germinal layer of the cyst wall,while Eg-EB1 was only observed in protoscoleces and the germinal layer.Eg-BAG3 and Eg-EB1m RNA levels were significantly up-regulated after treatment with H₂O₂,an agent known to trigger apoptosis.Moreover,the apoptotic rate was significantly higher than that in controls after interference of Eg-BAG3,while the apoptotic rate was lower after interference of Eg-EB1.In conclusion,Eg-BAG3 and Eg-EB1 might be involved in regulating the production of infertile cysts.Eg-BAG3 might enhance resistance to apoptosis induced by oxidative stress to preserve the fertility of hydatid cysts,while Eg-EB1 may trigger programmed cell death after sensing severe oxidative damage and thereby promote the formation of infertile cysts.2.Echinococcus granulosus Fis1 and PDCD6 genes expression and research of their functionHerein,the full-length c DNA of Eg-Fis1 and Eg-PDCD6 were amplified.The proteins were expressed and characterized.Bioinformatics analysis showed that the Eg-Fis1 gene encoded a 157 residue polypeptide and contained a transmembrane region which closely related to its function,while the Eg-PDCD6 gene encoded a putative protein of 174 amino acids and had a high conservatism among species.In western blotting assays,it suggested that both r Eg-Fis1 and r Eg-PDCD6 had good reactionogenicity.In immunofluorescence analysis,anti-Eg-Fis1 and anti-Eg-PDCD6 rabbit Ig Gs were applied for detection of their respective native proteins in adult worms,cyst walls（from fertile and infertile cysts）,and PSCs.Eg-Fis1 and Eg-PDCD6 m RNA expression were up-regulated at the beginning of H₂O₂ treatment,and levels continued to increase after an 8 h incubation,but the increase of Eg-PDCD6 was not significant.This might be that Eg-PDCD6 didn’t play a major role in the response to oxidative stress-induced apoptosis.To investigate whether Eg-Fis1 and/or Eg-PDCD6 are responsible for regulating apoptosis response to oxidative stress,RNAi and TUNEL assays were performed.The apoptotic rate was significantly lower than that in controls after interference of Eg-Fis1,while there was no difference between the apoptotic rate of the interference group for Eg-PDCD6 and the control group.We speculated that Eg-Fis1 might promote the apoptosis induced by oxidative stress,then further inducing the production of the infertile cysts.Eg-PDCD6 might be not not the key regulatory gene in the process of apoptosis.3.Molecular and biochemical characterization of citrate synthase from Echinococcus granulosusIn our study,citrate synthase from Echinococcus granulosus was firstly cloned,expressed and characterized.Eg-CS is a highly conserved protein,consisting of 466 amino acids.In western blotting,r Eg-CS could react with E.granulosus positive sheep sera and anti-r Eg-CS rabbit sera,which indicated that Eg-CS had a good antigenicity and immunoreactivity.Immunohistochemical localizations showed that r Eg-CS was ubiquitously expressed in the larva,germinal layer and adult worm sections of E.granulosus,which indicated that Eg-CS might play a crucial role in the growth and development of the parasite.To further study the function of CS in E.granulosus under the oxidative stress,PSCs were treated with H₂O₂.The m RNA expression level of Eg-CS was increased from the start of H₂O₂ exposure.The result suggested that apoptosis was an active and energy consuming process.With increasing exposure time,the activity and metabolic rate of PSCs declined and the apoptosis or death of PSCs was enhanced.The m RNA expression of Eg-CS also decreased.Thus,it was demonstrated that CS is related to the growth and metabolism of PSCs.In conclusion,as a key enzyme in metabolic pathway,citrate synthase play a key role in the growth and metabolism of E.granulosus,and may maintain the fertility of the cyst.The decrease of Eg-CS activity and expression may lead to the formation of the infertile cysts.Moreover,the preliminary serodiagnostic potential of Eg-CS based on indirect ELISA was assessed.Even though sensitivity（93.55%）and specificity（80.49%）were high,the use of r Eg-CS in the serodiagnostic ELISA lacked utility because of its high cross reaction with other parasties.The results revealed that r Eg-CS was not a suitable as a diagnostic antigen.