Protein Extract From Head-foot Tissue Of Oncomelania Hupensis Promotes The Growth And Development Of Mother Sporocysts Of Schistosoma Japonicum Via Regulation Of Parasite Aldolase Gene

Objective:Previous studies showed that protein extract from head-foot tissue of Oncomelania hupesis(O.hupensis)(PhfO),when cocultured with mother sporocysts of Schistosoma japonicum(S.japonicum),was benefit for parasite’s growth and development but the underlying mechanisms remain unclear.One possible strategy for PhfO to promote the growth and development of mother sporocysts of S.japonicum is to upregulate parasite’s survival genes.Fructose-1,6-bisphosphate aldolase(ALD),an essential enzyme of glycometabolism in the energy metabolism process,plays an important role in the survival and the growth and development of schistosoma.Using an in vitro coculture system and RNAi interference,in this study we analyzed the potential involvement of the ald gene in the growth and development of mother sporocysts of S.japonicum following coculture with PhfO.Methods:The conventional culture and coculture system were established in vitro to observe the growth and development of the mother sporocysts of S.japonicum,and we performed and measured the physiological growth parameters such as length,width and volume of mother sporocysts cocultured with PhfO on termed days,including the survival rate,detection of ATP consumption.The recombinant expression plasmid of PET28(a)-Sjald was constructed by molecular cloning method.The ALD protein was expressed and purified to immunize the New Zealand white rabbits to obtain ALD immune serum for detection the ecpression and distribution of ALD protein of mother sporocysts in subsequent Immunofluorescence histochemical staining.Using real-time PCR and immunofluorescence histochemical staining to detect the expression levels of ald mRNA and ALD protein on 3rd,4th,5th and 8th day.Custom-designed siRNA oligos against Sj-ald were synthesized,and transfected into parasites using the soaking method to silent ald gene expression,at day 6,parasites were collected to detect ald RNA level,ATP consumption,the length and width of larvae,or fixed for immunofluorescence staining.Results:Mother sporocysts in the PhfO coculture groups showed healthy growth statues with active movements,statistical analysis showed that the size of cocultured mother sporocysts increased from 97.42±7.65 to 114.40±8.78μm in length,from47.33±5.37 to 51.74±6.29μm in width,and from 144983±33834 to 207231±59232μm~3 in volume from the 3rd to 8th day of cultivation.While the size of the larvae in the control groups just increased from 93.34±8.99 to 102.3±7.51μm in length and from 145231±34030 to 161529±32410μm~3 in volume,and it didn’t change much in width,it was 49.04±7.00μm and 48.75±4.70μm respectly in width.On the 8th cultivation day,cocultured parasites even kept 92.91%survival rate,however,only 81.54%parasites survived in the control groups(p<0.05).The changes of length,width,volume and survival rate of cocultured mother sporocysts compared with those of the control groups on the 8th day had significantly statistical differences(p<0.05);And the ATP level in cocultured mother sporocysts was significantly increased from the 3rd cultivation day compared with that in the control groups,and on the 8th cultivation day,ATP level in cocultured parasite was about 20 times higher than that in the control groups.The result of Immunofluorescence histochemical staining shown that ALD protein express in cell cytoplasm as granular-like or mass precipitate,especially in tegumental cytoplasm in mother sporocysts.The expression levels of ald RNA in mother sporocysts cocultured with PhfO for 5 and 8 d were significantly higher than that of the control groups,ALD protein express has increasing staining of ALD was confirmed in cocultured mother sporocysts on the 8th day than 3rd day.The results of RNA interference experiments of mother sporocysts shown,ald siRNA treatment suppressed ALD expression at the RNA level in 72%(p<0.05),and subsequently,decreased the staining of ALD in the cytoplasm of larvae,and ATP level was reduced about 60%in mother sporocysts treated with siRNA to ald(p<0.05);The size of mother sporocysts in the ald siRNA groups decreased from 97.73±9.45 to 77.31±7.79μm in length,from 46.34±4.85 to 42.56±4.32μm in width,and from 139500±29504 to 90909±22626μm~3 in volume from the 3rd to 10th day of cultivation,while the size of larvae in the control siRNA groups increased from 96.53±9.65 to 105.49±6.19μm in length,from 45.34±6.61 to50.98±6.10μm in width,and from 132321±35505 to 182844±44689μm~3 in volume.The changes of length,width and volume of mother sporocysts in the ald siRNA groups compared with those of the control siRNA groups had significantly statistical differences(p<0.05),Furthermore,the survival rate of parasite in the ald siRNA groups decreased dramatically on the 10th cultivation day(p<0.05).The upregulation of ald gene expression at the RNA level in parasites induced by PhfO was partially suppressed by pretreatment of siRNA to ald,Increasing staining of ALD in cytoplasm of mother sporocysts induced by PhfO was attenuated by pretreatment of siRNA to ald on 6th day;the increasing ATP level in 1%PhfO+siRNA to ald group mother sporocysts was lower 26%than that in 1%PhfO+Control siRNA group(p<0.05);On the 10th day after treated with siRNA to ald,the growth of larvae in the1%PhfO+siRNA to ald group was significantly slower than the 1%PhfO+Control siRNA group.Compared with the 3rd day,The size of mother sporocysts in the 1%PhfO+siRNA to ald groups decreased from 100.02±11.27 to 92.37±8.16μm in length,from 49.69±5.30 to 47.99±3.95μm in width,and from 160474±33551 to 138387±23785μm~3 in volume,while the size of mother sporocysts in the 1%PhfO+Control siRNA groups increased from 99.53±10.83μm to 119.66±6.96μm in length,from47.79±6.81to 56.34±4.57μm in width,and from 150139±38629μm~3 to 252849±41534μm~3 in volume.PhfO cocultured mother sporocysts had significantly higher survival rate,which may maintain about 95%alive larvae during the whole 10 days test time.After pretreating with siRNA to ald,the survival rate of cocultured mother sporocysts decreased gradually to a similar level as the non-PhfO cocultured parasites.Conclusion:PhfO could promote the growth and development and the survival of mother sporocysts of S.japonicum,and increased parasites’ATP consumption.PhfO upregulated ald gene and protein expression in cocultured mother sporocysts of S.japonicum.ALD protein mainly expressed in cell cytoplasm of mother sporocysts of S.japonicum,especially in tegumental cytoplasm.Sjald could promote the ATP consumption and the growth and development,and the survival of mother sporocysts of S.japonicum.PhfO promoted the growth and development,survival and ATP consumption of mother sporocysts of S.japanicum via upregulation of parasite ald gene.