Studies On The Gene Function Of Serine Protease And Glutathione S-transferase In Trichinella Spiralis By Using RNA Interference

Serine protease are digestive enzymes of a variety of parasites.In addition to catalyzing and protein processing,it also plays an important role in the development and survival of parasites,including vesiculation,tissue invasion,nutrient uptake,immune escape and pathogenicity.Trichinella spiralis serine protease?TsSP,Genbank:ABY60762?may play an important role in the Trichinella spiralis invading host small intestinal epithelial cells?IECs?,and larvae development.T.spiralis glutathione-s-transferase?TsGST,Genbank:XM?003373603.1?is expressed in the life-cycle of worms,mainly located in the cuticle,stichosome and genital primordium.Anti-r TsGST immune serum had a significant inhibitory effect on larval invasion of IEC in vitro,indicating that TsGST was involved in larval invasion.In this paper,RNA interference?RNAi?technology was used to study the function of TsSP and TsGST genes,to observe the effect of RNAi on enzyme activity,and to further observe the influence of RNAi on invasion,development ability and fecundity of larvae.Designing small interfering RNA?siRNA?based on the mRNA sequences of the two genes,and the siRNA was transfected into the larvae by electroporation.To investigate the silencing effect of RNAi on genes by qPCR and Western blot in gene transcription and protein expression,respectively.Using gelatin zymogram and substrate degradation test to observe the RNAi influences on activity of natural TsSP and TsGST in soluble and excretory-secretory?ES?proteins of muscle larvae.In vitro experiments to observe the effect of RNAi on larvae invading IECs and intestinal mucosa.Animal experiments were used to observe the inhibitory effects of TsSP and TsGST gene silencing on larval invasion,developmental ability and fecundity.Materials and Methods1.Trichinella,experimental mice and cellsThe T.spiralis isolate?Trichinella spiralis,T.spiralis?kept by passage in BALB/c mice in our laboratory.BABL/c mice?female,15-20g?were obtained from the animal center of Zhengzhou University.Mouse intestinal epithelial cells were isolated and cultured from normal mouse small intestine by our laboratory,and used in vitro Trichinella spiralis-cells invasion model.2.Induced expression of TsSP and TsGST and preparation of antiserumThe recombinant bacteria TsSP and TsGST constructed in our laboratory were activated,and the r TsSP and r TsGST proteins were expressed and purified for immunizing mice to obtain immune serum against r TsSP and r TsGST.3.Design and synthesis of siRNAsComplete cDNA encoding TsSP and TsGST was utilized to design the siRNA sequences by using siDirect version 2.0.According to each siRNA screened,it was named TsSP specificity siRNA-156,TsSP-siRNA-171,TsSP-siRNA-436 and TsGST specificity siRNA-366 according to its'siRNA targeting site.At the same time,a control siRNA was designed and added FAM fluorescent label,as negative control and to observe whether the siRNA was transfection into Trichinella spiralis.4.The larvae were transfected by siRNA through electroporationAfter electroporation,1.0?M siRNA was introduced into Trichinella spiralis muscle larvae,and then cultured for 18 hours in vitro to observe whether the siRNA entered the larvae.5.Effects of RNAi on gene transcription and protein expression levelsThe best interference effect of TsSP-siRNA was determined by qPCR and Western blot technology.The best interference dose was obtained from 1.5,2.0,2.5,3.0 and 3.5?M siRNA.After transfection of siRNA to larvae 1,3,5 and 7d,the silencing effect of RNAi on TsSP gene in transcription and protein expression was observed.The best silencing effect of TsGST-siRNA was obtained at different concentrations?1.5,2.0,2.5 and 3.0?M siRNA?and transfection at different times?1,2,3,4 and 5d?.6.RNA interference gene specificityAfter TsSP-siRNA and TsGST-siRNA were transfection into Trichinella spiralis by electroporation,they were used as controls to analyze the specificity of RNAi to specific silencing of two genes.7.The effects of TsSP and TsGST gene silencing on their enzyme activityThe effect of TsSP-siRNA on nature TsSP enzyme activity was observed by gelatin zymography and degradation of serine-specific substrate azocasein.The effect of TsGST-siRNA on the activity of TsGST was observed by using glutathione S-transferase substrate CDNB.8.Assessing the RNAi on larva survival rate and intrusion of IECsAfter transfection of siRNA into muscle larvae,they were cultured for 7 days in vitro,the morphological changes of muscle larvae were observed,and the survival rate of the larvae was calculated.The larvae cultured for 1 day in vitro after RNAi were activated into intestinal infection stage 1 larvae?IIL1?by 5%bile,and then 100 IIL1 larvae were mixed with semi-solid medium and inoculated onto the IEC monolayer cultured in vitro,the number of larvae was observed and counted that invaded IECs after 2h incubation.9.RNAi-treated larva invasion of isolated enteral epithelium in vitroThe larvae were cultured in vitro for one day after RNAi,100 larvae were Injected into a small intestine bag 2cm long,and incubated at 37°C,5%CO₂ for 2 h.The small intestine was scissored,and the invasion rate was counted and observed.10.TsSP and TsGST gene silencing inhibited Trichinella spiralis larvae the invasion,development ability and female fecundity in miceEighty BALB/c mice were equally divided into 4 groups?20/group?,which set as:TsSP-siRNA interference group,TsGST-siRNA interference group,control siRNA group and PBS blank control group.Each group of each mouse was infected by 300 muscle larvae that siRNA was transfected into larvae.After infection for 6 days,10 mice were sacrificed in each group.and the intestinal worms were recovered.The adult worm?AW?reduction rate was calculated.10 female adults were selected separately and collected for incubation at 37°C,5%CO₂,and the newborn larvae?NBL?were collected for 72 hours.NBL was counted and photographed.And female fecundity was calculated.After 35 d of infection in the remaining mice,the mice were sacrificed after anesthesia,the muscle larvae?ML?were collected,and the number of muscle larvae per gram of muscle tissue and the larval reduction rate were calculated.Simultaneously,the morphology of the very period worm ware observed and the length were measured.Results1.FAM fluorescence signal trackingAfter siRNA was introduced into the muscle larva,green fluorescence was observed that existed in the larvae under the fluorescence microscope.2.Down-regulation of TsSP and TsGST gene transcription and expression by RNAi2.1 RNAi inhibited TsSP gene transcription and protein expression:qPCR and Western blot results showed that TsSP transcription levels in the siRNA-156,siRNA-171 and siRNA-436experimental groups were reduced by 50.54 and 36.36?P<0.05?and 13.41%,compared with the PBS group.The protein expression of TsSP decreased by 54.55,46.84 and 30.06%,respectively?P<0.05?.When siRNA-156 at 1.5,2.0,2.5,3.0 and 3.5?M TsSP decreased by50.49,51.49,55.48,50.14 and 48.47%?P<0.05?compared to the PBS group at the transcription level.Compared the PBS group,the protein expression reduction was 49.62,44.38,58.6,51.91,and 54.38%?P<0.05?.At 1,3,5 and 7 days of 2.5?M siRNA-156,the TsSP gene was reduced by 59.98,37.23,36.22 and 8.71%compared with the PBS group at the translation level?P<0.05?.Compared with PBS,TsSP gene protein expression was reduced by 48.35,36.68,35.01 and 21.75%compared with PBS?P<0.05?.The difference between the control siRNA group and PBS group was not statistically significant?P>0.05?.2.2 RNAi inhibited TsGST gene transcription and protein expression:qPCR and Western blot results showed that siRNA-366 was at 1.5,2.0,2.5,and 3.0?M TsGST at the transcription level was reduced by 19.53,20.24,21.39 and 49.38%?P<0.05?,compared to the PBS group,respectively.Compared with the PBS group,TsGST decreased in protein expression by 40.36,41.59,46.42 and 59.18%?P<0.05?.When 3.0?M siRNA-366 at 1,2,3,4 and 5 days,the TsGST gene was reduced by 49.09,32.65,15.46,12.62 and 12.14%?P<0.05?,compared to the PBS group at the transcription level.Compared with the PBS group,the TsGST protein expression was reduced by 65.22,41.00,19.11,12.97 and 1.97%?P<0.05?.The difference between the control siRNA group and PBS group was not statistically significant?P>0.05?.3.RNA interference gene specificitySi RNA was introduced into Trichinella spiralis muscle larvae by electroporation for 1 day.The TsSP-siRNA-156 treatment group had a 57.10%decrease in TsSP expression?P<0.05?but TsGST expression level change was no significant?P>0.05?.The TsGST-siRNA-366treatment group had a 56.09%decrease?P<0.05?but TsSP expression level change was no significant.?P>0.05?.4.Inhibitory effect of RNAi on enzyme activity4.1 RNAi inhibited TsSP activity:Zymography profiles showed that after the muscle larvae were treated with 2.5?M siRNA 156,the capacity of muscle larvae ES proteins to hydrolyze the gelatin was obviously decreased in comparison to control siRNA and PBS groups.The TsSP enzymatic activity following siRNA-156 treatment was detected by degrading azocasein.siRNA-156 treated group,control siRNA group and PBS control group,TsSP protease activity of siRNA-156 treated group muscle larvae ES protein for degrading azocasein was decreased by 61.38%relative to PBS group?F=1570.157,P<0.05?.4.2 RNAi inhibited TsGST activity:The enzymatic activity analysis revealed that the enzymatic activity of the TsGST to conject glutathione to CDNB was 0.55±0.35 U/mg protein in the siRNA-366 group,0.94±0.12 U/mg protein in the control siRNA group,and 1.00±0.078 U/mg protein in the PBS group.There was statistically significant in the enzymatic activity of the TsGST between the siRNA-366 group or the PBS group?F=18.063,P=0.003?.The control siRNA group and the PBS group had no statistically significant?P=0.445>0.05?.The results suggested that siRNA-366 interference could reduce the enzymatic activity of larvae TsGST.5.Effect of RNAi on survival rate of larvaeAfter RNAi for culture 1 day,the mortality of larvae in the TsSP-siRNA-156,control siRNA,and PBS groups was 4.67,4.33 and 3.33%?P>0.05?.At 7 days after silencing,the larvae of the TsSP-siRNA-156,control siRNA and PBS group died rates were 33.33,32.00 and31.67?P>0.05?.The results showed that was no significant change in the survival rate of larvae after siRNA-156.After RNAi for culture 1 day,the mortality of larvae in the TsGST-siRNA-366,control siRNA,and PBS groups were 4.33,4.00 and 3.67%,respectively?P>0.05?.At 7 days after RNAi,the larvae of the TsGST-siRNA-366,control siRNA and PBS groups died rates were35.67,34.33 and 32.33%?P>0.05?.The results showed that was no significant change in the survival rate of larvae after siRNA-366.6.Inhibition of larval invasion of IECs by RNAi6.1 Inhibition of siRNA-156 on the in vitro larva invasion of IEC:While the IIL1 were inoculated onto the IEC monolayer and cultivated for 2 h,and the IIL1 invaded and migrated in the monolayer.The larvae invasion rate?40.90%?of siRNA-156 group was significantly lower than the of control siRNA group?65.76%?and PBS group?64.47%???²=20.999,P=0.0001?.The results revealed that the knockdown of TsSP gene by siRNA-156 obviously suppressed the IIL1 invasion IEC.6.2 siRNA-366 treatment inhibited the larval invasion to IEC:When the IIL1 larvae were added onto the IEC monolayer and cultured for 2 h,the IIL1 intruded into the IEC and migrated in monolayer.The larvae invasion rate?42.93%?of siRNA-366 group was significantly lower than the of control siRNA group?64.33%?or PBS group?65.20%???²=10.502,P<0.05?.Compared with PBS group,the larval invasion of the siRNA-366 treated group was significant inhibited invasion IECs and the inhibition rate was 34.16%.There was no statistical significant in the control siRNA group compared with the invasion rate of the PBS group??²=0.10,P>0.05?.7.Inhibition of larval invasion of small intestinal mucosa by RNA interference7.1 Suppression of siRNA-156 on the larval invasion of isolated enteral mucosa:When IIL1 were perfused into the small intestine cavity and incubated for 2 h with Tyrode's solution,larvae intruded into intestinal mucosa.The larva invasive rate of siRNA-156,control siRNA and PBS blank group was 51.00,68.00 and 69.00%??²=8.748,P<0.05?.The results indicated that siRNA-156 treatment group had 26.09%inhibition of larva invasion of enteral mucosa in comparison to the PBS group.7.1 Suppression of siRNA-366 on the larval invasion of isolated enteral mucosa:When IIL1 were perfused into the small intestine cavity and incubated for 2 h with Tyrode's solution,larvae intruded into intestinal mucosa.The larva invasive rate of siRNA-366,control siRNA and PBS blank group was 36.00,56.00 and 58.00%??²=11.840,P<0.05?.The invasion rate of siRNA-366 treatment group was obviously below the control siRNA group or PBS blank group.8.Inhibition of larvae invasion,development and fecundity by RNAi in mice8.1 TsSP silence inhibited larvae invasion,development and fecundity in vivo:Compared with the PBS control group,the adult worm and muscle larva reduction rate were 58.85 and60.48%,respectively.At 6d after siRNA-156 treatment larvae infection mice,the adult worm load?49.91±6.47?in the siRNA-156 group was significantly lower than that in the control siRNA group?120.64±9.71?or the PBS group?121.27±8.96?(F_adults=256.630,P<0.05).The number of newborn larvae produced by the siRNA-156 experiment?60.78±6.01?was significantly lower than that of the control siRNA group?87.94±5.88?or the PBS group?87.47±4.85?(F_NBL=50.440,P<0.05).At 35d after siRNA-156 treatment larvae infection,the siRNA-156 treated group muscle larvae load were?1245.736±248.0253?lower control siRNA group?2942.374±245.7014?or PBS group?3152.836±565.38?(F_larvea=81.877,P<0.05).Measuring very period worm the length of Trichinella spirali,the female AW body length?2076.51±420.98??m of the siRNA-156 group was significantly lower than the control siRNA group?2489.28±374.71??m or PBS?2437.34±270.93??m(F_{f AW}=5.817,P<0.05).The male AW body length?930.67±88.92??m of the siRNA-156 group was significantly lower than that of the control siRNA group?1159.60±220.53??m or the PBS group?1191.67±138.93??m(F_{m AW}=9.617,P<0.05).The NBL length?87.694±19.34??m of the siRNA-156group was shorter than that of the control siRNA group?103.5±12.05??m or the PBS group?110.592±10.72??m(F_NBL=9.753,P<0.05).The ML length?923.54±115.61??m of the siRNA-156 group was lower than the control siRNA group?1144.54±114.14??m or the PBS group?1125.24±80.90??m(F_ML=40.933,P<0.05).8.2 TsGST inhibited larvae in vivo:Compared with the PBS control group,the reduction rate of siRNA-366 adults and muscle larvae was 62.82 and 65.07%,respectively.At 6d after siRNA-366 treatment larvae infection mice,the adult charge number?45.09±7.45?of siRNA-366 group was significantly lower than that of control siRNA group?120.64±9.71?or PBS group?121.27±8.96?(F_adults=15642.775,P<0.05).The number of new larvae produced by adults in siRNA-366 group?58.31±5.60?was significantly lower than in control siRNA group?87.94±5.88?or PBS group?87.47±4.85?(F_NBL=89.133,P<0.05).At 35d after siRNA-366treatment larvae infection,the siRNA-366 treated group muscle larvae load were?1101.268±144.5001?and lower control siRNA group?2942.374±245.7014?or PBS group?3152.836±565.38?(F_larvea=105.050,P<0.05).When worms length were measured,the female AW body length of the siRNA-366 group?1949.96±326.75??m was significantly lower than the control siRNA group?2489.28±374.71??m or PBS group?2437.34±270.93??m(F_{f AW}=12.425,P<0.05).The male AW body length?1019.82±74.222??m of the siRNA-366 group was significantly lower than the control siRNA group?1159.60±220.53??m or the PBS group?1191.67±138.93??m(F_{m AW}=4.095,P<0.05).The NBL length of the siRNA-366 group?87.77±16.19??m was shorter than the control siRNA group?103.5±12.05??m or the PBS group?110.592±10.72??m(F_NBL=11.751,P<0.05).The ML length?910.01±119.80??m of the siRNA-366 group was lower than that of the control siRNA group?1144.54±114.14??m or the PBS group?1125.24±80.90??m(F_ML=44.961,P<0.05).Conclusions1.Delivery specificity siRNAs of TsSP and TsGST into Trichinella spiralis muscle larvae through electroporation successfully.2.TsSP and TsGST gene of transcription and protein expression were silenced in T.spiralis larvae following transfection with siRNAs and enzymatic activity was reduced.3.Knockdown of TsSP and TsGST gene by RNAi significantly reduced the T.spiralis larvae intrusion of IECs and invasion of enteral mucosa,and reduced the female fecundity.4.TsSP and TsGST played an important role in the invasion,development and reproduction of Trichinella spiralis,and can be used as a potential molecular target for anti-Trichinella vaccine.