Characterization Of A Trichinella Spiralis Inorganic Pyrophosphatase And The Immune Protection Induced By Its Oral Vaccine

Trichinellosis is one kind of food borne zoonosis which widespread worldwide.Eating raw or semi-cooked meat which containing Trichinella spiralis is the mainly infective way.It makes sense to study the proteins which play role in the growth and development of Trichinella spiralis,and to study their functions and mechanisms for the prevention and control of trichinellosis,to screen the anti-Trichinella infection drugs and the research of related vaccines.Inorganic Pyrophosphatase(PPase)is an essential catalytic protease in the whole life cycle of parasites,which is involved in the synthesis of biological macromolecules.At present,the research of PPase related to parasites mainly focuses on protozoa,Schistosoma,Caenorhabditis elegans and Ascaris,and the conclusion is that PPase plays a key role in the growth,development and molting process,but the PPase role are still not clear in the molting process of T.spiralis.In this study,the PPase of Trichinella spiralis(TsPPase)was cloned and purified.The biological properties and enzyme activity of TsPPase were analyzed.RNAi was used to investigate the role of TsPPase in the molting process.The rTsPPase Lactobacillus vaccine was constructed to orally immunize mice and determine the immune protection.The results of this study are helpful to reveal the biological characteristics of TsPPase and its role in the development process of T.spiralis.Moreover,it provide a strong theoretical basis for the development of vaccines and drugs against Trichinella spiralis infection.Methods and materialⅠ.Parasites,experimental animals,cells and ethics approvalIn the research,Trichinella spiralis strain(ISS534)was gained from domestic swine in central China.KM mice were used to carry out the passage.Six-week-old female BALB/c mice were purchased from Henan Provincial Experimental Animal Center(Zhengzhou,China).Primary IECs were isolated from normal mouse intestines and sensitive to T.spiralis intrusion.Mouse striated muscle myoblast C2C12 was non-sensitive to T.spiralis invasion and used as the negative control.The animal experiment was in accordance with the regulations of the Institutional Life Science Ethics Committee of Zhengzhou University(No.SCXK 2017-0001).Ⅱ.Cloning,expression and biological characteristics of TsPPaseComplete TsPPase cDNA sequence was acquired from GenBank(Gene ID:10913356).The characteristics of TsPPase gene were analyzed using bioanalysis software and websites.The TsPPase full-length cDNA was amplified with PCR by specific primers,then cloned to pQE-80L.The recombinant TsPPase(rTsPPase)was induced and purified in E.coli system.rTsPPase was used to immunize BALB/c mice and after the last immunization,the mice tail blood was collected,and antiserum was isolated.RT-PCR,Real-time PCR,Western blot and IFA assays were performed to investigate the transcription,expression and location of TsPPase in different stages of T.spiralis.Ⅲ.Enzyme activity and site directed mutagenesisUltraviolet spectrophotometer was used to determine the rTsPPase activity by measuring the rate of release of Pi from PPi and ATP through the molybdenum-blue colorimetry.In order to get the best condition of the reaction,different temperatures,pH and Mg2+ concentrations were experimented.To further verify the activity of rTsPPase,the PPases inhibitors(NaF and inosine-5’-diphosphate trisodium salt.IDP)of were used to ascertain whether they could effectively inhibit the rTsPPase activity,NaCl was used as the control.The enzyme activity of rTsPPase was defined as μmol Pi/min/mg of protein.The native TsPPase protein was also detected the enzymatic activity.The rTsPPase played a catalytic role when its metal binding sites combine with divalent metal ions,in order to further study its enzyme activity,the site directed mutagenesis was performed in the study.Ⅳ.The binding between rTsPPase and IECsFar-western blot was performed to investigate the interaction between rTsPPase and IECs.IFA assay was used to detect the binding in rTsPPase with small intestine,muscle and liver sections of mice.The effects of rTsPPase protein and its immune serum on the invasion of T.spiralis larvae into IECs monolayer were also analyzed.Ⅴ.RNA interference experimentThe TsPPase-specific siRNA-279 was designed and synthesized.the control-siRNA was used to be the control,the ML were transfected with siRNA-279 by electroporation.The introduction of siRNA into the ML was observed by the fluorescence under the microscope.qPCR and western blot were used to detected the TsPPase transcription and expression level after RNAi.In order to detect the changes of native TsPPase activity in ML and IIL soluble protein after RNAi,ML and IIL were treated with 4μM siRNA-279 and cultured for 2 days at 37℃.The souble proteins were prepared with the worms after cultivation.All the soluble proteins were diluted to the same concentration(2.0 μg/μL)and incubated with PPi in the standard reaction mixture at the best conditions to detect the native TsPPase enzymatic activity.The worms treated without siRNA were used as the experimental control.Ⅵ.Larval molting inhibition assay by RNAiIn order to observe the larval development and molting in different experimental groups.the anti-rTsPPase serum,inhibitor NaF and siRNA were used to incubated with IIL in vitro,respectively,and the normal serum was as the control.In order to further verify the role of rTsPPase in the molting and development of T.spiralis,three experimental groups were set up to collect 12 h IIL(L2 larvae),24 h IIL(L4 larvae)and 3 d AW.Forty mice in each group were divided into 4 groups(10 animals per group),and each mouse was challenged orally with 500 ML which treated with NaF,siRNA-279,control-siRNA and PBS,respectively.The 12 h IIL,24 h IIL and 3 d AW were collected and observed with a microscope,the number,length of larvae and molting rate were compared among different groups.Ⅶ.Immune protection of rTsPPaseThe plasmid of pSIP-409--pgsA’-TsPPase was constructed and transferred into Lactobacillus NC8 by electroporation.In order to estimate the immune protection of TsPPase,the mice were immunized orally with recombinant plasmid,empty plasmid and MRS,respectively.ELISA was performed to detect the IgG,IgG1 and IgG2a in serum,the sIgA in intestine,the cytokines of spleen,lymph and Peyer patch cells.After 3 immunization,the mice were challenged orally with ML,in order to estimate the effect of immune protection,the 24 h IIL were collected to detected the molting rates of different groups,and the 6 d AW and ML burden were measured.Ⅷ.Statistical analysisSPSS 21.0 was used to analyze the data,One-way ANOVA and Chi-square test were used in the research,the appropriate statistical method was selected according to the data.P<0.05 indicated the statistical difference existed.ResultsⅠ.Cloning,expression and biological characteristics of TsPPaseComplete TsPPase coding sequence consisted of 1104 bp encoding 367 amino acids(aa),with a 41.48 kDa MW.The value of rTsPPase isoelectric point is 5.76.No signal peptide was identified by Signal P 4.1 Server.TsPPase has no transmembrane domain which detected by TMHMM prediction.The structure prediction revealed that TsPPase is consisted by 2α-helices and 12 β-strands,and it has metal binding sites and substrate binding sites.The SDS-PAGE indicated that the E.coli containing pQE-80L/TsPPase expressed an about 44 kDa fusion protein which was a little bigger than predicted.It may be due to the rTsPPase containing a His-tag and glycosylation.The results of qPCR and western blot indicated that all soluble and ES proteins of different stages contained native TsPPase,and it could be effectively probed by anti-rTsPPase serum.The IFA result showed that immuno-fluorescence staining was observed at all T.spiralis stages,mainly at the stichosome and around the embryo.Ⅱ.Determination of the rTsPPase enzyme activityIn order to obtain the optimal condition of the enzymatic reaction,various temperatures,pH and divalent cations were tested in reaction mixture.The absorbance value of reaction mixture was detected by ultraviolet spectrophotometer on 690 nm.Finally,the optimal condition of reaction was gained when the substrate was PPi,the best temperature,Mg2+concentrations and pH were 50℃,5 mM and 7.5.The effects of different divalent cations on the rTsPPase enzyme activity were also detected,the result showed that the ability of Mg2+for rTsPPase catalysis was the strongest.NaF,an anion,is the potent inhibitor of PPases and was able to inhibit the rTsPPase activity in a dose-dependent manner,IDP also could inhibit the activity of rTsPPase,but the inhibitory effect was not as strong as NaF.Furthermore,the PPi dependence of the maximum hydrolytic velocity(Vmax)of rTsPPase was shown to be 81.96μmol Pi/min/mg of protein with a Michaelis constant(Km)value of 170 μM.When ATP was used as substrate,the best temperature,pH and Mg2+concentrations were 50℃,6.0 and 8.0 mM.The ATP dependence of the maximum hydrolytic velocity(Vmax)of rTsPPase was shown to be 1.816 μmol Pi/min/mg of protein with a Michaelis constant(Km)value of 108 μM.The enzymatic activity of mutated TsPPase(M-TsPPase)was determined.The relative activity of rTsPPase was taken as 100%,the result showed that the M-TsPPase only exhibited 24.33%enzymatic activity,the enzyme activity was inhibited by 75.67%(χ2=122.581,P<0.05).The results revealed that the activity was obviously inhibited after the metal binding sites mutation.Ⅲ.Binding of rTsPPase and IECsThe result of Far western showed that rTsPPase could bind to IEC proteins,and the combination was strong.There was no binding between rTsPPase and C2C12 proteins,the binding of rTsPPase and IEC was specific.The results of IFA showed that the positive staining was obviously observed in the intestinal and muscular sections of immune and infection serum groups.No positive staining was found in intestinal and muscular section of normal serum and liver sections.In in vitro intrusion assay,the result showed that rTsPPase had no obvious facilitation on larval penetration,only when its concentration reached 15μg/mL,rTsPPase had a significant effect on invasion.The result also showed that the antiserum had inhibitory effect on larval intrusion when the serum dilution was 1:100 and 1:200.and the inhibition rate was 37.23%and 24.80%.These results indicated that TsPPase played a role in the larval invasion.Ⅳ.The expression and enzyme activity were suppressed by RNAiThe results of qPCR showed that the ML electroporated using 3,4 μM of siRNA-279 exhibited 28.95 and 37.76%reduction of TsPPase transcription level,which was significantly lower than that of PBS group(F=5.194,P<0.05).At 1,2 and 3 days following electroporation using 4 μM of siRNA-279,the transcription level reduced in 19.83.38.37 and 27.94%(F=49.025,P<0.05).Compared to the PBS group.the TsPPase expression level was inhibited by different concentrations of siRNA-279.and there was a significant reduction with a 33.34%reduction in 4 μM siRNA-279 treated larvae(χ2=39.521.P<0.05).Then the 4 μM siRNA-279 treated worms were cultured in RMPI-1640 for 1.2 and 3 days,the TsPPase expression level was significantly inhibited with a 37.55%reduction at 2 days post cultivation compared with the PBS group(χ2=46.914.P<0.05).Compared with the control group,the level of PPi degradation by soluble protein in 4μM siRNA-279 treated ML and 10 h IIL was reduced by 29.83%and 21.41%,respectively(P<0.05).Ⅴ.Suppression of siRNA-279 on larval moltingThe IIL was cultured in vitro and incubated with siRNA,inhibitor and antiserum.The results showed that the larval molting was inhibited by RNAi,NaF and antiserum,the difference was statistically significant,the inhibition rates were 47.32%,37.36%and 22.99%,respectively(P<0.05).The results of molting assay in vivo showed that NaF and siRNA-279 had the ability to inhibit the molting and development of T.spiralis.The molting rate of 24 h IIL in RNAi group was only 14%while that of PBS group was 34%,the difference was statistically significant.Due to the molting inhibition,the IIL could not develop into adult worm,the RNAi group exhibited a 32.30 and 42.20%reduction of 24 h IIL and 3 d AW compared to the control-siRNA group.All the results revealed that TsPPase played a crucial role in T.spiralis larval molting and development process.Ⅵ.Immune protection of rTsPPaseL.plantarum NC8/TsPPase was successfully constructed in the study,the level of specific IgG,IgG1 and IgG2a in serum,sIgA in intestinal juice were obviously raised after oral immunization.The transcription and expression of IFN-y and IL-4 produced by spleen,MLN and Peyer patch cells were obviously increased,the result indicated that a mixed Th1/Th2 immune response was triggered by immunization with TsPPase/NC8.After immunization,the mice were challenged with 300 ML,the reduction rates of 24 h IIL,6 d AW and ML in TsPPase/NC8 group were 67.18%,54.78%and 51.91%.In addition,the molting of 24 h IIL was inhibited by immunization with TsPPase/NC8,the inhibition rate was 45.45%.All the results showed that the mice immunized with TsPPase/NC8 had a protective effect against T.spiralis.ConclusionⅠ.TsPPase was transcribed and expressed in all stages of T.spiralis.It was mainly located the cuticle,stichosome and around the embryo.Ⅱ.The enzymatic activity of rTsPPase was determined,it can promote the degradation of PPi and ATP into Pi.There was a specific binding between rTsPPase and IECs,which played an important role in larval intrusion.Ⅲ.The results of RNAi revealed that TsPPase was involved in the T.spiralis larval molting and development.Ⅳ.The rTsPPase could induce Th1/Th2 mixed immune response after oral immunization in mice,which had a protective effect against T.spiralis.Therefore,TsPPase could be used as a promising candidate vaccine target molecular against trichinellosis.