Molecular Characterization Of Two Aspartic Proteases Of Trichinella Spiralis And Their Facilitation Role For Larval Invasion Of Host's Intestinal Epithelial Cells

Trichinellosis,a global distribution foodborne zoonosis,has caused huge threat to public health.Thus,the research on the mechanism of Trichinella penetration into hosts and identification of Trichinella invasive molecules is meaningful for developing anti-Trichinella medicines and preventive vaccines.Aspartic proteases(ASPs),the virulence factors identified in several parasitic helminthes,are essential to parasite development,larval penetration and immune evasion.However,the function of ASPs in Trichinella spiralis(T.spiralis)was rarely described.In the present study,two proteins(TsASP1 and TsASP2)containing aspartic protease domain from T.spiralis were cloned and expressed,and recombination proteins(rTsASP1 and rTsASP2)were obtained.Biological characteristics and functions of TsASP1 and TsASP2 were analyzed and the proteolytic activities of rTsASP1 and rTsASP2 were detected.Vaccination of mice with rTsASP1 and rTsASP2 was carried out to evaluate the protective immunity against T.spiralis infection.RNA interference(RNAi)was operated to ascertain the functions of TsASP1 and TsASP2 in larval invasion into intestinal epithelial cells(IECs).Results in this study are helpful for illustrating the biological characteristics and functions of TsASP1 and TsASP2,and developing anti-Trichinella medicines and vaccines.Material and Methods?.Experimental parasite,animals and cellsT.spiralis isolate(ISS534)used in this study was stored by serial passages in BALB/c mice.The specific pathogen-free(SPF)BALB/c mice were purchased from the Experimental Animal center of Henan Province.IECs were isolated from fetal mice,and mouse myoblast(C2C12)was kept in our laboratory.All animal experiments were reviewed and approved by the Life Science Ethics Committee of Zhengzhou University.?.Cloning,expression and biological characteristics of TsASP1 and TsASP2Two aspartic proteases(named as TsASP1 and TsASP2)were selected according to the whole genome of T.spiralis.The bioinformatics of TsASP1 and TsASP2 were first analyzed using software.The sequences of TsASP1 and TsASP2 were amplified by RT-PCR and cloned into the expression vector pQE80L(His tag)and pMAL-c2x(MBP tag).Then the recombinant proteins(rTsASP1 and rTsASP2)were obtained.The mice were immunized with rTsASP1 and rTsASP2(with Hig tag),then anti-rTsASPl/anti-rTsASP2 sera were obtained from the immunized mice.The expression of TsASP1 and TsASP2 in the different developmental stages were analyzed by western-blot.Immunofluorescent assay(IFA)was carried out to confirm the location of TsASP1 and TsASP2 at T.spiralis diverse stages.The transcription of TsASP1 and TsASP2 gene at different stages was observed by Real-time PCR.?.Enzyme activity and immune protection of rTsASP1 and rTsASP2Hemoglobin(Hb)and the fluorescent substrate were used as substrates to evaluate the proteolytic activity of rTsASPl and rTsASP2.Proteolytic activity of rTsASP2 was further detected by SDS-PAGE using other proteins(including serum albumin,IgG,IgM,fibrinogen and collagens IV).The inhibition of rTsASP2 proteolytic activity by anti-rTsASP2 serum and pepstatin A was also investigated.The enzymatic characteristics of rTsASP2 were identified by cleavage of the fluorescent substrate under different pH,temperature and inhibitors.To analyze the immune protection of rTsASPl and rTsASP2,200 mice were divided into five groups,40 mice per group.Those five group mice were vaccinated with PBS,MBP tag,ISA201 adjuvant,rTsASPl and rTsASP2,respectively.The level of specific total IgG,IgGl and IgG2a antibodies in the sera and IgA in the interior small intestine were detected by ELISA.To measure the specific cellular immune responses,the cellular cytokines in splenocytes and lymphocytes from immunized mice were also detected.Two weeks after last immunization,mice were orally challenged with 300 T.spiralis larvae.Then,mice were sacrificed at 6 days and 35 days after challenge,respectively.The numbers of adult worms,muscle larvae burden and the fecundity of female worms were counted and the immune response of anti-T.spiralis infection were evaluated.?.Establishing the method of silencing of TsASP1 and TsASP2 geneThe FAM labeled specific TsASPl siRNA,TsASP2 siRNA and Control siRNA were designed.The siRNA was delivered into ML larvae via electroporation,and the transformation efficiency was assessed according to the observation of fluorescent signal of ML.The transcription and expression of TsASP1 and TsASP2 in worms treated with different siRNA was detected by real-time PCR and western blot,respectively.The TsASPl siRNA and TsASP2 siRNA was used as mutual control to verify RNAi gene specificity,making sure the methods of RNAi are credible.?.Analyzing the effect of RNAi on the larval penetration into IECsThe interactions between rTsASPl/rTsASP2 and IECs proteins were first demonstrated by the Far-western blot,Enzyme-linked immunosorbent assay(ELISA)and IF A,and then the effect of RNAi on the interactions was also analyzed.The larval invasion assay was established to investigate the roles of TsASP1 and TsASP2 played in larval penetration into IECs with recombinant proteins and corresponding immune sera.The invasion rates and damage cells of monolayer were also detected after the TsASP1 and TsASP2 gene were silenced.Mice were infected with ML treated with different siRNA or PBS.The infection mice were sacrificed,then the histological sections of duodenal was observed by the PAS and HE staining.The 6 days post-infection(dpi)adult worms and 35 dpi ML were collected from infection mice,and the fecundity of female worms was detected.The lengths of worms were measured and the surface morphology of 6 dpi adult worms was observed by scanning electron microscope(SEM).Based on these results,the effect of RNAi on the larvae development in host and roles of TsASP1 and TsASP2 play in larvae invade into host intestinal was investigated?.Statistical analysisThe data analysis was performed with the aid of SPSS 19.0 software.The differences of data were analyzed by One-way ANOVA,Chi square test or linear regression.P<0.05 was defined as statistical significanceResults?.Cloning,expression and biological characteristics of TsASP1 and TsASP2Bioinformatics analysis results showed that both TsASP1 and TsASP2 have signal peptides and no trans-membrane domain,and belong to aspartic protease superfamily which contain two domains possessing similar topological features.TsASP2 contains conserved consensus sequences Asp-Thr(Ser)-Gly in the active site,while active domains of TsASPl was different from the conserved consensus sequences.The recombinant proteins were expressed in E.coli BL21(DE3),and the fusion proteins were 43 kDa(His tag)and 86 kDa(MBP tag)of rTsASPl,and 42.9 kDa(His tag)and 85.9 kDa(MBP tag)of rTsASP2,respectively.The results are consistent with the prediction.The transcription of TsASP1 and TsASP2 genes was detected in all developmental stages of T.spiralis,with highest at intestinal infection larval(IIL)stage and lowest at new born larval(NBL)stage.The native TsASP1 and TsASP2 protein were recognized by immune sera in all stage except the NBL.The IF A results suggested that TsASPl was mainly located at muscle cells,stichosome and around the embryos of AW,while TsASP2 was located at muscle cells,midgut,hindgut and around the embryos of AW.?.Enzyme activity and vaccination of mice by rTsASP1 and rTsASP2The proteolytic activity assay revealed that rTsASP1 could not cleave Hb,while rTsASP2 could cleave the Hbs originated from human,mouse and cattle,but could not hydrolyze the chicken Hb.The optimal pH for rTsASP2 to degrade mouse Hb is 2.5,while to degrade human and cattle Hbs is 4.5.The degradation efficiency of mouse Hb was higher than that of human and cattle Hbs.Besides,collagen IV and IgM could also be hydrolyzed by rTsASP2.The enzyme activity of rTsASP2 was confirmed under pH 2.0 to 6.0 when using the fluorogenic peptide as substrate.The maximum activity was detected at pH 3.0 and temperature 37?.The rTsASP2 enzymatic activity was significantly inhibited by pepstatin A.The enzymatic activity could be inhibited by Cu2+and Fe2+,while enhanced by Mg2+.The enzymatic kinetics was determined by Michaelis-Menten equation:Vmax=16.68±0.3465 nM/min,Km=2.254±0.1878 nM.After mice were injected subcutaneously with rTsASPl and rTsASP2,the specific IgG antibodies were elicited in the sera,and the level of IgG1 was higher than IgG2a.The specific mucosal IgA in the interior small intestine was increased,which revealed that the vaccination of rTsASP1 and rTsASP2 could induce the mucosal immune response of mice.The cytokines(IFN-? and IL-4)of spleen and MLN cells were increased,indicating the mixed Th1/Th2 immune response was triggered by immunization with rTsASP1 and rTsASP2.After mice were challenged with 300 T.spiralis worms,the reduction of 6 dpi adult worms was 49.79%from rTsASPl immunized group and 54.18%from TsASP2 immunized group,respectively.The reduction of 35 dpi larvae burden was 50.55%from rTsASPl immunized group and 54.59%from TsASP2 immunized group,respectively.These results suggested that protective immunity against T.spiralis infection in mice was triggered after vaccination of rTsASPl and rTsASP2.?.Establish the method of silencing of TsASP1 and TsASP2 geneResults showed that there was a clear fluorescence in the worms treated with siRNA.After the larvae were treated with PBS or different siRNAs,no significant difference in mortality was detected among these groups.Based on the gene transcription and protein expression level,worms treated with 5 ?M siRNA and cultivated for 5 days was the optimal condition.TsASPl transcription level and protein expression level in worms treated with TsASPl siRNA were reduced by 66.52%(P<0.05)and 63.60%P<0.05),compared to those of PBS group.While in TsASP2 siRNA group,TsASP2 transcription level and protein expression level were reduced by 58.69%(P<0.05)and 64.86%(P<0.05).The worms treated with Control siRNA cannot silence the TsASP1 or TsASP2.TsASP1 siRNA and TsASP2 siRNA was specific to silence each target genes.Thus,a method of silencing TsASP1 and TsASP2 gene was established.?.Analyzing the effect of RNAi on larval development and penetration into IECsAfter silencing TsASP1 and TsASP2,The aspartic protease activities of crude protein were investigated.Compared to PBS group,aspartic protease activities were reduced to 43.3%(P<0.05)in TsASP2 siRNA group and 93.1%(P>0.05)in TsASPl siRNA group,respectively.These results indicated that TsASP2 protein contribute to the aspartic protease activities of crude protein,while TsASPl owns no aspartic protease activity.After pre-incubated with rTsASPl or rTsASP2,about 14 bands and 17 bands of IEC proteins could be recognized by the anti-rTsASPl/anti-rTsASP2 sera,respectively.The interactions between TsASPl/TsASP2 and IEC lysates were dose-dependent on the protein concentration.The confocal microscopy revealed that TsASPl bound with cytoplasm of IECs,while TsASP2 mainly bound with cytomembrane of IECs.Furthermore,both the native TsASP1 and TsASP2 could bind with the IECs and the binding efficiency was significantly reduced after silencing of TsASP1 and TsASP2.Both rTsASPl and rTsASP2 could promote T.spiralis invasion into IECs monolayer,while the anti-rTsASPl/anti-rTsASP2 sera could inhibit the larval penetration into IECs monolayer.After silencing of TsASP1 and TsASP2 gene,the larval invasion rate was reduced significantly.When worms treated with TsASP1 siRNA or TsASP2 siRNA,the percentage of worms penetrating into monolayer was reduced by 29.45%(?2=17.407,P<0.05)and 35.22%(?2=13.926,P<0.05),respectively.The damage cells were counted after larvae invade into monolayer.Compared to PBS group,the damage cells was reduced by 42.92%(P<0.05)for TsASPl siRNA treated group and 66.24%(P<0.05)for TsASP2 siRNA treated group,respectively.These results revealed that both TsASPl and TsASP2 play important roles in the larval penetration into IECs monolayer.The pathological changes of small intestines were observed after mice infected with worms treated with different siRNA.When mice infected with worms treated with Control siRNA,edematous cells and goblet cells were observed and the structural damage of cells were also detected.However,no obvious pathological changes of IECs was observed in the mice infected with larvae transfected with TsASP1 siRNA and TsASP2 siRNA.Compared to control siRNA group,mice infected with ML transfected with TsASPl siRNA and TsASP2 siRNA showed significantly reduction of 6 dpi AW burden,35 dpi ML burden and NBL production.Additionally,the length of females and males collected from TsASP1 siRNA group at 6 dpi were reduced by 16.37%(P<0.05)and 11.92%(P<0.05),respectively.Similarly,the length of females and males recovered from TsASP2 siRNA group showed 20.46%(P<0.05)and 24.65%(P<0.05)reduction,respectively.Meanwhile,the SEM results showed that the surface morphology of 6 dpi adult worms recovered from Control siRNA group was smoother than that from TsASP1 siRNA group or TsASP2 siRNA group.Those results convinced that both TsASPl and TsASP2 were closely related with the T.spiralis penetration into host' s IECs and the larval development in host.Conclusion?.The recombinant proteins rTsASPl and rTsASP2 were successfully obtained.The transcription of TsASP1 and TsASP2 gene were detected in all developmental stages,with highest transcription in IIL and lowest transcription in NBL.Both two proteins were expressed in all stage except NBL.TsASP1 was mainly located at muscle cells,stichosome and around the embryos of AW,while TsASP2 was located at muscle cells,midgut,hindgut and around the embryos of AW.?.The aspartic protease activity of rTsASP2 was detected,while rTsASP1 has no enzyme activity.Vaccination of rTsASPl and rTsASP2 could both trigger the protective immunity against T.spiralis infection in mice,and the immune response is Thl/Th2 mixed.?.Both TsASP1 and TsASP2 could facilitate T.spiralis penetration into the IECs.After TsASP1 and TsASP2 gene were silenced,the protein expression levels of TsASP1 and TsASP2 were both reduced.Then the development of T.spiralis and larval penetration into host were inhibited.?.Both TsASPl and TsASP2 are invasive molecular,and can be used as potential target molecule for Trichinella vaccine.