Biological Characterization Of Trichinella Spiralis Cysteine Protease And Its Role In Invading Intestinal Epithelial Cells

Background and PurposeTrichinellosis is mainly caused by eating raw or semi-raw animal meat containing muscle larvae(ML)cysts.The larvae undergo excystation in the stomach of the host by the action of digestive juice,and then are activated by bile to develop to intestinal infective larvae(IIL).IL1 larvae could invade intestinal epithelium cells(IECs),and molt in the intestinal tract for 4 times to develop into adult worms.The male and female worms produce new born larvae(NBL)after mating.The NBL invade the skeletal muscle and develop into cystic muscle larvae by traveling through the veins or lymphatic vessels of the host body,which remaining in the host for a long time.The process of Trichinella spiralis invading IECs is the key stage to infect the host.The larvae can develop into adults only by successfully invading IECs.Therefore,the proteases that play a role in the process of invasion may be a new target for trichinellosis vaccines and drugs.Cysteine proteases play an important role in the growth and development,invasion and immune escape of parasites.Therefore,in this paper,we selected Gut-specific cysteine proteinase(TsGSCP,Gen Bank:XM＿003377192.1)from the Trichinella spiralis gene library as the research object.Using cloning expression,immunofluorescence antibody test(IFA),immunohistochemistry,Western blot,ELISA andRNAi to observe the biological characteristics of TsGSCP and its role in the invasion of Trichinella spiralis larvae into IECs.Materials and Methods1.Experimental animals,Trichinella species,cell lines and strains4-weeks-old SPF class female KM mice were purchased from Henan Experimental Animal Center.The Trichinella spiralis used in this experiment were Trichinella T1(ISS534)from pigs in Nanyang,Henan Province.The cell lines were IECs isolated from the small intestine of normal mice.The strain used in the experiment was Escherichia coli BL21.2.Bioinformatics analysis of TsGSCPThe mRNA sequence of TsGSCP were obtained from NCBI.Bioinformatics analysis software was used to analyze the physicochemical properties of TsGSCP.Multi-sequence alignment and phylogenetic tree analysis of TsGSCP were performed by using Bioedit and Mega.3.Expression,purification and antigenicity analysis of rTsGSCPRecombinant expression plasmid pMAL-c2x/TSGSCP was constructed and introduced into BL21.After induction by 1 m M IPTG at 25℃for 6 h,the fusion protein rTsGSCP with MBP label was purified and the anti-rTsGSCP serum was prepared by immunzied mice.IIL of Trichinella spiralis were collected to prepare soluble antigens and ES antigens,and the antigenicity of rTsGSCP was detected by Western blot.4.Expression and localization of TsGSCP in different stages of Trichinella spiralisThe cDNA of Trichinella spiralis in different stages(ML,IIL,AW and NBL)were prepared for q PCR to detect the transcription of TsGSCP in different stages.Soluble antigens of different stages were prepared,and the expression and localization of TsGSCP in different stages were observed by Western blot,IFA and immunohistochemical assay.5.The binding of rTsGSCP to IECs and intestinal mucosa was detected by Far-western and IFAIECs soluble protein was prepared,and the binding of rTsGSCP to IECS protein was detected by Far-Western and Far-ELISA.The combination and localization of rTsGSCP and IECs were observed through IFA.The specific binding of rTsGSCP to intestinal mucosal tissue was observed by IFA.6.The promoting effect of rTsGSCP on Trichinella spiralis invasion of IECs and intestinal mucosaAfter IL1 larvae were co-incubated with monolayer IECs for 2 h,the effects of rTsGSCP and anti-rTsGSCP serum on the invasion of IECs were observed.In addition,the muscle larvae incubated with anti-rTsGSCP serum were orally infected with mice,and the load,morphology and fecundity of the adult larvae were observed at 5 days after infection.7.Effects ofRNA interference with TsGSCP gene on invasion,growth,development and fertility of Trichinella spiralisThe TsGSCP primers containing T7 promoter were designed,and dsRNA interfering with TsGSCP gene was obtained by inversion in vitro.The dsRNA was delivered into the muscle larvae of Trichinella spiralis by electroporation.The transcription and expression levels of TsGSCP were detected by Real-time PCR and Western blotto analyze the optimal condition of dsRNA-TsGSCP interference.In addition,the activity of natural cysteine protease in soluble protein of Trichinella spiralis was determined by using specific substrates,and the inhibitory effect of TsGSCP gene silencing on larval invasion of IECs and intestinal mucosa were observed.Finally,the effects ofRNA interference with TsGSCP gene on the growth and fertility of Trichinella spiralis were observed.8.Statistical treatmentThe experimental data were processed by SPSS and Graph Pad software.Select the appropriate statistical analysis method according to the type of data.The statistical methods used in this study include ANOVA and chi-square test.The test level wasα=0.05.Results1.Bioinformatics analysis of TsGSCPBioinformatics software was used to analyze the physicochemical properties of TsGSCP(Gen Bank:XM＿003377192.1).The results showed that TsGSCP gene sequence encoded 325amino acids,and the isoelectric point was 6.88,indicating that TsGSCP had a signal peptide also could be found in its sequence.The secondary structure of TsGSCP has 8α-helices and 7β-folds.The tertiary structure of TsGSCP was predicted to have four enzyme active sites,which were histidine(His),glutamine(Gln),cysteine(Cys)and asparagine(Asn).This protease family has the functional domain of the C1 peptidase family(cathepsin B family).2.Antigenicity analysis of rTsGSCPSDS-PAGE results showed that the molecular weight of rTsGSCP was 74.2 k Da(cDNA clone 34.2 k Da+MBP-tag 40 k Da).Western blot results showed that rTsGSCP could be recognized by Trichinella infected serum and anti-rTsGSCP serum,but could not be recognized by normal serum,indicating that rTsGSCP had good antigenicity.The anti-rTsGSCP serum can recognize the natural TsGSCP in the crude protein of IIL,but not the TsGSCP in IIL ES,suggesting that TsGSCP is a somatic protein rather than a secreted protein.3.Expression and localization of TSGSCP in different stagesWestern blot showed that TsGSCP was expressed in soluble proteins of all stages of Trichinella spiralis,and the expression level of TsGSCP in the stage of IIL was significantly higher than that in other stages(P<0.05).IFA and immunohistochemical results showed that TsGSCP was expressed in different stages(ML,IIL,AW and NBL)of Trichinella spiralis and was mainly localized in the epidermis and female embryos.4.Specific binding of rTsGSCP with IECs and intestinal mucosaFar Western results showed that 7 bands(96.2,70.8,63.6,56.4,47.4,38.8 and 36.4 k Da)of IECS protein and rTsGSCP were recognized by anti-rTsGSCP serum after incubation,and6 bands(97.2,75.0,62.7,57.9,46.4 and 41.9 k Da)were recognized by infection serum.ELISA results showed that the OD value increased with the increase of IECs protein concentration(F=397.007,P<0.05),and was correlated with the dose of IECs protein(r=0.994,P<0.05).OD value increased with the increase of rTsGSCP protein concentration,and was correlated with the dose of rTsGSCP protein(F=99.967,P<0.05;r=0.976,P<0.05).IFA results showed that rTsGSCP could specifically bind to small intestinal mucosa,but could not bind to liver and lung tissues of mice.The rTsGSCP can only bind to IECs but cannot bind to C2C12,and the binding site of rTsGSCP and IECs is in the cytoplasm.5.Promotive effect of rTsGSCP on in vitro invasion of IECs and intestinal mucosa by Trichinella spiralis larvaeIL1 larvae were co-incubated with rTsGSCP(or IIL ES and MBP tag protein),anti-rTsGSCP serum(or infected serum,normal serum,Ani-MBP serum)and monolayer cells of IECS for 2 h,and the invasion of larvae into monolayer cells was observed.The results showed that the invasion rates of IL1 larvae were 80.95%,84.49%and 86.74%at TsGSCP concentrations of 9,12 and 15μg/m L,respectively.Compared with the PBS control group,the invasion rates were higher,and the differences were statistically significant(P<0.05),the larval invasion rate was correlated with the dose of rTsGSCP(r=0.95,P<0.05),and the invasion was enhanced with the increase of rTsGSCP concentration(F=228.325,P<0.05).Anti-rTsGSCP serum has obvious inhibited effect of invasion to IECs monolayer(χ~2=16.743,P<0.05),when the Anti-rTsGSCP serum dilution degree is 1:100,the inhibition rate of the larvae invade was 41.47%,the inhibition function and serum dilution degrees are dose dependent(r=0.969,P<0.05),With the increase of serum dilution,the inhibition of Trichinella invasion gradually decreased(F=136.542,P<0.05).After ML incubated with anti-rTsGSCP serum was orally infected with mice,the intestinal adult worm reduction rate was 38.20%at 5 days after infection,which was significantly higher than that of normal serum and anti-MBP-tag serogroup(F=46.013,P<0.05),but there was no significant difference in adult length,fecundity and newborn larva length between different serogroups(P>0.05),indicating that anti-rTsGSCP serum has a significant inhibition effect on Trichinella invading intestinal mucosa.6.Inhibition effect of interfering TsGSCP gene on invasion,growth,development and fertility of Trichinella spiralisThe qPCR and Western blot results showed that when the dsRNA-TsGSCP concentration was 20,40,and 60 ng/μL,the transcription and protein expression levels of TsGSCP gene were significantly reduced(P<0.05),the transcription level of the TsGSCP gene decreased by14.82%、33.30%and 62.13%,respectively.The TsGSCP protein expression was decreased by22.24%、45.28%and 60.51%,respectively.And the protein expression level were decreased with the increase of dsRNA concentration.When the larvae were cultured for 2,4,and 6 days after the TsGSCP gene was silenced,the transcription level of the TsGSCP gene decreased by24.25%,2.21%,and 1.89%,respectively.The TsGSCP protein expression was decreased by46.82%,1.73%and 2.26%,respectively.After cultured 2 days,the expression level of TsGSCP in the dsRNA treatment group was significantly reduced(χ~2=61.438,P<0.05),indicating that the optimal interference time of dsRNA-TsGSCP was 2 days.The enzymatic activity of the cysteine protease in the natural soluble protein of ML and IIL was reduced by37.39%and 28.00%,respectively,and the inhibition rate of larvae invading IECs afterTsGSCP gene silencing was 25.02%,which was significantly higher than that 3.24%of dsRNA-GFP control group(χ~2=5.578,P<0.05).In addition,after silencing TsGSCP gene,the AW reduction rate reached 35.15%at 5 d,the length of female larvae was significantly shorter(F=21.082,P?0.05),and the fertility was decreased(F=18.728,P?0.05).Conclusion1.The recombinant expression plasmid pMAL-c2x/TsGSCP was constructed,and rTsGSCP was expressed and purified.2.TsGSCP is expressed in different stages of Trichinella spiralis(muscle larva,intestinal infectious larva,adult,newborn larva)and the highest expression level is in the intestinal infection larvae,which mainly located in the cortex of the worm body and the female embryo.3.rTsGSCP can specifically bind to mouse intestinal epithelial cells and small intestine tissues to promote the invasion of Trichinella spiralis,while the anti-rTsGSCP serum can inhibit the invasion of larvae.Interfering with the TsGSCP gene can significantly reduce the expression and enzyme activity of TsGSCP,inhibit the invasion of intestinal epithelial cells,and affected the growth and fecundity of Trichinella spiralis.TsGSCP is an invasion-related protein of Trichinella spiralis and can be used as a candidate target molecule for anti-Trichinella infection vaccine.