Studys On The Role Of Cathepsin L In The Invasion Of Trichinella Spiralis Larvae Into The Intestinal Epithelium Of Mice

Trichinella spiralis can infect various carnivores and omnivores including humans,and may cause global public health risks.To ensure meat safety,the global demand for reliable methods of diagnosis and control of Trichinella spiralis in food animals is increasing.The process of Trichinella spiralis invading host intestinal epithelial cells（IEC）is the key to its development and infection.Some proteolytic enzymes may help Trichinella spiralis to invade,and the study of related proteases is crucial to the study of vaccines against Trichinella infection.Cysteine proteases play a key role in parasite immune evasion,virulence,tissue and cell invasion,and molting of parasitic nematodes.The Trichinella spiralis cathepsin L（Ts CL,Gen Bank:XM＿003375022.1）studied in this article is a moulting-related protease selected from the intestinal infective larvae（IIL）of Trichinella spiralis in the early stage of our research group.It is a cysteine protease of the papain type and belongs to the C1 family.In this paper,Ts CL was cloned and expressed and recombinant Trichinella spiralis cathepsin L（rTsCL）was prepared.Western blot and immunofluorescence test（IFA）were used to analyze its expression and location in different stages.Through Western blot,ELISA,RNAi,in vitro and in vivo experiments,the role of Ts CL in the process of Trichinella spiralis invading the intestinal epithelium was verified.Materials and Methods1.Parasite species,laboratory animals,strains and cellThe Trichinella species used in this experiment was Trichinella spiralis T1（ISS534）of Henan Nanyang pig origin from Kunming Mouse Protection in our laboratory.The experimental animals were KM rats purchased from Henan Experimental Animal Center.The cloned and expressed strain is BL21.Mouse small intestinal epithelial cells（IEC）were stably preserved in our laboratory.2.Bioinformatics analysis and prokaryotic expression of Ts CLThe basic physicochemical properties of Ts CL were analyzed by using NCBI,Signal P,SMART and other online websites.Sequence alignment between T.spiralis Ts CL and cathepsin L of other species was performed using Bioedit software to construct the evolutionary tree.The Ts CL gene was amplified by PCR,and the recombinant expression plasmid p QE-80L/Ts CL was constructed.The rTsCL was purified by nickel column containing His tag.3.Transcription,expression and location of Ts CL in various stage of Trichinella spiralisThe c DNA of muscle larva（ML）,IIL,adult（AW）and newborn larva（NBL）was collected from each stage,and use RT-PCR to analyze the transcription of Ts CL in various worm stage.Western blot and immunofluorescence techniques were used to analyze the expression and location of Ts CL in various stage.4.The role of Ts CL in the invasion of intestinal epithelial cells by TrichinellaWestern blot,ELISA and IFA were used to verify the binding of Ts CL and IEC.Using the Trichinella larvae invasion model in vitro,the rTsCL or anti-rTsCL immune serum and IIL larvae were incubated on a monolayer IEC for 2 hours to observe the larvae invasion.Set up BSA irrelevant protein control group,IIL ES positive protein control group,Trichinella infection mouse serum and normal mouse serum control group.Mice were orally inoculated with 500 muscle larvae incubated with infected serum,anti-rTsCL immune serum and normal serum,with 10 mice in each group.After 5 days,the mice were sacrificed to collect adult,and the number,length,fecundity and embryo development of different groups were observed.5.The effect of silencing Ts CL gene on the invasion of IEC by Trichinella spiralisDesign the ds RNA primers of Ts CL gene,and use T7 reverse transcription kit to prepare ds RNA-Ts CL.The optimal concentration and time of ds RNA-Ts CL action were analyzed by Western blot.The transcription level after silencing the Ts CL gene and the change of the natural Ts CL enzyme activity in the body protein were measured.The interfered IIL was added to the single-layer IEC and incubated for 2 hours to observe the invasion of IIL after silencing the Ts CL gene.Set up green fluorescent protein（GFP）and PBS control group.6.Effects of silencing Ts CL gene on the development of larvae and female fecundity30 mice were divided into 3 groups,and each mouse was orally with 500 ML treated with 60 ng/μL ds RNA-Ts CL,GFP and PBS.After 5 days,the mice were sacrificed to recover the adult worms from the small intestine.Count the amount of recovered AW and measure the length.The fecundity of female was observed after 3 days of culture.7.Statistical AnalysisThe data in this study was analyzed by SPSS 21.0 software.One-way ANOVA and chi-square test were used,and the data used the mean±marked difference,and the test level wasα=0.05.Results1.Bioinformatics analysis and prokaryotic expression of Ts CLTs CL（Gen Bank:XM＿003375022.1）,the gene sequence is 1491bp in length,encoding496 aa,molecular weight is 56.64 k Da,p I is 7.81.Ts CL contains signal peptide,the N-terminal has obvious hydrophobicity,contains a transmembrane region（a transmembrane protein）,and the subcellular location is mainly extracellular.The protein has a cystatin-like domain at 24-138 aa,a cathepsin propeptide inhibitor domain（I29）at 196-253 aa,and a papain family cysteine protease at 279-494 aa.There are 4 active sites（glutamine,cysteine,asparagine,histidine）.The Ts CL gene was successfully amplified using ML c DNA as a template.The recombinant plasmid p QE-80L/Ts CL was induced by IPTG and purified by nickel column.2.Transcription,expression and location of Ts CL in various stage of Trichinella spiralisThe RT-PCR results showed that Ts CL was transcribed in each worm stage.Western blot results showed that the anti-rTsCL immune serum could recognize the natural Ts CL protein in the soluble protein of each stage.IFA results showed that Ts CL was mainly located in the stichosome,muscle cells,midgut,epidermis and embryo of Trichinella spiralis.3.Ts CL promotes Trichinella invasion of IECWestern blot results showed that the anti-rTsCL immune serum recognized 10 bands of the IEC protein（89.5,59.1,55.7,52.0,44.6,41.9,38.4,35.2,32.3,29.9 k Da）,and the infected serum recognized 4 bands（89.5,52.0,44.6,35.2 k Da）,normal mouse serum cannot recognize IEC protein,indicating that rTsCL can specifically bind to IEC protein,which may be related to Trichinella invading IEC.The ELISA plate was coated with different concentrations of IEC protein and incubated with different doses of rTsCL.The results showed that rTsCL can bind to the IEC protein,and the OD value is correlated with the IEC protein concentration（r=0.9243,P<0.01）,and the concentration of rTsCL are also correlated（r=0.9059,P<0.01）.IFA results show that rTsCL can specifically bind to IEC,and the binding site is mainly in the cytoplasm.Different concentrations of rTsCL and IIL were incubated on the IEC for 2 hours and the invasion was observed.The results showed that the larval invasion rate of the rTsCL different doses（8,12,16μg/m L）group was statistically significant compared with the control group(χ²₈=4.102,χ²₁₂=8.596,χ²₁₆=14.286,P<0.01).The promotion rates of the three groups of rTsCL on the invasion of Trichinella spiralis were 21.37%,29.99%and 36.72%,respectively.BSA has no effect on the invasion of larvae.Different dilutions of anti-rTsCL immune serum and IIL larvae were incubated on the IEC for 2 hours and the invasion was observed.The results showed that the invasion rate of the larvae in the anti-rTsCL immune serum（1:50,1:100,1:200）group was statistically significant compared with the control group(χ²_1:50=14.566,χ²_1:100=8.012,χ²_1:200=5.207,P<0.03).The inhibition rates of the three groups of anti-rTsCL serum on the invasion of Trichinella spiralis were 40.84%,30.83%and25.72%,respectively.Muscle larvae incubated with infected serum and anti-rTsCL immune serum,the number of adult intestinal worms in the five days after oral infection of mice was significantly reduced,and the adult worms load of the two groups was reduced by 39.88%and25.82%,respectively.There was no statistically significant difference in the length and fecundity of each group adult worms（P>0.05）.4.The effect of silencing Ts CL gene on the invasion of IEC by Trichinella spiralisIntroduce ds RNA-Ts CL and GFP into muscle larvae by electroporation.PBS was used as the control.After three days of culture,there was no significant difference in the mortality of the three groups（P>0.05）.It shows that when the concentration of ds RNA-Ts CL is 60 ng/μl and interference time is 3 d,the effect of silencing the Ts CL gene is best.Real-time PCR results showed that the concentration of ds RNA-Ts CL was 60 ng/μl,and the transcription level of Ts CL gene was reduced by 26.46%（P<0.01）after 3 days of interference.After silencing the Ts CL gene,the natural Ts CL enzyme activity in the somatic proteins of Trichinella spiralis ML and IIL decreased by 34.13%and 22.07%,respectively（P<0.01）.In addition,after silencing the Ts CL gene,the larvae’s inhibition rate of IEC invasion was36.58%（F=22.614,P<0.001）.5.Effects of silencing Ts CL gene on larval development and adult fecundityThe number of recovered adults in ds RNA-Ts CL group was lower than that in PBS group,and the reduction rate was 31.79%（F=32.501,P<0.001）.The length of the adults was measured,and the length of the females became significantly shorter（F=16.939,P<0.001）,while the length of the males did not change significantly.After 72 h culture,the number of new larvae of ds RNA-Ts CL was lower than that of PBS（F=182.927,P<0.001）.Conclusion1.Recombinant Trichinella cathepsin L（rTsCL）was cloned and expressed.Ts CL is transcribed and expressed in all stages of Trichinella spiralis,and is mainly located in the stichosome,muscle cells,midgut,epidermis,and embryo of the worm.2.rTsCL can specifically bind to the IEC and promote the invasion of Trichinella spiralis into the IEC,and the anti-rTsCL immune serum can inhibit the invasion of Trichinella spiralis into the IEC.3.RNAi confirmed that Ts CL is involved in the invasion of the intestinal epithelium of Trichinella and the development of the worm in the intestine.