The Biological Properties Of A Novel Pyruvate Kinase From Trichinella Spiralis And Its Participation In Sugar Metabolism,Larval Molting And Development

Trichinellosis is a common zoonotic parasitic disease caused by Trichinella spiralis in the host.It can be divided into 12 species,and Trichinella spiralis（T1）is the dominant species in China.Searching for proteins that play an important role in the growth and development of Trichinella spiralis is of great significance for the screening of drugs against trichinellosis.Pyruvate kinase widely exists in parasites and is the key enzyme in glycolysis pathway.ATP produced by glycolysis directly provides energy for the body.Pyruvate can participate in subsequent aerobic and anaerobic oxidation to produce more ATP.At the same time,it completes the conversion of sugar,fat and amino acids under the action of acetyl coA and citric acid cycle,It plays an important impact on the growth,development and reproduction of parasites.Pyruvate kinase may be a potential drug target for killing parasites.However,the biological characteristics and functions of pyruvate kinase in the life cycle of Trichinella spiralis have not been reported.The aim of this study was to evaluate the biological characteristics of TsPKM and its role in glucose metabolism,larval molting and development of Trichinella spiralis.From the gene sketch of Trichinella spiralis,TsPKM（KRY:30732.1）was cloned,expressed and purified.The transcription and expression of TsPKM in different stages of Trichinella spiralis were analyzed by RT-PCR and Western blot,and the localization of TsPKM in different stages of Trichinella spiralis was analyzed by indirect immunofluorescence assay.The enzyme activity conditions and kinetic parameters of rTsPKM were analyzed by 2,4-dinitrophenylhydrazine method.The role of TsPKM in glucose metabolism,growth and development of Trichinella spiralis was verified by RNA interference.Materials and methods1.Experimental animals,Trichinella spiralis species and cellsBALB/c mice required for the experiment are purchased from Henan animal experiment center;The species of Trichinella spiralis（ISS534）is T1 species;The IEC cells were isolated and cultured from the small intestine of fetal rats in the early stage by our research group.2.Strains and plasmidsThe cloning vector used for cloning and expression is pMD-19T,the expression vector is pQE-80L,and the strain is E coli BL21.3.Bioinformatics analysis and cloning and expression of TsPKMThe physical and chemical properties and biological characteristics of TsPKM were predicted through the online bioinformatics analysis website.Sequence alignment of pyruvate kinase from different species of Trichinella genus was carried out by Cluster Omega.Based on the neighbor-joining method,the evolutionary tree was constructed by Mega 7.0.Then TsPKM gene was amplified by PCR using the cDNA of ML as template,and the pQE-80L/TsPKM expression vector was constructed to induce the expression of rTsPKM.Mice were immunized with purified rTsPKM to prepare anti rTsPKM serum.4.Antigenicity analysis of rTsPKM and transcription,expression and localization of TsPKM in different T spiralis phasesThe antigenicity was analyzed by Western blot.The transcription and expression of TsPKM in different stages of Trichinella spiralis were analyzed by RT-PCR and Western blot,and the localization of TsPKM in different stages of Trichinella spiralis was analyzed by indirect immunofluorescence assay.5.Kinetic analysis of enzymatic reaction of rTsPKMThe optimum conditions of rTsPKM under different protein concentration,temperature and pH were detected by 2,4-dinitrophenylhydrazine method.Under the optimum enzyme activity conditions,the effects of metal ions,tannin,ethyl pyruvate and enzyme inhibitors commonly used in the laboratory on rTsPKM enzyme activity were analyzed.The enzyme kinetic parameters of rTsPKM were detected by different concentrations of substrates catalyzed by rTsPKM.6.RNA interference and tannin on the growth and development of Trichinella spiralisThe best inhibitor was selected by analyzing the effects of different concentrations of tannin and ethyl pyruvate on ML PKM activity after ML was treated for 2 hours.Three pairs of specific primers were designed by selecting 1-420,328-807 and 616-1062 bp of TsPKM DNA sequence as target sequences,and the best target sequence was screened by Real Time PCR and Western blot.Then the optimal interference concentration and interference time of the optimal target sequence were analyzed by Real Time PCR and Western blot.In vitro,ML was treated with RNA interference under the best conditions.The effects on ATP,total sugar,fat content and molting of ML were analyzed by ATP content detection kit,anthrone sulfuric acid method and acetylacetone method.The distribution of ML glycogen and lipid droplets between different groups treated with RNA interference were analyzed by PAS staining and oil red O staining;In vivo experiments,ML was treated by RNA interference under the best conditions,and then gavaged into mice to collect the residual 24 h IIL and 3 d AW in the intestine.The 24 h IIL and 3 d AW collected from each group were counted,the morphology was observed and the length of the worm was measured.The TsPKM activity of 24h IIL and 3 d AW was measured through soluble protein.The effects of anthrone sulfuric acid method and acetylacetone method on the content of total sugar and lipid in 3 d AW were analyzed.The distribution of glycogen and lipid droplets in 3 d AW treated with RNA interference and tannin were analyzed by PAS staining and oil red O staining.7.Statistical analysis of dataThe obtained experimental data was statistically analyzed by SPSS 21.0.The differences of TsPKM mRNA transcription level,protein expressionenzym,enzymatic activity,larval glycolipid content,larval load and length were analyzed by one-way ANOVA and t-test.Chi square test was used to analyze the difference of larval molting rate in different groups.The test level was P<0.05.Results1.Bioinformatics analysis of TsPKMTsPKM has a total length of 1629 bp and encodes 542 amino acids.The molecular weight is 58.48 kDa and pI is 7.16.The N-terminal has obvious hydrophilicity,no transmembrane region,no signal peptide,belonging to transferases,with 2 PK domains and secondary structures has 25 a spiral,32 β folding,18 β Corner,15 irregular curls.The tertiary structure is tetramer,and there are 8 enzyme active sites（Arg71,Asn73,Asp110,Phe241,Lys267,Glu269,Asp293,Thr325）.2.Cloning,expression and antigenicity analysis of TsPKMSDS-PAGE analysis showed that the largest amount of recombinant bacteria was induced at 25℃ and 0.2 mM IPTG for 23 hours,with a molecular weight of 58.48kda,and a single band could be purified.Solubility analysis showed that rTsPKM existed in both supernatant and inclusion bodies,but the content of rTsPKM was higher in supernatant.At 2 weeks after the final immunization,the anti rTsPKM serum IgG antibody titers was detected by ELISA.The results showed that after four immunization,the IgG titer of anti rTsPKM serum reached 1:105,indicating that rTsPKM had good antigenicity.Western blot analysis showed that rTsPKM was recognized by anti rTsPKM serum,infected serum and Hi s-tag monoclonal antibody（McAb）,but not by normal pre immune serum.3.Transcription,expression and localization of TsPKM in different T.spiralis phasesThe results of RT-PCR and Western blot showed that TsPKM gene was transcribed and expressed in different T.spiralis phases.Native TsPKM in somatic crude proteins of various worm phases（ML,IIL,3 d AW and NBL）was identified by anti-rTsPKM serum,and the native TsPKM in ES proteins of various phases（ML,IIL and 6 d AW）was also detected by anti-rTsPKM serum.Indirect immunofluorescence assay showed that TsPKM was mainly distributed in cuticle,muscle,stichosome,midgut and intrauterine embryos of the female adult worms.4.Enzymatic activity of rTsPKMThe enzymatic activity of rTsPKM gradually increased with increasing rTsPKM concentration,and stabilized at a concentration of 10 ng/μL.The optimum temperature of rTsPKM for catalyzing the substrate reaction is 37℃,and the optimum buffer pH is 8.0.Metal ions K⁺and Mg²⁺ enhanced the enzyme activity of rTsPKM,whereas Ni²⁺,Fe²⁺,Co²⁺,Ca²⁺,Mn²⁺,Zn²⁺and Cu²⁺inhibited the enzyme activity of rTsPKM.Tannin,ethyl pyruvate,EDTA and 1.10-Phe have obvious inhibitory effects on rTsPKM activity and tannin has the strongest inhibitory effect on rTsPKM activity.The inhibitory effect of EDTA and 1.10-Phe is due to the chelation of metal ions.The suppressive role of Tannin（r=0.933,P<0.001）and ethyl pyruvate（r=0.989,P<0.001）on rTsPKM activity is dose-dependent.The reaction conforms to the simple Michaelise-Menten kinetics.The kinetic parameter Vmax of PEP is 0.9469 mM/min and the Km value is 1.51 mM.The kinetic parameter Vmax of ADP is 1.84 mM/min and the Km value is 3.33 mM.5.Screening of tannin and ethyl pyruvate and analysis of RNA interference effectDifferent concentrations of tannin and ethyl pyruvate also inhibited TsPKM activity in a dose-dependent manner.Compared with ethyl pyruvate,tannin could inhibit TsPKM at a lower concentration.The best target sequence of RNA interference is target sequence 2,and the best interference concentration is 60 ng/μL.The best interference time is 3 days.After 60 ng/μL and one day,compared with PBS group,the relative expression of TsPKM protein in dsRNA-TsPKM2 group decreased by 22.49%（P<0.01）,and the relative expression of TsCRT protein did not change significantly（P>0.05）.The interference of dsRNA-TsPKM2 on TsPKM was specific.The results of enzyme activity test showed that compared with PBS group,Native TsPKM activity in ML decreased by 26.06%after RNA interference（P<0.001）.6.Effects of RNA interference and tannin incubation on ML metabolism and moltingCompared with no inhibitor group,the content of ATP,total sugar and lipid in ML in tannin group decreased by 43.99,22.71 and 54.88%respectively;Compared with PBS group,the content of ATP,total sugar and lipid in ML in dsRNA TsPKM2 group decreased by 20.29,19.86 and 33.02%respectively.Glycogen is mainly distributed in the rod body and around the intestine in ML.Small lipid droplets are distributed all over the body in ML,but there are large lipid droplets in the intestine and tail.After ML in different treatment groups was activated by 5%bile and cultured in semi-solid medium（DMEM+1.75%agarose）+IECs for 3 days,the molting rates of no inhibitor,tannin,PKM,GFP and PBS groups were 22.67,10.67,13.33,20.67 and 22.00%respectively.Compared with no inhibitor group,the molting rate of tannin group decreased by 12%（χ²=7.776,P<0.01）,compared with PBS group,the molting rate in PKM group decreased by 8.67%（χ²=3.873,P<0.05）。7.RNA interference and tannin in vivo to verify the effect of TsPKM on the growth and development of Trichinella spiralisCompared with no inhibitor group,the 24 h IIL and 3 d AW of tannin group recovered insect load decreased by 43.63%（P<0.001）and 44.51%（P<0.001）,the 24 h IIL,3 d AW female and male recovered insect length decreased by 17.14%（P<0.001）,9.30%（P<0.001）and 7.09%（P<0.001）,and the enzyme activity decreased by 24.10%（P<0.001）and 5.33%（P>0.05）respectively;Compared with PBS group,the 24 h IIL and 3 d AW of PKM group recovered insect load decreased by 27.69%（P<0.001）and 48.57%（P<0.001）,the 24 h IIL,3 d AW female and male recovered insect length decreased by 11.37%（P<0.001）,21.40%（P<0.001）and 16.75%（P<0.001）,enzyme activity by 14.76%（P<0.001）and 28.68%（P<0.001）respectively.For the recovered 3-d AW,glycogen was mainly distributed in the lateral line and around the embryo,and lipid droplets were mainly distributed around the female embryo,male testis and intestine.Compared with no inhibitor group,the contents of total sugar and lipid in tannin group basically had no change;Compared with PBS group,the total sugar content and lipid content in PKM group decreased by 14.59%（P<0.001）and 23.28%（P<0.001）.Conclusion1.The prokaryotic expression system pQE-80L/TsPKM/BL21 was successfully constructed,and a single rTsPKM was induced and purified;2.TsPKM was transcribed and expressed in different stages of Trichinella spiralis（ML,6 h IIL,3 d AW,NBL）,mainly in the epidermis and muscle cells of ML and 6 h IIL,around the intestine of ML and female embryos;3.2,4-dinitrophenylhydrazine detected that rTsPKM had enzyme activity,and the activity was the highest in the buffer with K⁺and Mg²⁺,at 37℃,pH=8;4.RNA interference and tannin can significantly inhibit TsPKM enzyme activity,reduce the level of ATP,total sugar and lipid in Trichinella spiralis.TsPKM is of great significance to the metabolism,growth and development of Trichinella spiralis.