| The plerocercoid larva（sparganum）of Spirometra erinaceieuropaei,can parasitize in humans and animals causing a parasitic zoonosis known as sparganosis.Despite their significance in medical tapeworms,our knowledge about the molecular basis of S.erinaceieuropaei remains fragmentary.During the comparative analysis of genomes and transcriptomes,we found that several gene families related to stress resistance were specifically high expression in S.erinaceieuropaei,indicating that these gene families might be close related to the adaptive parasitism of S.erinaceieuropaei.However,we nearly know nothing about the protein motif structures,molecular features and evolution patterns of these gene families.Here,we selected two gene families:universal stress proteins（USPs）gene family and Glutathione S-transferases（GSTs）gene family for further analysis through comprehensive evaluation.Therefore,two objectives were addressed in this study.First,all members of the universal stress protein family of S.erinaceieuropaei（SeUSP）were screened and identified based on the WormBase ParaSite database and our transcriptomic data.Then protein motif structures,gene expressions and phylogenetic patterns of all identified SeUSP members were analyzed.Finally,sequence characteristics and phylogenetic reconstruction of USP sequences of all known medical tapeworms were carried out to explore the gene structure and evolutionary patterns of USPs in S.erinaceieuropaei.In the second part,all members of the Gluathione S-transferase family of S.erinaceieuropaei（SeGST）were screened,identified and analyzed.Then one member of SeGSTs was selected for further cloning,expression and protein function analysis.In the end,all GSTs of parasitic tapeworms were screened and the phylogenetic patterns of cestode GST family were explored to provided theoretical foundations for elucidating the molecular mechanism of SeGST.The achievements of this study will be helpful for deeply understanding the molecular characteristics of stress resistance related gene families of S.erinaceieuropaei,so that shedding light on revealing the mystery of adaptive parasitism mechanism of S.erinaceieuropaei.Materials and methods1.Parasite and experimental animalsThe plerocercoid larvae were initially collected from infected frogs and identified as S.erinaceieuropaei by molecular analysis based on the mitochondrial cytochrome c oxidase subunit 1（cox1）marker.The collected plerocercoids were orally administered to female specific pathogen-free（SPF）Kunming mice.And the plerocercoids were maintained by serial passage in mice every 10-12 months.Twenty female BALB/c mice（4～6-week-old）purchased from Henan Experimental Animal Center（Zhengzhou,China）were used to immunize by antigens of recombinant glutathione S-transferase to obtain the anti-rSeGST serum.An adult cestode representing S.erinaceieuropaei was obtained from an infected domestic cat.The collected immature proglottids and mature proglottids were used for the subsequent experiments.2.Screening and identification of SeUSP and SeGST familiesGenes encoding proteins that contain the universal stress protein domain in the S.erinaceieuropaei were searched using the NCBI Conserved Domains database,which obtained both from the WormBase ParaSite database and our transcriptomic data（GJLF00000000）.These extracted sequences were identified belong to universal stress protein family by querying for genes annotated with the Pfam domain accession PF00582.All of the identified candidates were analyzed using the HMMER tool to confirm the presence of UspA domains in their protein structure.For our transcriptomic data,the nucleotide sequences were translated into amino acid and corresponding coding DNA sequences using the NCBI’s ORF finder tool and using BLASTX for homology searches.In addition,these retrieved sequences have been corroborated by cloning and sequencing of S.erinaceieuropaei cDNAs.Sequence analysis of SeGST were carried out according to those of SeUSP.The query reference sequences annotated with the Pfam domain accessions PF02798,PF14497 and PF00043 for cytosolic GSTs,and PF01124 for MAPEG.3.Sequence analysis and quantitative RT-PCR analysisBasic physical and chemical properties were predicted using the ExPASy server and NCBI.The conserved protein motif analysis and gene features of SeUSPs were performed with MEME and GSDS,respectively.Multiple sequence alignments of SeUSPs were generated by DNAMAN software,using the protein sequence MJ0577 as template.The phylogenetic tree was inferred using maximum likelihood（ML）method.Secondary structure prediction was performed using PSIPRED server.The 3D structure of a selected SeUSP was determined using homology modeling available at the SwissModel server,the quality of the model was examined using Ramachandran plot analysis in PROCHECK.Visual analysis was carried out using PyMol software.The three-dimensional structure of a selected SeGST（AEI16476.1）was determined using homology modeling available at the SwissModel server,and visualized by the Swiss-PdbViewer.Quantitative RT-PCR（qRT-PCR）analysis was performed to monitor the expression levels of SeUSPs and SeGSTs in different life cycle stages:plerocercoid,as well as different segments of the adult（immature proglottide,mature proglottide and gravid proglottide）.Relative gene expression levels were analyzed whose internal control was GAPDH.4.The evolutionary patterns of USP and GST families in CestodeUSP sequences were retrieved from the WormBase ParaSite database using the key word "universal stress protein" based on the following criteria:synteny,the presence of a single exon in most sequences（or the conserved position of the intron where present）,and phylogenetic relationships between orthologs.For each species,each USP protein sequence against the entire genome were blasted applying an e-value threshold of 1e-1,and using opening and extending gap penalties of 14 and 2,respectively.In order to understand the origin and regulation of USPs,conserved motif was analyzed with the obtained sequences of all medical tapeworms.The expected maximization mixed model（MEME）was used for motif analysis with the default parameters,maximum width was set 50 amino acids and any number of motif repeats was allowed.Protein sequences were aligned with MAFFT v7.Phylogenetic analyses were performed using the Bayesian Inference（BI）and Maximum Likelihood（ML）probabilistic methods.Protein sequences were aligned with MAFFT v7 using the FFT-NS-I method,and any columns containing more than 95%gaps were deleted using Gap Strip/Squeeze.The best substitution model for our data set was defined with the Smart Model Selection（SMS）tool incorporated in PhyML.The ML tree was generated with PhyML using the aLRT-SH method for branch support.The BI tree was generated with BEAST v1.8.4,using two independent runs of 50.000.000 chains and sampling at every 5.000 generations.The software TRACER v1.6 was used to check the convergence of Monte Carlo Markov Chains（MCMC）.The maximum clade credibility tree was estimated with TreeAnnotator,and the tree was visualized using Figtree v1.4.3.The evolutionary pattern analysis of GST gene family is the same as that of USP family.5.Strains and plasmidsThe Escherichia coli DH5α and BL21 was used as strain.The pMD19-T（Takara）and pQE-80L（New England Biolabs）were used as cloning vector and expression vector,respectively.6.Cloning,expression and identification of SeGSTPCR amplification was carried out with the cDNA of plerocercoid as the template.GST cloned into pMD19-T vector and pQE-80L expression vector in turn after gel recovery.It was transferred into E.coli for prokaryotic expression by heat shock method.Twenty BALB/c mice were immunized with 20 μg of purified rSeGST in every two weeks,a total of 3 times.The titer of immune serum was determined by ELISA,and the antigenicity of rSeGST,ES and soluble protein was analyzed by Western blot.The fixed tissues were embedded and sliced,and the location of rSeGST was determined by IFA.7.Enzyme assays and inhibition studies of rSeGSTActivity was determined in potassium phosphate buffer with 1-chloro-2,4-dinitrobenzene（CDNB）and GSH as substrates.The initial rates were determined under conditions where one substrate was held constant while the other one was varied,and then enzyme activity was assayed immediately.The effect of temperature and pH on rSeGST was carried out by keeping the enzyme in different temperature gradients and different pH buffer gradients.In addition,the effects of Rose Bengal（RB）,TPT（Tripheniltin chloride）,BSP（Bromosulfophtalein）and CB（Cibacron Blue）inhibitors on rSeGST activity were also determined to compare their inhibitory effects.8.Analysis of the abiotic stresses in rSeGSTFor evaluating their degree of cold and heat tolerance,the samples of BL21/pQE-80L-rSeGST and BL21/pQE-80L were exposed to-20℃ for 24 h and 50℃ for 40 min respectively before being inoculated into an LB liquid medium（1:100 ratio）and cultured at 37℃ for 24 h.OD600 value was measured at 2-h intervals to produce a growth curve.The effects of hypertonicity and high salinity on the growth of recombinant bacteria were determined by liquid medium with 1 mol/L sorbitol and 0.8 mol/LNaCl respectively.Result1.Sequence characteristics of SeUSP familyTotal 17 SeUSP gene members that contain the UspA domain were identified based on the search criteria.The molecular weights of the predicted S.erinaceieuropaei USPs vary from 10.69 kDa to 24.77 kDa.A phylogenetic analysis using the S.erinaceieuropaei USPs revealed that these 17 SeUSPs can be arranged into four groups.Subfamily A has the most,i.e.,8 SeUSPs,while subfamily B,C and D have six,one and two members,respectively.The result of MEME tool motif analysis shows that all SeUSP members have the ATP-binding structure,and contain four α-helices and five β-chains that are closely related to ATP binding.Analysis of SeUSPs gene structures revealed that 15 members contain only single exon without introns.Whereas,SPER0000951901 contains two introns,and SPER0002066001 contains one intron,respectively.qPCR result showed that 15 genes were expressed in both plerocercoid and adult stages,12 genes were highly expressed in the plerocercoid stage,and 3 genes were highly expressed in the adult stage.2.The evolutionary pattern of cestode USP familyA total of 305 USP family members of tapeworms were identified based on the public database.Among them,the largest number is D.latus（44）,followed by H.diminuta（32）,M.corti（25）,H.nana（23）,S.solidus（23）,H.taeniaeformis（21）,H.microstoma（18）,S.erinaceieuropaei（17）and so on.Among all 305 sequences,121 members did not contain ATP motif（mainly in D.latus and M.Corti）and 184 members did.The 184 sequences are further divided into two categories:those with no change in ATP motif（conserved sequences）and those with variation in ATP motif（changed sequences）.The analysis of conserved sequences showed that all the sequences contained motif 2+motif 3+motif 1 except for 9 sequences.46 members were changed at the ATP-binding sites,such as inserted,deleted or altered.The USP gene of medical tapeworms can be divided into Clade Ⅰ,Clade Ⅱ and Clade Ⅲ,of which Clade Ⅰ is located at the base,including a few Hymenolepis.Clade Ⅱ only includes Dibothriocephalus,while Clade Ⅲ has more taxa and can be divided into three subgroups:sub-Cladel,sub-Clade2 and sub-Clade3.Sub-clade3 can be further divided into four groups:Group A,Group B,Group C and Group D.The 17 USP sequences of S.erinaceieuropaei were scattered in Group D,indicating that the USP family of S.erinaceieuropaei was differentiated late and highly diversified,which may be in the process of further differentiation.3.Sequence characteristics of SeGSTTotal 27 SeGST gene members were identified,whose molecular weights vary from 10.92 kDa to 30.80 kDa.Phylogenetic analysis showed that SeGST could be divided into four groups.MEME analysis showed that motif 1 contained N-terminal cap sequence（S/T）XXD.Motif 2 contains FPNLPY,which only exists in GST-mu.The analysis of exon/intron gene structure showed that 15 members contained introns（ranged from 1 to 6）.The 3D modeling results show that SeGST contains N-terminal and C-terminal structures,and the N-terminal domain mainly containsβ1α1β2α2β3β3α3,while the C-terminal domain is all of α helix.The results of qPCR showed that 9 genes were highly expressed in the stage of plerocercoid stage and 12 genes were highly expressed in the adult stage.4.Cloning,expression and identification of SeGSTAccording to the prediction results of online tool ExPASY,SeGST（GenBank No:16476.1）encodes 221 amino acids,with molecular weight of 25767.39 Ku,isoelectric point of 5.98 and total average hydrophilicity of-0.504,belonging to hydrophilic protein.No transmembrane region and signal peptide.The results of conservative domain prediction showed that the protein belonged to GST-mu.3D modeling results showed that the protein had typical N-terminal and C-terminal domains.That is,the N-terminal contains 3 α helixs and 4 β sheets,C-terminal consists of 6 α helixs.The prediction of phosphorylation sites showed that the protein could be phosphorylated in several positions.The molecular docking showed that two hydrogen bonds were formed between GST and ligand GSH and one hydrogen bond was formed with CDNB.The recombinant colony of BL21/pQE-80L-rSeGST was successfully obtained.Maximal production of functional recombinant GST was obtained when the expression plasmid was induced with 0.5 mM IPTG for 4 h at 37℃.The induced cells were sonicated and centrifuged,SDS-PAGE results showed that the recombinant protein existed in both supernatant and precipitation.Those expressed in supernatant was chosen for purified.And the results showed that the target band was single,indicating that the purification effect was good.The results of ELISA showed that the serum titer of 20 mice immunized could reach 1:104,indicating that the immune effect was good.The results of Western blot showed that the immune serum could recognize the recombinant protein rSeGST,and the ES antigen and soluble antigen of plerocercoid could be recognized by the serum of infected mice.According to the result of IFA,GST was located in subcutaneous and some parenchymal tissues in the plerocercoid stage,and fluorescent staining can be observed in epidermis,parenchymas,uterus and eggshells of adult worms.5.Enzyme assays and inhibition studies of SeGSTThe results of enzyme kinetic experiments showed that the binding of GST to substrate GSH and CDNB conforms to Michaelis equation,which means the reaction speed increases with the increase of substrate concentration in the initial stage of the reaction.When the substrate reaches a certain concentration,it reached the saturation point.The optimum temperature of enzyme activity was 25℃.The optimum pH of enzyme activity was 6.5.The corresponding IC50 values of TPT,CB,BSP and RB are 12.1,11.66,289.5 and 33.95 μM respectively.Among them,RB and CB are anti-competitive inhibitors,while BSP and TPT are non-competitive inhibitors.6.Analysis of the abiotic stressesIt reveals that no significant difference without any stress treatments,indicating that the expression of GST protein in E.coli did not affect the growth of bacteria.After treatment at-20 and 50℃,the growth state of the recombinant bacteria was better than that of non-recombinant bacteria;while,in the medium containing NaCl,the growth state of non-recombinant bacteria was significantly better than recombinant bacteria,after treatment with sorbitol,in the first 10 hours,the growth state of non-recombinant bacteria was slightly better than recombinant bacteria,but after 10 hours,the situation completely changed.Indicating that GST gene plays an important role in some abiotic stress conditions.7.Molecular evolution analysisA total of 192 GST sequences were obtained based on public databases.The results of conserved motif analysis showed that motif 9 only existed in MAPEG,and there are 15 members scattered in each tapeworm species contained the MAPEG domain.The GST gene of medical tapeworms can be divided into two major branches:Clade Ⅰ and Clade Ⅱ.Clade Ⅱ contains only a few sequences of Spirometra,Dibothriocephalus,Echinococcus,Taenia,Mesocestoides and Hymenolepis.The Clade Ⅰ group has a large number and can be further divided into two subgroups:Sub-cladel and Sub-clade2,of which Sub-clade2 consists of a monophyletic clade（Spirometra,Dibothriocephalus and Schistocephalus）.Sub-cladel includes three groups:Group A,Group B and Group C,in which Group A is dominated by tapeworms of Cyclophyllidea,and part of Pseudophyllidea is located at the base.Group B was highly diverse which included 8 Taenia sequences and Group C consisted of a few sequences from Taenia and Hymenolepis.The GST sequences of S.erinaceieuropaei were distributed in all clades,indicating that the SeGSTs had a high level of diversity and different degrees of differentiation,including conserved sequences and family sequences which differentiated later.Conclusions1.A total of 17 new members of SeUSP were screened and identified,and all SeUSPs contain conserved ATP-binding structures and secondary structures related to ATP binding;SeUSP genes were expressed in both plerocercoid and adult stages,and 12 genes were highly expressed in the plerocercoid stage.2.Mutations in conserved ATP motifs were detected in 46 USP memebers of all medical tapeworms;phylogenetic analysis showed that the SeUSPs were highly diversified and lately differentiation.3.A total of 27 new members of SeGST were identified;The 3D modeling showed that the N-terminal domain of SeGSTs mainly contains the β1α1β2α2β3β4α3 structure;qPCR analysis revealed that 9 genes were highly expressed in the plerocercoid stage,12 genes were highly expressed in the adult stage,suggesting that GST gene may be involved in the whole life cycle of S.erinaceieuropaei.4.The recombinant expression vector pQE-80L-rSeGST was successfully constructed;the expression of SeGST in egg,plerocercoid and adult stages were validated with the highest expression in the adult stage;IFA test revealed that SeGST exists in the epidermis and parenchyma of plerocercoid,and in the epidermis,parenchyma,uterus and egg shell of adult worm.The GSH showed high enzyme affinity to GST,and TPT displayed as a powerful enzyme inhibitor of GST.5.rSeGST contributes to the survival of host bacteria under abiotic stress conditions in low temperature,high temperature and hypertonic,indicating that GST gene plays an important role in the resistance of abiotic stresses of S.erinaceieuropaei.6.The phylogenetic analysis showed that the GST family of medical tapeworm showed a high level of diversity. |
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