Transcriptome Of Spirometra Erinaceieuropaei And Molecular Characterization Of Its Fatty Acid Binding Protein (FABP)

The plerocercoid larva of Spirometra can parasitize in human and animals and cause parasitic zoonosis,sparganosis.Little is known about the biological characteristicis and parasitic mechanism of Spirometra erinaceieuropaei.The developments in omics sequencing and analysis technics provide new methods to study the mechanisms of adaptive evolution of parasites.However,there is no transcriptome data for different life cycle stages of S.erinaceieuropaei,and it is not known which gene families play a key role in the adaptive evolution of the Spirometra.In this study,the following two objectives were addressed.First,transcriptomes of plerocercoid stage and adult stage of S.erinaceieuropaei were sequenced using RNA sequencing method（Illumina technology）.Next,the comparative transcriptomic analysis was performed between the two life cycle stages to identify differentially expressed genes（DEGs）,stage-specific genes and up-regulated genes.Finally,GO and KEGG enrichment analysis were performed for the DEGs to further explore their function.According to the results of comparative genomic and transcriptome analysis,the fatty acid binding protein（FABP）family of S.erinaceieuropaei was highly expressed.Therefore,in the second part,we analyzed the FABP of S.erinaceieuropaei in detail:first,the SeFABP gene was cloned and expressed to obtain recombinant rSeFABP,then RT-PCR,Real-time PCR,immunofluorescence technique（IFA）and Western Blot were used to analyze the molecular characteristics of rSeFABP,respectively.Then,the function of rSeFABP was identified by fatty acid binding assay.Finally,the phylogenetic pattern of FABP family sequences was analyzed.The achievement of this study not only provide high quality transcriptome data of S.erinaceieuropaei,but also lay the foundation for the study of the molecular characteristics and evolutionary model of fatty acid binding proteins.Materials and methods1.Parasites and experimental animalsPlerocercoid:the plerocercoid larvae were collected from infected frogs（Pelophylax nigromaculatus）from Zhengzhou city of Henan province in central China.The Kunming mice were infected though mouth with the scolex of plerocercoid.Then the plerocercoids were kept in the infected mice.Adult:a single plerocercoid from the same host was perorally administered to a 6-month-old specific pathogen free tabby cat.After 40 days from the infection,the cat was pre-anaesthetized then euthanized and the adult worm recovered from the small intestine.The 4%paraformaldehyde was used to fix the immature proglottids,the mature proglottids and the gravid proglottids after washing 3 times with normal saline.Then a few amount of TRIzol was added for the cryopreservation of immature proglottids and mature proglottids.And the freeing immature proglottids and mature proglottids were used for the subsequent experiments such as RNA extraction.The adult was identified as S.erinaceieuropaei according to the morphological characters and molecular identification.Experiment and conservation mice:Female BALB/c mice（4～6-week-old）and Kunming mice were collected from Henan Experimental Animal Center.2.Strains and plasmidsThe Escherichia coli BL21 frozen for laboratory was used as strain.The pMD19-T（Takara）and pMAL-c2X（New England Biolabs）were used as cloning vector and expression vector respectively.3.Illumina sequencingTotal RNA was isolated each from pooled plerocercoids or pooled adults of S.erinaceieuropaei respectively,using TRIzol Reagent and RNeasy Mini Kit,following the manufacturer’s protocol.All RNA samples were treated with DNase prior to m RNA isolation and sequencing.Total RNA of each sample was qualified by Agilent2100 Bioanalyzer.The RNA concentration and purity were assessed by Nano Drop.RNA integrity was determined by electrophoresis on a 1%agrose gel.Each sample was biologically repeated three times.Next generation sequencing library preparations were constructed according to the Illumina’s recommendations.Oligo（d T）was used for purifying poly（A）-containing m RNA from total RNA.The purified m RNA was fragmented into short fragments.Then cDNAs were synthesized using the m RNA fragments as templates.The short fragments were connected with adapters in both ends.The products were purified and enriched using PCR to create the final indexed double-stranded cDNA library.The libraries was then qualified by Agilent2100 Bioanalyzer,and quantified by Qubit 2.0 Fluorometer.Subsequently,the libraries were sequenced by paired-end sequencing in an Illumina Hi Seq 2000sequencer at the Genwiz Company.4.Assembly of the transcriptome and functional annotationThe raw reads sequences were filtered by the software Cutadapt v.1.9.After that,high quality clean data was obtained.Transcriptome de novo assembly is carried out by the program Trinity.The open reading frame（ORF）in each transcript was predicted using Trans Decoder.Quantification of transcription was analyzed using RSEM.Unigene sequences were annotated using BLASTp and BLASTx and inferred for amino acid sequence similarity to molecules submitted to the protein databases Nr（NCBI non-redundant protein database）,Swiss-Prot,GO（Gene Onotology）,KEGG（Kyot encyclopedia of genes and genomes）and COG（Cluster of Orthologous Groups of proteins）.In addition,simple sequence repeats（SSRs）in the nucleotide sequences were identified using the MISA-web tool.The poly-A and poly-T sequences at the terminal regions of the UTs were removed before SSR identification.5.Differentially expressed genes（DEG）analysis and qRT-PCRThe FPKM method（Fragments per kb per Million reads）was used to compare the levels of differentially expressed genes（DEG）between plerocercoid larvae and adult worms.Furthermore,DEGs were subjected to GO functional analysis and KEGG pathway analysis.The enrichment analysis of DEGs was conducted with the GO database,and the gene number for each GO term was calculated.The main pathways of biochemical and signal transduction significantly associated with DEGs were determined via KEGG pathway analysis.DEGs in each plerocercoid stage（5genes）and adult stage（5 genes）were measured using quantitative real-time PCR analysis to confirm the transcription levels of genes identified by RNA-seq.6.The bioinformatic analysis of SeFABPThe online software ExPASy-Port Param was used to analyze the isoelectric point,molecular weight,amino acid composition and other physical and chemical properties of SeFABP.The hydrophilic/hydrophobic of SeFABP were predicted by Kyte&Doolittle method of the online website ExPASy-Port Scale.TMHMM Serever was used to analyze the transmembrane region of fatty acid binding proteins.The signal peptide was predicted by the Signal P server platform.Targetp 1.1 Server was used for subcellular localization analysis.Protein secondary structure was predicted by software PSIPRED,the tertiary structure of FABP was analyzed in Phyre2 online software.7.Cloning and characterization of SeFABPUsing cDNA of plerocercoid as template,the SeFABP gene was amplified by RT-PCR.SeFABP was attached to the clone vector pMD19-T after purification by gel extraction.The validation of SeFABP was conformed by sequencing.Then SeFABP sequence was ligated into the expression vector pMAL-c2X.Next,colony PCR,plasmid PCR and sequencing verification were performed sequentially.After that,the induction temperature,IPTG concentration and induction time were optimized.The recombinant protein of rSeFABP was induced and expressed under the optimal conditions.Then it was purified by affinity chromatography using the amylose resin column.Twenty BALB/c mice aged 4 to 6 weeks were immunized with purified rSeFABP protein to prepare anti-rSeFABP immune serum.The quality of immune serum was determined by ELISA and Western Blot.RNA from egg stage,plerocercoid stage and adult stage were extracted by TRIzol method.Then the RNA was reverse transcriptedi into cDNA.RT-PCR and Real-time PCR were performed to detect the transcription level of SeFABP gene in different life cycles of S.erinaceieuropaei.The fresh body of plerocercoid and adult were collected to make paraffin sections.Using anti-rSeFABP immune serum,immunofluorescence analysis was performed to determine the expression and localization of SeFABP in different life cycles of S.erinaceieuropaei.8.Fatty acid-binding assayThe binding property of recombinant protein rSeFABP was tested using palmitic acid（C16:0）and oleic acid（C18:1）.2μg of purified recombinant SeFABP protein were preincubated for 30 min at room temperature with each fatty acid at a ratio of1:4 in buffer containing 50 m M Tris–HCl,50 m M DTT,2 m M EDTA and 5 m M Ca Cl₂.Then,1μg/ml of clostripain（Arg C）was added to each reaction and incubated further at 37°C.The reactions were collected at 1h and 15 h after incubation.Then,the reactions were subjected to analysis by SDS-PAGE.Bovine Serum Albumin（BSA）was added as a positive control.9.Phylogenetic analysisFABP sequences from Cestode,Trematode and Nematode were selected for performing phylogenetic analysis.These sequences were firstly retrieved from theWorm Base Parasite database,then checked through TBLASTN method.The alignment of FABPs was performed in CLUSTALW and manually edited in MEGA software.The phylogenetic pattern of all FABPs was estimated through maximum parsimony（MP）and Bayesian inference（BI）.The MP analysis was performed in MEGA.BI was performed in Mr Bayes.Result1.Transcriptome assembly and annotationIllumina high-through-put sequencing produced 43,544,371（6,531,655,700bases）and 42,579,785（6,386,967,800 bases）raw reads for plerocercoids and adults,respectively.After quality filtration,43,151,508（6,377,133,505 bases,plerocercoid）and 42,274,079（6,264,471,514 bases,adult）high quality clean reads were obtained for assembly.Based on the pooled clean reads,a total of 241,564 unigenes were produced from all samples（plerocercoids and adults）after removing short unigenes（<200 nt）,with an average length of 573 nt and N50 of 794 nt.A total of 38,647unigenes（16%）were longer than 500-1000 nt,and 12,695 unigenes（5.26%）were longer than 1000-1500 nt.Of the 241,564 unigenes,67,412（27.91%）were annotated by BLAST searches against the public databases,and the majority（72.09%）unigenes were found to be non-homologous sequences.These 67,412 protein-encoding transcripts were retained for further analysis.Of these 67,412 unigenes,67,057 were annotated by Nr（99.47%）,20,753 by COG（30.79%）,18,540 by Swiss Prot（27.50%）and 1,897 by KEGG（2.81%）,respectively.For the GO analysis,67,412 unigenes were assigned to 61 functional groups of three GO categories:molecular function（MF）,cellular component（CC）and biological process（BP）.All assembled unigenes were further classified into 25 functional categories based on COG category.Among these categories,T（signal transduction mechanisms）included 6.35%（4,284/67,412）unigenes,followed by R（general function prediction only）and O（post-translational modification,protein turnover,chaperones）.Based on the KEGG database,a total of8,585（12.74%）uningenes were classified into six categories that mapped to 326KEGG pathways.2.Comparative transcriptome analysisIn the plerocercoid stage,highly transcribed genes included cytoplasmic antigen genes,antigenic polypeptide,peptidase inhibitor,kunitz-type serine protease inhibitor,cysteine protease,cysteine protease inhibitor,ADP-ribosyl cyclase,phosphoenolpyruvate carboxykinase,glutathione S-transferase,aspartate aminotransferase,senescence associated protein,dynein and heat shock proteins.In the adult stage,highly transcribed genes included transcription factor protein,fatty acid binding protein,hydrophobic ligand binding protein,Na（+）/H（+）exchange regulatory cofactor,peptidyl-prolyl cis-trans isomerase,hydatid disease diagnostic antigen,glyceraldehyde-3-phosphate dehydrogenase,leucyl aminopeptidase,malate dehydrogenase,ornithine aminotransferase,calreticulin,gelsolin,thioredoxin,titin,annexin,translation elongation factor and tetraspanin.Several cytoskeletal housekeeping genes（action,tubulin and ribosomal proteins）,metabolic enzymes（pyrophosphatase/phosphodiesterase,Co A,cytochrome c oxidase）,and signaling proteins（polyubiquitin,Calcium-binding protein）were abundant transcripts in both stages.A total of 25,120 distinct genes were found differentially expressed between plerocercoid and adult,of which 12,349 genes were up-regulated and 12,771 genes were down-regulated in plerocercoid versus adult,respectively.Among the DEGs,4,512 unigenes were identified to be expressed specifically in plerocercoid and 4,339unigenes were identified to be expressed specifically in adult.The GO and KEGG analysis revealed that the molecular functions of upregulated genes in plerocercoid were mainly enriched for protein degradation and amino acid metabolism,while DEGs enriched in the adult stage were associated with metabolic activity.Using GAPDH as a reference gene,expression levels determined by qRT-PCR were consistent with those obtained by RNA-seq,confirming the accuracy and reliability of the RNA-seq results.3.The sequence analysis of SeFABPThe SeFABP gene（Gen Bank:JQ919795）encodes 130 amino acids with a relative molecular weight of 14.5 k Da and a p I of 5.88.The instability index of SeFABP is 13.24,indicating that it is a stable protein.The aliphatic coefficient was88.46.The total average hydrophilicity was-0.283.SeFABP has no signal peptide,does not contain transmembrane region,N terminal has hydrophobic region.Second-order structure prediction showed that SeFABP have 2αhelices,10βfolds and 13 random coils.Three-stage modeling results show that theβfolds into a"bucket"structure,which can bind different hydrophobic ligands through van der Waals forces and hydrophobic interactions.Twoαhelices at the nitrogen end form a"hat"structure,which controls the flow of hydrophobic ligands in and out.4.The molecular identification of SeFABPThe SeFABP gene was transferred into the clone vector pMD19-T for expression.Sequencing results showed that the sequence similarity between SeFABP gene and Gen Bank SeFABP gene（JQ919795）was 99%.The recombinant plasmid pMD19-T-FABP was successfully constructed.After double digestion,SeFABP gene was linked to the expression vector pMAL-c2X to construct the recombinant expression plasmid,which was verified by double digestion and sequencing.After optimization of induction conditions,1m M IPTG was added,induced at 33℃for 6 h,and the bacteria were collected for SDS-PAGE.Compared with the uninduced sample,an obvious protein band appeared at the molecular weight of 56.5k Da（MBP label）,which was consistent with the theoretical prediction,indicating that the target protein rSeFABP was successfully induced and expressed.Solubility analysis showed that most of rSeFABP was expressed in the supernatant.Anti-rSeFABP immune sera were prepared by immunizing mice with purified rSeFABP.The titers of anti-sera were all over 10⁵detected by indirect ELISA.Western Blot analysis showed that anti-rSeFABP mouse sera could specifically recognize the recombinant protein.RT-PCR and Real-time PCR results showed that SeFABP was transcripted in egg,plerocercoid and adult stages of S.erinaceieuropaei.And the difference of transcriptional level in different insect stages was statistically significant.The transcriptional level of the adult stage was the highest,followed by the egg stage and the lowest at the plerocercoid stage.The results of immunofluorescence assay showed that there was green fluorescence in the scolex of pleroceroid,the cross section of the body,the vertical section of the body,the immature proglottids,the mature proglottids,the gravid proglottids and the eggs in the uterine of the gravid proglottids.The results showed that SeFABP was expressed in pleroceroid,adult worms and eggs,and was mainly localized in egg shell,tegument,parenchymal tissue and adult uterus of S.erinaceieuropaei.5.Assay of rSeFABP binding with fatty acidsWhen BSA in the positive control group was not bound to fatty acids,it was significantly degraded after incubation at 37℃for 15h with or without the addition of Arg C enzyme.While BSA combined with fatty acid showed weak degradation after incubation at 37℃for 15h with or without the addition of Arg C enzyme.It is suggested that BSA binds to fatty acids to form a relatively stable form,but the stability of the conformational change after such binding is limited.Before and after the combination of the recombinant protein and fatty acid,there was no significant change in whether Argc enzyme was added.It may be caused by misfolding ofprokaryotic expression.6.Phylogenetic patternThe topologies of the phylogenetic trees obtained by the maximal parsimony and the Bayesian method were identical.In general,the FABP gene of all helminths can be divided into two large clades:Clade I and Clade II.Clade I was composed with two sub-clades:Clade Cestode and Clade Trematode.Clade II was also composed with two sub-clades.One of the sub-clades is composed with Nematode FABP（Clade Nematode）.The other sub-clades contained nematodes,flukes,and cestode groups,which named Clade Complex.Phylogenetic analysis showed that most members of the fatty acid binding protein family were conserved and species-specific,but some of them were still in the process of differentiation.For Spirometra erinaceieuropaei,4 of the 13 fatty acid binding protein sequences were inserted into the Clade Complex.Although the other nine inserted in the cestode branch,they didn’t group together,instead of inserting into 4 different branches.These results indicated that the FABP sequence of Spirometra erinaceieuropaei was highly diversified,and there might be several different family members,and each family member was in the different evolutionary process.Conclusion1.Several protease-related genes were highly expressed in plerocercoid,while lipid-related genes and cystoskeleton proteins were highly expressed in adult.A total of 25,120 distinct genes were found differentially expressed between plerocercoid and adult,of which 12,349 genes were up-regulated and 12,771 genes were down-regulated in plerocercoid versus adult.Among the DEGs,4,512unigenes were identified to be expressed specifically in 274 plerocercoid and 4,339unigenes were identified to be expressed specifically in adult.2.The GO and KEGG analysis revealed that the molecular functions of upregulated genes in plerocercoid were mainly enriched for protein degradation and amino acid metabolism,while DEGs enriched in the adult stage were associated with metabolic activity.3 The recombinant expression plasmid pMAL-c2X/SeFABP/BL21 was constructed by prokaryotic expression system and the rSeFABP was expressed and purified.Fatty acid binding protein of S.erinaceieuropaei（SeFABP）is expressed in egg,pleroceroid and adult stages,and it is mainly located in the egg shell,tegument,parenchymal tissue and uterus.4.High diversification of FABPs was found in S.erinaceieuropaei,and multi-family members of FABP might be existed in S.erinaceieuropaei.