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Transcriptome sequencing and analysis of the zoonotic parasite Spirometra erinacei spargana (plerocercoids)
- Dae-Won Kim†1,
- Won Gi Yoo†1,
- Myoung-Ro Lee†1,
- Hye-Won Yang2,
- Yu-Jung Kim1,
- Shin-Hyeong Cho1,
- Won-Ja Lee1Email author and
- Jung-Won Ju1Email author
© Kim et al.; licensee BioMed Central Ltd. 2014
Received: 12 March 2014
Accepted: 22 July 2014
Published: 15 August 2014
Although spargana, which are the plerocercoids of Spirometra erinacei, are of biological and clinical importance, expressed sequence tags (ESTs) from this parasite have not been explored. To understand molecular and biological features of this parasite, sparganum ESTs were examined by large-scale EST sequencing and multiple bioinformatics tools.
Total RNA was isolated from spargana and then ESTs were generated, assembled and sequenced. Many biological aspects of spargana were investigated using multi-step bioinformatics tools.
A total of 5,634 ESTs were collected from spargana. After clustering and assembly, the functions of 1,794 Sparganum Assembled ESTs (SpAEs) including 934 contigs and 860 singletons were analyzed. A total of 1,351 (75%) SpAEs were annotated using a hybrid of BLASTX and InterProScan. Of these genes, 1,041 (58%) SpAEs had high similarity to tapeworms. In the context of the biology of sparganum, our analyses reveal: (i) a highly expressed fibronectin 1, a ubiquitous and abundant glycoprotein; (ii) up-regulation of enzymes related with glycolysis pathway; (iii) most frequent domains of protein kinase and RNA recognition motif domain; (iv) a set of helminth-parasitic and spargana-specific genes that may offer a number of antigen candidates.
Our transcriptomic analysis of S. erinacei spargana demonstrates biological aspects of a parasite that invades and travels through subcutaneous tissue in intermediate hosts. Future studies should include comparative analyses using combinations of transcriptome and proteome data collected from the entire life cycle of S. erinacei.
Spargana, the plerocercoid form of Spirometra erinacei, are the larvae of intestinal tapeworms of the order Diphyllobothriidea in the class Cestoda. Sparganosis has been reported in many countries, including the United States and Europe. Human sparganosis occasionally occurs by ingestion of water contaminated with Copepods that have been infected with procercoids or by invasion of plerocercoids from hosts such as frogs and snakes.
The ingested sparganum has the ability to invade various organs, which include eyes, subcutaneous tissues, abdominal walls, brains, spinal cords, lungs, and breasts, among others[3–5]. Human sparganosis can cause diverse symptoms, such as non-specific irritation, uncertain pain, apparent masses, and headaches. Although radiologic examinations have been introduced, using techniques such as ultrasonography, CT, and MR, it is difficult to confirm a correct diagnosis. Because expensive equipment and experts are necessary, this approach is not appropriate as a practical method for field diagnosis. Furthermore, sparganosis cannot even be deciphered by autopsy because of restrictions, which include many latent infections, unexpected locations of the worm in the body and a low predicted infection rate.
Serodiagnostic tests using sparganum antigen proteins could be good alternative techniques for diagnosing sparganosis. These tests include enzyme-linked immunosorbent assays (ELISA) and immunoblotting. Several antigenic proteases are reportedly present in spargana, including 31/36 kDa excretory-secretory (ES) proteins, a 27 kDa cathepsin S-like protease, and a 53 kDa neutral protease. ES proteins in crude extracts have been shown to be highly specific and sensitive in sera from patients with sparganosis. However, preparation of sufficient amounts of ES proteins is labor-intensive and time-consuming. Therefore, recombinant antigens were employed to overcome the disadvantages of ES protein preparation. Recently, multiple antigen mixtures using combinations of these antigenic proteins have been recommended because an absolute antigen with high sensitivity and specificity does not yet exist.
As mentioned above, the first definitive treatment is surgical resection of the worm from the infected tissues. The second choice for treating sparganosis is two drugs, praziquantel or mebendazole, which are also recommended for treatment of trematode or nematode infections, respectively[14, 15]. Although these drugs are currently orally administrated for treatment, low cure rates and high recurrence rates have already been observed[16, 17]. Because novel therapeutic targets against sparganosis are not studied, with the exception of these drugs, development of anti-helminthics should be actively encouraged.
Large-scale sequencing data can be applied to gene-based discovery of drug targets and diagnostic antigens. Recently, genomes or transcriptomes from other cestode parasites have been sequenced and functionally analyzed, including data from Taenia solium[19–21], Echinococcus multilocularis, E. granulosus[21, 22] and Hymenolepis microstoma. This genetic information has been applied to understanding a number of metabolic mechanisms used for parasite growth and during host-parasite interactions. Furthermore, monitoring fluctuations in gene expression is indispensable for finding drug targets, predicting secretory proteins, and elucidating evolutionary relationships[18, 21, 23]. Currently, however, knowledge regarding the genome or transcriptome of various developmental stages in S. erinacei is restricted to adult worms.
In this study, a major expressed sequence tags (ESTs) sequencing project on S. erinacei spargana was carried out to improve a basic genetic resource. This transcriptome profile is presented with the abundant transcripts, frequently occurring functional domains and antigen candidates.
Spargana of S. erinacei were collected from naturally infected Rhabdophis tigrinus snakes in Gyeong-sangnam-do province, South Korea. All worms were washed with physiological saline several times and either used directly for RNA preparation or stored at -70°C until use.
RNA isolation and cDNA library construction
After separating the mycelia from S. erinacei spargana, the worms were submerged in liquid nitrogen in pre-chilled grinding jars and a grinding ball on a bed of dry ice. Spargana in pre-chilled grinding jars were pulverized using a Mixer Mill MM301 (Retsch GmbH, Germany). Spargana were transferred to 15 ml polypropylene tubes filled with liquid nitrogen and stored at -80°C. Total RNA was extracted from the fragmented frozen tissues using TRI reagent (MRCgene, OH, USA). Total RNA was purified (100 μg) using the absolutely mRNA Purification Kit (Stratagene, CA, USA) according to the manufacturer’s instructions. To construct the cDNA library, a directional λ ZAP cDNA synthesis/Gigapack III gold cloning kit (Stratagene, CA, USA) was used. Reverse transcription of mRNA for first stand cDNA synthesis was primed from the poly-A tail using an oligo-dT linker primer containing an Xho I cloning site. Following second strand synthesis, EcoR I linkers were ligated to the 5′-termini. Xho I digestion released the Eco RI adapter and residual linker primer from the 3′ end of the cDNA. These two fragments were separated on a drip column containing Sepharose® CL-2B gel filtration medium. The fractionated cDNA (above 500 bp) was then precipitated and ligated into the ZAP Express vector (pBK-CMV). The primary library was produced by in vitro packaging of the ligation product with a ZAP Express cDNA Gigapack III Gold cloning Kit.
cDNA clones were plated onto LB-kanamycin plates (Rectangle, 23.5 cm × 23.5 cm) with X-gal and IPTG for blue/white selection. White colonies were randomly and manually picked, inoculated into 15 384-well plates (Corning, NY, USA) containing 40 μl TB/kanamycin and incubated for 16 h at 37°C with fixation culture. Sequences of the cDNA inserts were determined from the 5′ end of clones using the BigDye Terminator Sequencing Kit ver. 3.1 (Applied Biosystems, Foster City, CA, USA) and a 3730XL DNA analyzer (Applied Biosystems).
EST cleaning and clustering
Homology search and functional annotation
To assign putative functions to S. erinacei ESTs, we took into account the BLASTX best hit descriptions and subsequent alignments with E-value cutoffs below 1e-10 and compared them to the non-redundant (NR) protein database at NCBI. Because a large portion of these ESTs have not yet been annotated, we further characterized domains/families of the SpAEs using InterPro database version 27 (HMMPfam, HMMSmart, HMMTigr, HMMPanther and SuperFamily; flagged as true by InterProScan with E-value ≤ 1e-4). We also classified our SpAEs with Gene Ontology (GO) terms at the protein level using BLAST2GO (cut-off E-value ≤ 1e-10). These GO terms were further mapped and classified at the third level to two GO categories: ‘molecular function,’ and ‘biological process.’ Because some predicted proteins were assigned to more than one GO term, the percentages of each category add up to one hundred percent. SpAEs also were mapped to the Enzyme Commission (EC) database via BLAST2GO.
Comparative transcriptome analysis
Gene sequences of spargana were globally compared to those of other species using TBLASTX (E-value 1e-5) and the results were displayed using the SimiTri program (BLAST score cut-off score: 50). Sequences of the comparator species were downloaded from the GenBank databases.
From the ORFs inferred from SpAEs, secreted proteins were predicted using a combination of four programs (ORFpredictor, SignalP, TMHMM and YLoc) to minimize the number of false positive predictions. Firstly, we identified protein-coding regions of ORFs in SpAEs by starting exactly at the initiation codon encoding the amino acid methionine (Met) with ORFpredictor. Secondly, SignalP 3.0 was used to predict the presence of secretory signal peptides and signal anchors for each predicted SpAE protein, using both neural networks and Hidden Markov models with default option. To exclude erroneous predictions of putative transmembrane (TM) sequences as signal sequences, TMHMM, a membrane topology prediction program, was applied. We further validated the list of secreted proteins with extracellular localization using YLoc.
Results and discussion
Overview of sparganum EST analysis
Transcriptome features of S. erinacei spargana
Total sequence reads
Total analyzed reads (average size)
5,634 (687 bp)
Total number of assembled sequences (average size)
1,794 (715 bp)
Total annotated genes (BLASTX or InterProScan)
Functional annotation of SpAEs
To identify likely S. erinacei sparganum genes through sequence similarity, BLASTX analyses and InterProScan domain searches were performed on all SpAEs against the NCBI NR protein databases and the InterProScan database (Figure 1B). The two alignment algorithms were used to annotate 1,351 SpAEs (75%), and most matches were to tapeworms, such as E. granulosus and H. microstoma (Additional file2: Figure S1). After removing redundant protein hits, 1,335 unique reference proteins were identified within public databases. Among them, 1,268 (95%) of the annotated SpAEs had E-values of ≤ 1e-10 (Additional file1: Table S1). In our study, 443 SpAEs (30%) did not share sequence similarity with any other predicted or known molecules in public databases. These SpAEs potentially represent novel genes with unknown functions in S. erinacei spargana.
Biological process and molecular function GO terms with the 15 highest scores
Cellular macromolecule metabolic process
Cellular protein metabolic process
Cellular macromolecule biosynthetic process
Macromolecule metabolic process
Protein metabolic process
Regulation of cellular process
Cellular biosynthetic process
Macromolecule biosynthetic process
Purine ribonucleoside binding
Purine ribonucleoside triphosphate binding
Nucleoside phosphate binding
Nucleic acid binding
Adenyl ribonucleotide binding
Metal ion binding
Purine nucleoside binding
Purine ribonucleotide binding
Adenyl nucleotide binding
Highly abundant genes
The most abundant transcripts in S. erinacei spargana
No. of reads
PREDICTED: fibronectin isoform X1
Cytoplasmic antigen containing repeat epitope, partial
Elongation factor 1 alpha
AF418991_1 cytoplasmic antigen 4
ATP-dependent RNA helicase UAP56/SUB2
40S ribosomal protein S24
Programmed cell death protein 4
Tubulin beta-2C chain
Nervous system adducin
14332_ECHGR RecName: Full = 14-3-3 protein homolog 2
Synaptic vesicle membrane protein VAT 1
Plerocercoid growth factor/cysteine protease
Heat shock protein 90 alpha
Heat shock 70 kDa protein 4
gtp binding protein 2
40s ribosomal protein s15
Ubiquitin conjugating enzyme E2 G1
Excitatory amino acid transporter 3
Fructose 16 bisphosphate aldolase
Signal peptidase complex subunit 3
A parasite should adapt to a variety of biological stresses in the host environment, including thermal shock, oxidative stress and other forms of stress. Hence, proteins that allow spargana to survive stresses are important components for infection establishment. We found stress response-related proteins, such as HSP70, HSP40, HSP90, HSP71, HSP105, HSP60 and HSPA8. HSPs are highly conserved and abundant proteins in many parasitic organisms[21, 41, 42] and are essential for cellular viability and activity under both normal and stress conditions. The top 3 most abundant genes are HSP70 (55 reads), HSP40 (47 reads) and HSP90 (24 reads). It has been previously observed that HSP70 and HSP80 in T. solium cysticerci were highly induced under temperature stress. Recently, expansion of HSP70 was described in tapeworms and points out the importance of such proteins for the parasite life cycle. HSP40 gets involved in the prevention of protein aggregation and the regulation of protein refolding for parasitic development. HSP90 functions downstream of the HSP70/HSP40-chaperone system and serves as an important determinant in regulating protein conformation and cell signal transduction.
The 25 most frequent Pfam domains in S. erinacei spargana
Protein domain family
No. of SpAEs
Protein kinase domain
RNA recognition motif domain
EF-hand domain pair
Small GTPase superfamily
Heat shock protein 70 family
Kelch repeat type 1
Fibronectin, type III
Calponin homology domain
Ubiquitin-conjugating enzyme, E2
Zinc finger, C2H2
Leucine rich repeat 4
Collagen triple helix repeat
Dynein light chain, type 1/2
Aminotransferase, class V/Cysteine desulfurase
K Homology domain, type 1
The 10 most abundant enzymes in S. erinacei spargana
No. of reads
No. of SpAEs
ATP dependent rna helicase ddx1
EPA018LGAA12C000070, EPA018LGAA12C000238, EPA018LGAA12C000503, EPA018LGAA12C000561, EPA018LGAA12C000680, EPA018LGAA12S005500
Heat shock protein 90 alpha
EPA018LGAA12C000009, EPA018LGAA12C000086, EPA018LGAA12C000157, EPA018LGAA12C000209, EPA018LGAA12C000367, EPA018LGAA12C000500, EPA018LGAA12C000591, EPA018LGAA12S002094, EPA018LGAA12S004373, EPA018LGAA12S005358
Phosphoserine aminotransferase 1
2 amino 3 ketobutyrate coenzyme a ligase
Diagnostic candidate genes based on secretome analysis
Putative secretory proteins predicted by ORFpredictor, SignalP, TMHMM and YLoc
No. of reads
PREDICTED: ADP-ribosyl cyclase-like
DNA-binding protein HEXBP
T-cell immunomodulatory protein
Collagen alpha-1(III) chain
Elongation factor 1 alpha
Hypothetical protein EgrG_001045000
Collagen alpha 2(I) chain
PREDICTED: transforming growth factor-beta-induced protein ig-h3
Hypothetical protein EgrG_001037900
Collagen alpha(iv) chain
Cysteine-rich with egf-like domains protein
Leucine rich repeat typical subtype
Heat shock protein DnaJ N terminal
Type II collagen B
TPA: endonuclease-reverse transcriptase
AF523312_1 oncosphere-specific antigen
PREDICTED: c-C motif chemokine 4-like
Transcriptome-wide comparison and parasitism
We identified 28 SpAEs, which were predicted to be helminth-parasitic genes in the intersection between cestode-parasitic genes (a) and trematode-parasitic genes (b) in the Figure 3 (Additional file3: Table S2). These proteins in parasitic helminth were absent from the corresponding molecules in the free-living S. mediterranea (Turbellaria, outside of Neodermata). Of these, 9 showed sequence similarity neither to a gene/protein of known function nor to an identifiable protein domain. Due to the presence of these gene products only within parasitic helminths, and although their full characterization is needed, they may be good candidates for the development of potentially novel parasitic helminth drug targets. From the BLAST analyses, 537 SpAEs did not have any homologs in the analyzed species (Additional file4: Table S3). These gene products can be explored as potential species-specific antigen candidates against sparganosis.
This study is the first to analyze and characterize the transcriptome of S. erinacei spargana. This project provides an all-inclusive overview and preliminary analyses for genomic research on S. erinacei spargana and is a useful starting point for gene discovery, new drug development, novel antigen identification, and comparative analyses of genomes. In addition, this study will help facilitate whole genome sequencing and annotation.
This work was supported by grant 2006-N54002-00 and the Pathogenic Proteome Management Program (4800-4847-300) from the Korea National Institute of Health, Korea Centers for Disease Control and Prevention.
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