Gene discovery for the carcinogenic human liver fluke, Opisthorchis viverrini
- Thewarach Laha†1,
- Porntip Pinlaor†2,
- Jason Mulvenna†3,
- Banchob Sripa2,
- Manop Sripa2,
- Michael J Smout3,
- Robin B Gasser4,
- Paul J Brindley5 and
- Alex Loukas3Email author
© Laha et al; licensee BioMed Central Ltd. 2007
Received: 08 March 2007
Accepted: 22 June 2007
Published: 22 June 2007
Cholangiocarcinoma (CCA) – cancer of the bile ducts – is associated with chronic infection with the liver fluke, Opisthorchis viverrini. Despite being the only eukaryote that is designated as a 'class I carcinogen' by the International Agency for Research on Cancer, little is known about its genome.
Approximately 5,000 randomly selected cDNAs from the adult stage of O. viverrini were characterized and accounted for 1,932 contigs, representing ~14% of the entire transcriptome, and, presently, the largest sequence dataset for any species of liver fluke. Twenty percent of contigs were assigned GO classifications. Abundantly represented protein families included those involved in physiological functions that are essential to parasitism, such as anaerobic respiration, reproduction, detoxification, surface maintenance and feeding. GO assignments were well conserved in relation to other parasitic flukes, however, some categories were over-represented in O. viverrini, such as structural and motor proteins. An assessment of evolutionary relationships showed that O. viverrini was more similar to other parasitic (Clonorchis sinensis and Schistosoma japonicum) than to free-living (Schmidtea mediterranea) flatworms, and 105 sequences had close homologues in both parasitic species but not in S. mediterranea. A total of 164 O. viverrini contigs contained ORFs with signal sequences, many of which were platyhelminth-specific. Examples of convergent evolution between host and parasite secreted/membrane proteins were identified as were homologues of vaccine antigens from other helminths. Finally, ORFs representing secreted proteins with known roles in tumorigenesis were identified, and these might play roles in the pathogenesis of O. viverrini-induced CCA.
This gene discovery effort for O. viverrini should expedite molecular studies of cholangiocarcinogenesis and accelerate research focused on developing new interventions, drugs and vaccines, to control O. viverrini and related flukes.
Throughout East Asia, there is a strikingly high prevalence of cholangiocarcinoma (CCA – cancer of the bile ducts) in regions where the human liver fluke is endemic. No stronger link occurs between a human malignancy and infection with a eukaryotic parasite than that between CCA and infection with the liver fluke, Opisthorchis viverrini (Digenea) . Indeed, the International Agency for Research on Cancer (IARC) recognizes O. viverrini as a 'category I carcinogen' [2, 3]. CCA is highly prevalent in Northeast Thailand, areas where uncooked cyprinoid fish are a dietary staple. Due to poor sanitation practices and inadequate sewerage infrastructure, O. viverrini-infected people pass the trematode's eggs in their feces into natural bodies of fresh water. Aquatic snails, which represent the first intermediate hosts of O. viverrini, ingest the eggs from which the miracidia undergo asexual reproduction before a population of the free swimming larval stage, called a cercaria, is shed from the infected snails. The cercaria then locates a cyprinoid fish, encysts in the fins, skin and musculature of the fish, and becomes a metacercaria. The metacercarial stage is infective to humans and other fish-eating mammals. Infection is acquired when people ingest raw or undercooked fish. The young adult worm escapes from the metacercarial cyst in the upper small intestine and then migrates through the ampulla of Vater into the biliary tree, where it develops to sexual maturity over four to six weeks, thus completing the life cycle. The adult worms, which are hermaphrodites, can live for many years in the liver, even decades, shedding as many as 200 eggs per day which pass out via bile into the chyme and feces .
In Thailand, ~6 million people are infected with O. viverrini. Despite widespread chemotherapy with the compound, praziquantel, the prevalence of O. viverrini in some endemic areasapproaches 70% (reviewed in ). Moreover, in Thailand, liver cancer is the most prevalent of the malignant/fatal neoplasias, and the prevalence of CCA in regions in which the parasite is endemic is unprecedented .
While sexual reproduction takes place in the mature adults of O. viverrini within the bile ducts, asexual reproduction in the snail leads to a massive increase in the number of infectious cercarial stages exiting and swimming off to locate then infect the fish host. The adult fluke is a diploid organism which reproduces by meiosis; self fertilization of the male and female organs occurs, but it is believed that cross-fertilization between adjacent adult worms is the normal pattern. Although the genome size of O. viverrini has not yet been reported, it is known to have six pairs of chromosomes, i.e. 2n = 12 , distinct from the closely related liver fluke, Clonorchis sinensis, which possesses 2n = 56 chromosomes .
Despite its public health importance, only a small number of O. viverrini sequences (mostly ribosomal genes) have been available in public databases prior to the present study. Characterization of the genes expressed in this organism should provide a foundation for elucidating the immunopathogenesis of CCA, particularly the molecular mechanisms by which infection with this parasite induces cancer. Indeed, secreted proteins of O. viverrini induce hyper-proliferation of cells (or hyperplasia) in vitro , implying that carcinogenesis may not be just a consequence of chronic inflammation, but that the parasite actively secretes gene products which initiate neoplasia.
Here, we undertake gene discovery for O. viverrini after the construction of a cDNA library and characterization of ~5,000 expressed sequence tags (ESTs) from this carcinogenic parasite. A similar dataset exists for C. sinensis , which, despite its widespread prevalence , is not recognized as a 'class I carcinogen' . Therefore, we compared the available transcriptomic dataset from O. viverrini with those from C. sinensis, and from several other flatworms, both free-living and parasitic in humans.
Results and Discussion
Features of the dataset
Features of the Opisthorchis viverrini EST catalogue.
1995 (1632 singletons; 363 clusters)
Contigs after clean-upb
Contigs identical to known proteinsc
Contigs similar to other proteinsd
Contigs with gene ontology assignments
Novel contigs with signal sequences
75 (29 signal peptides; 46 signal anchors)
Average insert size
548 bp (ESTs); 660 bp (contigs)
Percentage of recombinant clones
Number of ribosomal seqs
1184 ESTs; 136 clusters
Abundantly expressed transcripts
The 10 most abundant contigsa from the Opisthorchis viverrini EST dataset.
Closest homologue in GenBank nr (accession no.)
%identities (no. of aa)
Closest homologue in dbEST (accession no.)
%identities (no. of aa)
vitelline B precursor, O. viverrini (AAL23712)
C. sinensis cDNA clone CSAD-01-D02 (AT007557)
17 kDa myoglobin, Clonorchis sinensis (AAM18464)
C. sinensis cDNA clone CSAD-29-A12 (AT009373)
hypothetical protein, C. sinensis (AAM55183)
C. sinensis cDNA clone CS30 (AT006763)
egg protein, C. sinensis (AAN64160)
C. sinensis cDNA clone CSAD-20-B05 (AT008604)
hypothetical protein, Macaca fascicularis (BAE73006)
SJA_AAF_D11.T3 SJA S. japonicum (CX857852)
histone H1, Schistosoma japonicum (AAP06509)
C.sinensis cDNA clone CSAD-25-H03 (AT009091)
egg protein, C. sinensis (AAN64160)
C. sinensis cDNA clone CSAD-01-B01 (AT007532)
retrotransposon gag region, Monascus pilosus (ABC24965)
translationally controlled tumor protein, C. sinensis (AAX84199)
C. sinensis cDNA clone CSAD-24-E07(AT008979)
glutathione-S-transferase, O. viverrini (AAL23713)
C. sinensis cDNA clone CSAD-32-E05 (AT009695)
Gene ontology assignments of ESTs from O. viverrini and related flukes
Evolutionary relationships between O. viverrini and other platyhelminths
Secreted and membrane proteins
Selected Opsithorchis viverrini contigs that encode families of secreted/membrane proteins that potentially interact with or are exposed to host tissues. Genera of the closest homologues from BLAST × (nr) searches are shown. Where the closest homologue was from a vertebrate (bold font), a tBLASTx search against dbEST was conducted.
Examples/comments and genera of closest orthologues/paralogues
bone morphogenic protein receptor type I (Sus nr/ Macaca est)
Seven transmembrane receptor
DC-STAMP (Strongylocentrotus); laminin receptor (Bos nr/ Clonorchis est)
stabilize cell membranes – expressed in the tegument of schistosomes (Schistosoma)
C1 family papain-like cysteine protease
cathepsin L (Paragonimus), cathepsin B (Fasciola; Clonorchis)
OvAE1795, OvAE813, OvAE1171, OvAE532, OvAE1070, OvAE1613, OvAE1711, OvAE615, OvAE398
C13 family asparaginyl endopeptidase
legumain (O. viverrini)
S1 family serine protease
HtrA-like (Macaca nr/ Schistosoma est) and kallikrein-like (Schistosoma) peptidases
A1 family aspartic protease
cathepsin D-like; digestive enzyme in helminths (Clonorchis)
M41 family metalloprotease
mitochondrial membrane proteinase (Schistosoma)
mitogen associated with cancer (Bos nr/ Clonorchis est)
water channel protein (Schistosoma)
critical for S. mansoni egg shell production (Schistosoma)
similar to vertebrate venom proteins; (Heloderma nr/ Clonorchis est)
immunomodulatory in fasciolosis (Schistosoma)
EF-hand secreted Ca2+-binding protein
calumenin (Rattus nr/ Xenopus est)
pore forming; similar to fluke cytolysins (Clonorchis)
Pathogenesis related protein
similar to helminth venom allergen homologues (Schistosoma)
detoxification of heme and free radicals (Clonorchis)
OvAE1057, OvAE1892, OvAE1601, OvAE1729
neurotransmission/vesicular docking – vesicle associated (Schistosoma)
integral membrane protein forming gap junctions (Schistosoma)
Fibroblast growth factor (FGF) receptor substrate 2
host FGF is essential for growth of schistosomes (Schistosoma)
Immune cell differentiation antigen
As with other parasitic helminth transcriptomes [11–13, 25, 26], proteins with catalytic activity were abundantly represented in the O. viverrini dataset (27.9% of contigs that were assigned GO molecular functions). Many of these enzymes encoded endo- and exo-proteases belonging to established families (MEROPS classification), but which have not yet been described from liver flukes (Table 3). Of particular interest were members of the S1A serine protease family with sequence similarity to kallikrein and chymotrypsin, and, therefore, potentially involved in feeding or tissue migration . Other proteases included homologues of enzymes that digest hemoglobin in blood-feeding helminths, including cathepsin D-like aspartic and cathepsin B-like cysteine proteases [28–30] as well as an asparaginyl endopeptidase, which is known to activate the gastrodermal cathepsin B enzyme, and probably other gut proteases in S. mansoni . We also identified O. viverrini homologues of the cell death enzyme, caspase-2, and the neutral cysteine protease from the tegument of schistosomes, calpain.
Multiple membrane-spanning proteins
Host-parasite "cross talk"
Molecules associated with cancer?
O. viverrini is the major cause of CCA in South-East Asia . The molecular mechanisms underlying induction of O. viverrini-induced CCA are thought to be multi-factorial (reviewed in ), but recent evidence suggests that O. viverrini secretes mitogenic proteins into host tissues [1, 8]. OvAEs encoding secreted proteins with prospective mitogenic activity were identified in the EST dataset. Of note, first, progranulin (pgrn) is a pluripotent secreted growth factor that mediates cell cycle progression, cell motility  and wound repair . We identified an OvAE (OvAE1732) that shared sequence identity with pgrn (data not shown). Of particular importance is that pgrn has been implicated in regulating the proliferation of tumour cells, and its expression is up-regulated in more aggressive cancers (reviewed in ). The kallikrein-like serine proteases are another family of enzymes whose over-expression has been linked to cancer. The expression of some kallikreins in prostate cells leads to changes indicative of an epithelial to mesenchymal transition, an important process in cancer progression . An OvAE (OvAE1918) with sequence identity to kallikrein-like secreted proteases is present in the new O. viverrini gene catalogue. Phospholipase A2 (PLA-2) regulates the provision of arachidonic acid to both cyclooxygenase- and lipoxygenase-derived eicosanoids (reviewed by ), and the upregulation of cyclooxygenase-2 is thought to be an important feature of cholangiocarcinogenesis in both humans and experimental rodent models [49, 54, 55]. We identified an OvAE (OvAE1644) that encodes a secreted PLA-2 which shared greatest sequence identity with PLA-2 from venom of Heloderma (Gila monster) and an EST from C. sinensis (Table 3). Parasites utilize secreted serine proteases  and PLA-2s  to invade host tissues, and homologues of these proteins (and granulin) are potentially secreted by O. viverrini into host tissues where they might promote cell proliferation, mutagenesis and ultimately carcinogensis. Ongoing studies in our laboratories are now focused on the physiological roles of these putative carcinogens in the host-parasite relationship and in cholangiocarcinogenesis induced by O. viverrini infection.
Opisthorchis viverrini ESTs with sequence identity to mRNAs encoding proteins efficacious as vaccines against other flatworm and nematode parasites.
Predicted protein family
Vaccine antigen and helminth targeted
APR-1 for hookworm; cathepsin D for Schistosoma japonicum
Ac-GST-1 for hookworm; bilvax for schistosomes
TSBP for Haemonchus contortus; Ac CP1 for hookworm; Cathepsins L1 and L2 for Fasciola hepatica
TSP-1, TSP-2 and Sm23 for Schistosoma mansoni
Pathogenesis related protein
ASP-2 for hookworm; ASP-1 for Onchocerca volvulus
Smp80 for schistosomes
Fatty acid binding protein
Sm14 for S. mansoni and F. hepatica
FhSAP-2 for F. hepatica
Sm14-3-3 for schistosomes
22.6 (unknown function)
Sm22.6 for S. mansoni
This report provides the first description of gene discovery for the liver fluke O. viverrini. Infection with O. viverrini is an important tropical health issue, but even more important and enigmatic is that chronic O. viverrini infection leads to the development of CCA. Indeed, there is no stronger link between a human parasite and cancer than that between O. viverrini and CCA . The new gene catalogue for O. viverrini represents the largest EST dataset in the public domain for any species of liver fluke, and provides a platform for explorations into the molecular basis of host-helminth parasite interactions. We  and others  are interested in the molecules secreted into host tissues by O. viverrini that induce hyper-proliferation of biliary cells which can subsequently undergo malignant transformation. Given the number of O. viverrini ESTs sequenced herein, it is possible that mRNAs corresponding to these parasite mitogens are already present in the current dataset. Proteomic analysis of proteins secreted by adult O. viverrini maintained in vitro also is underway in our laboratories, and linking peptide sequences to corresponding mRNAs can be expected to be facilitated by this gene discovery program . Finally, this gene discovery information for O. viverrini should expedite molecular studies of cholangiocarcinogenesis and accelerate research focused on developing new interventions, drugs and vaccines, to control O. viverrini and related flukes.
Adult O. viverrini were collected from experimentally infected hamsters (Mesocricetus auratas) maintained at the animal facility of the Khon Kaen University Faculty of Medicine. Protocols approved by the Khon Kaen University Animal Ethics Committee were used for all animal research conducted in this study. Briefly, metacercariae of O. viverrini were collected from naturally infected cyprinoid fish by pepsin digestion. Metacercariae (100 per hamster) were administered intragastrically to hamsters. Hamsters were euthanazed 6 weeks after inoculation, and adult worms were flushed with saline from the bile ducts . Worms were washed extensively with sterile phosphate-buffered saline (pH 7.2), after which they were snap frozen and stored in liquid nitrogen or employed immediately as the source of fluke RNA.
Construction and mass excision of cDNA library
Total RNA from adult O. viverrini was extracted using Trizol (Invitrogen), following the manufacturer's instructions. Ten μg of O. viverrini total RNA was used as a template for the synthesis of double-stranded cDNA using the SMART cDNA kit (BD Bioscience), after which the cDNA modified with adapters was cloned into the Sfi I site of the pTriplEx2 plasmid (BD Bioscience) and packaged into λ arms. The titer and percentage of recombinant phages in the library were determined using the protocols recommended by the manufacturer. Escherichia coli strain BM25.8 cells were transduced with recombinant phage, from which the excision of the pTriplEx phagemid library was accomplished.
Clone selection, sequencing and annotation
Five thousand clones were randomly selected from the phagemid library and grown overnight in Luria Bertani (LB) broth supplemented with ampicillin to a final concentration of 25 μg/ml. Overnight cultures were shipped at 4°C in LB broth/ampicillin to the University of Melbourne (Department of Veterinary Science). The sequencing was performed by AgGenomics Inc., Australia, using a 3730xl DNA analyzer (Applied Biosystems). The TempliPhi™ DNA Sequencing Template Amplification system (GE Healthcare) was used to sequence each clone using the 5'λ TriplEx2 sequencing primer.
Edited sequences were condensed into contigs or singletons using TGICL  with the default parameters of 40 bp overlap, a minimum of 95% identity and a 30 bp maximum mismatched overhang. Sequences of less than 100 nt were discarded. Sequences were named using the same convention as that used for the human blood fluke, Schistosoma mansoni ; OvAE for O. v iverrini A ssembled E ST. Sequences were compared with those available in the NCBI non-redundant protein and nucleotide databases using BLASTx and BLASTn. searches, respectively in October 2006. The dbEST database was queried using BLASTn and tBLASTx searches. BLAST alignments with an E-value of ≤ 1.0 × 10-5 were reported. OvAEs were functionally categorized by querying a local copy of the Gene Ontology (GO) database  (downloaded November, 2006) with an E-value cutoff of 1.0 × 10-5. All ESTs from C. sinensis  and Schistosoma japonicum [11, 12] were downloaded from NCBI , and the same methodology was used to derive ontology classifications for the C. sinensis ESTs. ORF predictions were performed using GENSCAN  using the HumanIso parameter set. Signal sequence prediction was accomplished using SignalP 3.0 , incorporating both hidden Markov models and neural networks. Positive signal sequence predictions from either method and positive signal anchor predictions using Markov models were reported. Predictions of transmembrane domains were conducted using TMPred . All multiple sequence alignments were carried out using ClustalW. Clan and family assignments of proteolytic enzymes were analyzed via the MEROPS protease database . Putative phosphorylation sites were predicted using the NetPhos 2.0 server .
Multiple sequence alignments were assembled using ClustalW. Only regions which completely overlapped with partial ORFs of O. viverrini ESTs were used for tree construction. Alignments were imported into PAUP version 4.0 beta  to construct trees using the neighbour joining and maximum parsimony methods. Robustness was assessed by bootstrap analysis using 100 replicates. Clades with more than 50% support were denoted with bootstrap values on the branches.
Cross-taxon similarity analysis
OvAEs were compared with all entries for other organisms in the NCBI dbEST database using tBLASTx. The highest BLAST scores (above a cut-off value of 50) were used to generate SimiTri plots  using software developed in-house (J. Mulvenna, unpublished).
We are grateful for grant support from the Sandler Foundation, NIH-NIAID (award number AI065871), Khon Kaen University Research Fund, Thailand-Tropical Diseases Research Program and the Australian Research Council (LP0667795 and DP0665230). JM was supported by a Peter Doherty Training Fellowship from the National Health and Medical Research Council of Australia (NHMRC). AL was supported by an R. Douglas Wright Career Development Award from NHMRC. We thank Ian Smith, Jacqui Batley, Nonglack Kewgrai, Bronwyn Campbell and David Blair for advice or support.
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