Gene discovery for the carcinogenic human liver fluke, Opisthorchis viverrini

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Summary of predicted gene product function and location using gene ontology terms. Gene ontology (GO) terms for annotated Opisthorchis viverrini assembled ESTs were extracted, if present, from the GO database and sorted into the immediate subcategories for molecular function, cellular component and biological process. The GO subcategory and percentage relative to the total number of extracted terms is indicated in the legend. Although cellular and physiological processes, structural proteins and catalytic activity were strongly represented other categories of interest include the caspases and transporter activity that may represent proteins important for a parasitic lifestyle. The large number of unknowns in each of the three categories highlights the lack of knowledge regarding many of the proteins found in these parasites.

Figure 2

Comparison of the gene ontology molecular function terms for expressed sequence tags from Opisthorchis viverrini, Clonorchis sinensis and Schistosoma japonicum. Expressed sequence tags from C. sinensis and S. japonicum were downloaded from NCBI and subjected to the same analyses used for O. viverrini sequences. A comparison of the percentage of terms correlating to the molecular function subcategory for each organism shows a broad similarity, although in some cases, such as categories for structural or motor proteins, categories are over- or under-represented in certain species.

Figure 3

Evolutionary relationships between Opisthorchis viverrini and related platyhelminths based on similarities of protein coding genes using SimiTri. Similarity of O. viverrini ORFs (1,932 ESTs) to those from the liver fluke Clonorchis sinensis (2,679 ESTs), the blood fluke Schistosoma japonicum (107, 427 ESTs) and the free-living turbellarian Schmidtea mediterranea (171,472 ESTs). SimiTri [20] was used to plot 1,932 O. viverrini contigs against related species database entries (A). Each spot represents a unique contig and its sequence similarity to each of the three selected databases as determined by tBLASTx scores. Sequences showing similarity to only one database are not shown. Sequences showing sequence similarity to only two databases appear on the lines joining the two databases. Spots are coloured by their highest tBLASTx score to each of the databases. O. viverrini sequences with homologues in the parasitic flukes only (not in Schmidtea) are highlighted in the dotted region and the identities of selected examples are shown in the table (B). The entire list (105) of these putative parasite-specific proteins is shown in Table S1.

Figure 4

Distribution of Opisthorchis viverrini assembled ESTs (OvAEs) that contain predicted signal peptides or signal anchors. OvAEs that had BLAST hits greater than 1 × 10^-5 were sorted into conserved (those matching entries for species other than platyhelminths), phylum Platyhelminthes-specific (only matching platyhelminth entries) and novel (no significant homology to any database entry). The sequences in each category were then analysed for the presence of a signal sequence using SignalP. The relative percentages of each category are indicated along with the sub-category of signal sequence positive contigs.

Figure 5

An Opisthorchis viverrini homologue of Sm-TSP-2, a vaccine antigen expressed in the tegument of Schistosoma mansoni. Multiple sequence alignment comparing the ORF of OvAE953 with Sm-TSP-2 from S. mansoni (GenBank AF521091) and human CD63 (NM_001780). Both Sm-TSP-2 and CD63 sequences shown here are truncated at the C-terminus (fourth transmembrane domain and C-terminal tail are not shown) for comparative purposes because OvAE953 is a partial sequence. Black boxes denote identical residues shared by two or more of the sequences. Grey boxes denote conservative substitutions. Dashed lines denote the predicted transmembrane (TM) domains of Sm-TSP-2; the solid line represents the extracellular (EC) loop 2 region of Sm-TSP-2 [33].

Figure 6

A TGF-β receptor type I from Opisthorchis viverrini. Multiple sequence alignment of the ORFs of OvAE22 with homologues from Schistosoma mansoni (SmRK-I – GenBank AF031557), the hydatid tapeworm Echinococcus multilocularis (TR1 – AJ841786) and human (TGF-β receptor type I – L11695) (A). The overlined region denotes the putative serine-threonine kinase active site in SmRK-I [44]. Residues highlighted in red font in OvAE22 are putative sites of serine/threonine phosphorylation. Both SmRK-I and human TGF-β receptor type I sequences shown here are truncated at the N-terminus and SmRK-I is truncated at the C-terminus for comparative purposes with the partial sequence from O. viverrini. Black boxes denote identical residues shared by two or more of the sequences. Grey boxes denote conservative substitutions. Neighbour joining phylogenetic tree showing the relationship between the ORF of OvAE22 and other members of the TGF-β receptor type I family (B). Numbers on branches denote bootstrap values from 100 samplings. The nominated outgroup was the type 2 receptor, SmRK-2. GenBank accession numbers not already provided above are as follows: pig bone morphogenic protein (BMP) receptor type I (AY065994); dog hookworm Ancylostoma caninum S/T kinase (AY053388); Caenorhabditis briggsae CBG02627 (CAAC01000012); filarial nematode Brugia pahangi trk-1 (AF013991); S. mansoni SmRK-2 (AY550912).