Summary Tapeworms cause debilitating neglected diseases that can be deadly and often require surgery due to ineffective drugs. Here we present the first analysis of tapeworm genome sequences using the human-infective species Echinococcus multilocularis, E. granulosus, Taenia solium and the laboratory model Hymenolepis microstoma as examples. The 115-141 megabase genomes offer insights into the evolution of parasitism. Synteny is maintained with distantly related blood flukes but we find extreme losses of genes and pathways ubiquitous in other animals, including 34 homeobox families and several determinants of stem cell fate. Tapeworms have species-specific expansions of non-canonical heat shock proteins and families of known antigens; specialised detoxification pathways, and metabolism finely tuned to rely on nutrients scavenged from their hosts. We identify new potential drug targets, including those on which existing pharmaceuticals may act. The genomes provide a rich resource to underpin the development of urgently needed treatments and control.

BackgroundThe cestode Echinococcus granulosus - the agent of cystic echinococcosis, a zoonosis affecting humans and domestic animals worldwide - is an excellent model for the study of host-parasite cross-talk that interfaces with two mammalian hosts. To develop the molecular analysis of these interactions, we carried out an EST survey of E. granulosus larval stages. We report the salient features of this study with a focus on genes reflecting physiological adaptations of different parasite stages.Methodology/Principal FindingsWe generated ∼10,000 ESTs from two sets of full-length enriched libraries (derived from oligo-capped and trans-spliced cDNAs) prepared with three parasite materials: hydatid cyst wall, larval worms (protoscoleces), and pepsin/H+-activated protoscoleces. The ESTs were clustered into 2700 distinct gene products. In the context of the biology of E. granulosus, our analyses reveal: (i) a diverse group of abundant long non-protein coding transcripts showing homology to a middle repetitive element (EgBRep) that could either be active molecular species or represent precursors of small RNAs (like piRNAs); (ii) an up-regulation of fermentative pathways in the tissue of the cyst wall; (iii) highly expressed thiol- and selenol-dependent antioxidant enzyme targets of thioredoxin glutathione reductase, the functional hub of redox metabolism in parasitic flatworms; (iv) candidate apomucins for the external layer of the tissue-dwelling hydatid cyst, a mucin-rich structure that is critical for survival in the intermediate host; (v) a set of tetraspanins, a protein family that appears to have expanded in the cestode lineage; and (vi) a set of platyhelminth-specific gene products that may offer targets for novel pan-platyhelminth drug development.Conclusions/SignificanceThis survey has greatly increased the quality and the quantity of the molecular information on E. granulosus and constitutes a valuable resource for gene prediction on the parasite genome and for further genomic and proteomic analyses focused on cestodes and platyhelminths.

The tissue-dwelling larval stages of the cestode Echinococcus granulosus are intimately associated with the host, implying that a range of molecular mediators may be secreted by the parasite into the host environment. These mediators are being sought through a transcriptome-based analysis, using recombinant cDNA libraries. Conventional cDNA libraries of E. granulosus contain high levels of mitochondrial transcripts, as well as host (bovine) genomic DNA. In particular, 60% of a conventional protoscolex stage cDNA library corresponds to the large subunit (LSU) of mitochondrial rRNA. We attribute the presence of LSU rRNA copies to its polyadenylation in E. granulosus. To circumvent this problem, we adapted the 5' Rapid Amplification of cDNA Ends (RNA-ligase mediated RACE) technique that excludes all polynucleotides missing the 7-methyl-guanosine (7MG) cap specific to the 5' end of full-length mRNA. By ligating a specific oligonucleotide (oligo-cap) to 7MG-bearing mRNA, three cDNA libraries were made by PCR from oligo-cap and oligo-dT primers. Analysis of these libraries showed that mitochondrial RNA contaminants had been excluded. Moreover, no bovine genomic sequences were detected. In parallel, we constructed three cDNA libraries using the newly described trans-spliced leader (SL) from Echinococcus. Although these represent a smaller subset of parasite genes, mitochondrial and genomic contributions were again excluded. In both cases, a majority of cDNAs (61-92%) were judged to contain the initiation ATG codon, and 11-27% of inserts included potential N-terminal signal sequences. The 5' UTR tracts of most oligo-capped cDNAs were <100 nt, although approximately 8% were longer than this. Among the trans-spliced cDNAs, 43% potentially utilise the AUG donated by the SL, and in only 6% was the SL separated from an endogenous putative start site by >60 nt. Sequence analysis of randomly selected clones shows virtually no overlap between the oligo-capped and SL libraries, indicating that trans-spliced E. granulosus mRNAs appear to be insensitive to the enzymatic treatments used to 'oligo-cap' unspliced mRNAs. The oligo-capped and SL strategies represent efficient and complementary pathways to isolate full-length cDNA clones from this cestode parasite and, possibly, from related parasitic flatworms.

The present work describes the purification and characterization of antigen B (AgB), the thermostable lipoprotein from E. granulosus. Native AgB was purified to homogeneity by a new strategy involving adsorption on DEAE-Sepharose, followed by immunopurification. The purified antigen was analysed using mapped monoclonal antibodies (MoAbs) and peptide isolation by in situ digestion in gels after SDS-PAGE. Epitope mapping of 7 MoAbs using PEPSCAN, synthetic peptides and competition studies, revealed that six of them defined epitopes which clustered the N-terminal extension of a 8 kDa subunit of AgB, whilst the remaining one reacted against the stretch RGLIAEGE, corresponding to the C-terminus. The epitopes defined by the seven MoAbs were found to be present in all the subunits. Furthermore, the similarities of the peptide finger prints obtained by HPLC analysis and amino acid sequencing of tryptic peptides isolated from the 8, 16 and 24 kDa subunits, indicated that they have most if not all the amino acid sequence in common. We also found evidence that the band representing a component of an apparent molecular weight of 8 kDa in SDS-PAGE, believed to be the smallest subunit of AgB, contained at least two components, which may constitute the building blocks of the higher molecular weight subunits.