Full-length-enriched cDNA libraries from Echinococcus granulosus contain separate populations of oligo-capped and trans-spliced transcripts and a high level of predicted signal peptide sequences

Abstract

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.

Contingent, non-neutral evolution in a multicellular parasite: natural selection and gene conversion in the Echinococcus granulosus antigen B gene family

“…Experiments of RT-PCR followed by cloning (Arend et al, 2002) and the construction of Echinococcus EST libraries (Fernández et al, 2002) have indicated the presence of sequences with nucleotide substitutions, some of them identical and in the same sites as the alleles characterized here. Our previous attempts to screen total or subtracted genomic libraries failed to isolate any EgAgB-related clone.…”

Alternative mRNAs Arising from Trans-splicing Code for Mitochondrial and Cytosolic Variants of Echinococcus granulosus Thioredoxin Glutathione Reductase

“…We have previously reported the existence of E. granulosus genes encoding leaderless thioredoxin and thioredoxin peroxidase and the capacity of the larval worm to be metabolically competent toward high hydrogen peroxide concentrations (23) (47). An examination of a recent E. granulosus EST data base (www.nematodes.org/Lopho/LophDB.php) (22) seems to reinforce the idea that antioxidant defenses are also critical in the mitochondria. Both a thioredoxin and a thioredoxin peroxidase with putative mitochondrial trans-location signal were identified.…”

“…Although the function of trans-splicing in these organisms is still unknown, it has already been used to construct Echinococcus cDNA libraries from trans-spliced mRNAs (22). The 5Ј-end of the EgTGR gene was cloned by a PCR splice leader strategy using an oligonucleotide derived from the E. granulosus SL exon and genespecific reverse primers (see "Experimental Procedures").…”

Usefulness of Four Different Echinococcus granulosus Recombinant Antigens for Serodiagnosis of Unilocular Hydatid Disease (UHD) and Postsurgical Follow-Up of Patients Treated for UHD

“…With this ELISA, we first compared the diagnostic performance of the N-terminal-truncated recombinant B1 and B2 antigens. We also obtained and assayed for the first time the usefulness of two additional recombinant antigens (E14t [23,25] and C317 [5]) as diagnostic molecules, and their usefulness was compared to that of HF by ELISA. In addition, a comparison of the performance of these antigens was made regarding cyst location.…”