Analysis of differential gene expression in Echinococcus multilocularis larval stages by means of spliced leader differential display.
All parasitic helminths process a large subset of their mRNA molecules via spliced leader trans-splicing. Here we describe a rapid and efficient method, 'spliced leader differential display', to generate comprehensive expression fingerprints of trans-spliced factors on silver-stained acrylamide gels. The method allows systematic screening of the entire trans-spliced transcriptome of parasitic organisms using only minute amounts of starting material. Spliced leader differential display selectively displays 5' end-fragments of trans-spliced messages thus facilitating the identification of associated proteins and the full-length characterisation of corresponding mRNAs. We employed spliced leader differential display to analyse gene expression patterns of in vitro cultivated protoscoleces and metacestode vesicles from Echinococcus multilocularis. These analyses revealed that the majority of trans-spliced mRNAs are expressed at equal amounts in both larval stages whereas about 25% of the identified transcripts displayed stage-specific differential expression. Full-length transcripts for several of the obtained fragments were isolated and their constitutive or differential expression was verified by competitive PCR and Virtual Northern blotting. Spliced leader differential display was further used to identify genes which are induced in the E. multilocularis metacestode stage under growth promoting conditions in the presence of host hepatocytes. One mRNA, encoding a putative member of the epidermal growth factor family of mitogens, was shown to be 10-fold induced upon co-incubation of metacestode vesicles with host cells which indicates a possible involvement of parasite-determined, epidermal growth factor-like signal transduction systems in metacestode proliferation and development. The successful application of spliced leader differential display to gene expression analyses of echinococcal larvae, combined with easy handling and a low rate of false positives, recommends this method not only for studies on the host-parasite interplay during alveolar echinococcosis, but also for investigations on other parasitic organisms.
D000293:Adolescent; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern / Q000379:methods; D004444:Echinococcosis, Hepatic / Q000469:parasitology*; D004815:Epidermal Growth Factor / Q000235:genetics*; D015870:Gene Expression; D015801:Helminth Proteins / Q000235:genetics*; D006790:Host-Parasite Interactions; D006801:Humans; D007814:Larva / Q000235:genetics; D008099:Liver / Q000469:parasitology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics*; D020038:RNA, Spliced Leader; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D016211:Transforming Growth Factor alpha / Q000235:genetics
C115843:Em6 protein, Echinococcus multilocularis; D015801:Helminth Proteins; D012333:RNA, Messenger; D020038:RNA, Spliced Leader; D016211:Transforming Growth Factor alpha; D004815:Epidermal Growth Factor