Intern
    Institut für Hygiene und Mikrobiologie

    Genomics/Proteomics

    Modern omics-approaches (genomics/transcriptomics/proteomics) already proved highly useful for molecular characterization of pathogenic organisms and the analysis of host-parasite interaction mechanisms. Due to the possibility to cultivate its larvae and stem cells under host-cell free conditions ( see  in vitro cultivation), E. multilocularis constitutes a suitable model for the generation of cDNA-libraries or genomic DNA that are essentially free of host-contamination. We could demonstrate that E. multilocularis, E. granulosus and T. solium trans-splice a significant part of their transcriptome, which is therefore exceptionally well accessible for cDNA library construction (Brehm et al., 2000; Brehm et al., 2002, 2003).
    In collaboration with researchers of the Wellcome Trust Sanger Institute and colleagues from Mexico, Uruguay, Argentina and China, we were recently successful in the determination of the whole genome sequence of E. multilocularis and three other cestodes (E. granulosus, Taenia solium, Hymenolepis microstoma) (Tsai et al., 2013; Olson et al., 2012). These analyses led to a high quality genome assembly and annotation, particularly for the model cestode E. multilocularis which can be accessed through GeneDB (http://www.genedb.org/Homepage/Emultilocularis). A very recent collaboration also revealed that Echinococcus and other flatworms do employ DNA cytosine methylation (Geyer et al., 2013) which is highly relevant for epigenetic analyses on the parasite.

    In parallel to genome sequencing and assembly, transcriptomes of different life cycle stages of E. multilocularis are currently being characterized using next-generation sequencing (NGS). These include primary cell cultures at different stages (mimicking the oncosphere-metacestode transition), metacestode vesicles without and with brood capsules (developing protoscoleces), dormant and activated protoscoleces as well as adult worms (Tsai et al., 2013; ongoing collaborations). Thus, transcriptome data that almost completely cover the E. multilocularis life cycle will soon be available, although it will still be difficult to obtain material of activated E. multilocularis oncospheres in amounts that are sufficient for RNA sequencing.
    Due to the completed and ongoing genome/transcriptome sequencing projects for E. multilocularis, E. granulosus, H. microstoma and several Taenia species, a wealth of cestode sequence data are currently available (or are expected in the very near future). This will be helpful in further improvement of transgenic techniques for E. multilocularis and will yield valuable data on factors that are involved in parasite development and immune-evasion.
    In a project which we started very recently, the genomics/transcriptomics approaches are complemented by proteomic analyses that concentrate on excretory/secretory products of the parasitic larvae and on factors that are present in hydatid fluid. Due to their secretion towards host tissue, these factors are likely to be involved in host-parasite interaction and immuno-evasion (Nono et al., 2012). One of the factors which we found to be secreted by the metacestode, EmABP, interacts with host apolipoprotein AI and could, thus, be involved in cholesterol uptake by the parasite (Bernthaler et al., 2009). A second, EmTIP, was found to be important for early parasite development and promoted the release of IFN-gamma by CD4+-Tcells (immunomodulation; Nono et al., in press). The E. multilocularis proteomics project is also carried out in close cooperation with Dr. Henrique Ferreira (Instituto de Biosciencias, Porto Alegre, Brazil).

    The spliced leader RNA of Echinococcus multilocularis

    References:

    Nono, J.K., Lutz, M.B., and Brehm, K. EmTIP, a T-cell immunomodulatory protein secreted by the tapeworm Echinococcus multilocularis is important for early metacestode development.
    PLoS Negl. Trop. Dis., in press.

    Geyer, K.K., Chalmers, I.W., MacKintosh, N., Hirst, J.E., Geoghegan, R., Badets, M., Brophy, P.M., Brehm, K., Hoffmann, K.F. (2013) Cytosine methylation is a conserved epigenetic feature found throughout the phylum Platyhelminthes.
    BMC Genomics 14: 462.

    Tsai, I.J., Zarowiecki, M., Holroyd, N., Garciarrubio, A., Sanchez-Flores, A., Brooks, K.L., Tracey, A., Bobes, R.J., Fragoso, G., Sciutto, E., Aslett, M., Beasley, H., Cai, X., Camicia, F., Clark, R., Cucher, M., De Silva, N., Day, T.A., Deplazes, P., Estrada, K., Fernández, C., Holland, P.W.H., Hou, J., Hu, S., Huckvale, T., Hung, S.S., Kamenetzky, L., Keane, J.A., Kiss, F., Koziol, U., Lambert, O., Liu, K., Luo, X., Luo, Y., Macchiaroli, N., Nichol, S., Paps, J., Parkinson, J., Pouchkina-Stantcheva, N., Riddiford, N., Rosenzvit, M., Salinas, G., Wasmuth, J.D., Zamanian, M., Zheng, Y., The Taenia solium Genome Consortium, Cai, J., Soberón, X., Olson, P.D., Laclette, J.P., Brehm, K., Berriman, M. (2013) The genomes of four tapeworm species reveal adaptations to parasitism.
    Nature 496: 57-63.

    Nono, J.K., Pletinckx, K., Lutz, M., Brehm, K. (2012) Excretory/secretory products of Echinococcus multilocularis larvae induce apoptosis and tolerogenic properties in dendritic cells in vitro.
    PLoS Negl. Trop. Dis. 6: e1516.

    Bernthaler, P., Epping, K., Schmitz, G., Deplazes, P. & Brehm, K. (2009) Molecular characterization of EmABP, an apolipoprotein AI binding protein secreted by the Echinococcus multilolcularis metacestode.
    Infect. Immun. 77: 5564-5571.

    Brehm, K., Wolf, M., Beland, H., Kroner, A. & Frosch, M. (2003) Analysis of differential gene expression in Echinococcus multilocularis larval stages by means of spliced leader differential display.
    Int. J. Parasitol. 33: 1145-1159

    Brehm, K., Hubert, K., Sciutto, E., Garate, T. & Frosch, M. (2002) Characterization of a spliced leader gene and of trans - spliced mRNAs from Taenia solium. Mol. Biochem. Parasitol. 122: 100 – 105

    Brehm, K., Jensen, K. & Frosch, M. (2000) mRNA trans-splicing in the human parasitic cestode Echinococcus multilocularis.
    J. Biol. Chem. 275: 38311-38318

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