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Genomic, Transcriptomic and Functional Studies of Diplomonads

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Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 990 Hidden Diversity Revealed Genomic, Transcriptomic and Functional Studies of Diplomonads JON JERLSTRÖM-HULTQVIST ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-554-8520-7 UPPSALA urn:nbn:se:uu:diva-182831 2012 Dissertation presented at Uppsala University to be publicly examined in B22, Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, Friday, December 14, 2012 at 09:00 for the degree of Doctor of Philosophy. The examination will be conducted in English. Abstract Jerlström-Hultqvist, J. 2012. Hidden Diversity Revealed: Genomic, Transcriptomic and Functional Studies of Diplomonads. Acta Universitatis Upsaliensis. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 990. 104 pp. Uppsala. ISBN 978-91-554-8520-7. The diplomonads are a diverse group of eukaryotic microbes found in oxygen limited environments such as the intestine of animals were they may cause severe disease. Among them, the prominent human parasite Giardia intestinalis non-invasively colonizes the small intestine of humans and animals where it induces the gastrointestinal disease giardiasis. Two of the eight genetic groups of G. intestinalis, assemblage A and B, are known to infect humans and have zoonotic potential. At the start of project, genome scale data from assemblage B-H was either sparse or entirely missing. In this thesis, genome sequencing was performed on the assemblage B isolate GS (Paper I) and the P15 isolate (Paper III) of the hoofed-animals specific assemblage E to investigate the underlying components of phenotypic diversity in Giardia. Comparisons to assemblage A isolate WB revealed large genomic differences; entirely different repertoires of surface antigens, genome rearrangements and isolate specific coding sequences of potential bacterial origin. We established that genomic differences are also manifested at the transcriptome level (Paper VIII). In a follow up analysis (Paper IV) we concluded that the Giardia assemblages are largely reproductively isolated. The large genomic differences observed between Giardia isolates can explain the phenotypic diversity of giardiasis. The adaptation of diplomonads was further studied in Spironucleus barkhanus (Paper II), a fish commensal of grayling, that is closely related to the fish pathogen Spironucleus salmonicida, causative agent of systemic spironucleosis in salmonid fish. We identified substantial genomic differences in the form of divergent genome size, primary sequence divergence and evidence of allelic sequence heterozygosity, a feature not seen in S. salmonicida. We devised a transfection system for S. salmonicida (Paper VI) and applied it to the study of the mitochondrial remnant organelle (Paper VII). Our analyses showed that S. salmonicida harbor a hydrogenosome, an organelle with more metabolic capabilities than the mitosome of Giardia. Phylogenetic reconstructions of key hydrogenosomal enzymes showed an ancient origin, indicating a common origin to the hydrogenosome in parabasilids and diplomonads. In conclusion, the thesis has provided important insights into the adaptation of diplomonads in the present and the distant past, revealing hidden diversity. Keywords: Giardia intestinalis, Spironucleus salmonicida, Spironucleus barkhanus, intestinal parasite, hydrogenosome, mitosome, lateral gene transfer, horizontal gene transfer, diplomonad, metamonad, sexual recombination, transfection, protein complex purification Jon Jerlström-Hultqvist, Uppsala University, Department of Cell and Molecular Biology, Microbiology, Box 596, SE-751 24 Uppsala, Sweden. © Jon Jerlström-Hultqvist 2012 ISSN 1651-6214 ISBN 978-91-554-8520-7 urn:nbn:se:uu:diva-182831 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-182831) ”The anus was a prerequisite for intelligence; without it, heads and brains would not have evolved.” - T. Cavalier-Smith (2006), evolutionary biologist List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Franzén O, Jerlström-Hultqvist J, Castro E, Sherwood E, Ankarklev J, Reiner DS, Palm D, Andersson JO, Andersson B, Svärd SG. (2009) Draft genome sequencing of Giardia intesti- nalis assemblage B isolate GS: is human giardiasis caused by two different species? PLoS Pathogens 5(8) II Roxström-Lindquist K, Jerlström-Hultqvist J, Jørgensen A, Troell K, Svärd SG, Andersson JO. (2010) Large genomic dif- ferences between the morphologically indistinguishable diplo- monads Spironucleus barkhanus and Spironucleus salmonicida. BMC Genomics. 11:258. III Jerlström-Hultqvist J, Franzén O, Ankarklev J, Xu F, Nohýnko- vá E, Andersson JO, Svärd SG, Andersson B. (2010) Genome analysis and comparative genomics of a Giardia intestinalis as- semblage E isolate. BMC Genomics. 11:543. IV Xu F, Jerlström-Hultqvist J, Andersson JO. (2012) Genome- Wide Analyses of Recombination Suggest That Giardia intesti- nalis Assemblages Represent Different Species. Molecular Bi- ology and Evolution. 29(10): 2895-98. V Jerlström-Hultqvist J, Stadelmann B, Birkestedt S, Hellman U, Svärd SG. (2012) Plasmid vectors for proteomic analyses in Giardia: purification of virulence factors and analysis of the proteasome. Eukaryotic Cell. 11(7):864-73. VI Jerlström-Hultqvist J, Einarsson E, Svärd SG. (2012) Stable transfection of the diplomonad parasite Spironucleus salm- onicida. Eukaryotic Cell. 11(11):1353-61 VII Jerlström-Hultqvist J , Einarsson E, Xu F, Hjort K, Ek B, Stein- hauf D, Bergqvist J, Andersson JO, Svärd SG. Spironucleus mi- tochondrial remnants suggest that hydrogenosomes are ancient organelles. Submitted. VIII Franzén O*, Jerlström-Hultqvist J*, Einarsson E, Ankarklev J, Ferella M, Andersson B, Svärd SG. Transcriptome Profiling of Giardia intestinalis Using Strand-specific RNAseq. Submitted. *These authors contributed equally Reprints were made with permission from the respective publishers. Review articles or other publications, not included in the thesis. I Ankarklev J, Jerlström-Hultqvist J, Ringqvist E, Troell K, Svärd SG. (2010) Behind the smile: cell biology and disease mechanisms of Giardia species. Nature Reviews Microbiolo- gy. 8(6):413-22. (Review article) II Jerlström-Hultqvist J, Ankarklev J, Svärd SG. (2010) Is human giardiasis caused by two different Giardia species? Gut Mi- crobes. 1(6):379-82. (Review article) III Hertz HM, von Hofsten O, Bertilson M, Vogt U, Holmberg A, Reinspach J, Martz D, Selin M, Christakou AE, Jerlström- Hultqvist J, Svärd S. (2012) Laboratory cryo soft X-ray mi- croscopy. Journal of Structural Biology. 177(2):267-72. Contents Sammanfattning på svenska (Summary in Swedish) .................................... 11 Genetiska studier av Giardia (Paper I, III & IV) ..................................... 12 Transkriptomstudier av Giardia (Paper VIII) .......................................... 12 Miljöanpassning hos diplomonader (Paper II & VIII) ............................. 13 Funktionella studier av diplomonader (Paper V, VI, VII) ........................ 13 1. Introduction ............................................................................................... 15 1.1 The eukaryotic cell ........................................................................ 15 1.1.1 Origins ........................................................................................ 15 1.1.2 The demise of the Archezoan hypothesis ................................... 16 1.1.3 Hydrogenosomes: discovery and origins .................................... 17 1.2 Excavata ............................................................................................. 19 1.3 Fornicata ............................................................................................. 20 1.4 The Giardia cell ................................................................................. 21 1.4.1 Giardia classification and assemblages ...................................... 22 1.5 The Giardia life cycle ........................................................................ 23 1.5.1 The trophozoite ........................................................................... 23 1.5.2 Differentiation............................................................................. 28 1.6 Cell biology and metabolism of Giardia ............................................ 30 1.6.1 Cellular metabolism .................................................................... 31 1.6.2 Sex in Giardia? ........................................................................... 33 1.7 Giardia pathogenesis .......................................................................... 34 1.7.1 Giardiasis .................................................................................... 35 1.7.2 Host-parasite interactions ........................................................... 35 1.7.3 Antigenic variation ..................................................................... 37 1.7.4 Treatment .................................................................................... 41 1.8 The G. intestinalis genome and transcriptome ................................... 41 1.8.1 Transcriptional landscape ........................................................... 42 1.8.2 Gene families .............................................................................. 45 1.9 Spironucleus ....................................................................................... 47 1.9.1 Taxonomy and host association .................................................
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  • Identification of the Meiotic Life Cycle Stage of Trypanosoma Brucei in The

    Identification of the Meiotic Life Cycle Stage of Trypanosoma Brucei in The

    Identification of the meiotic life cycle stage of Trypanosoma brucei in the tsetse fly Lori Peacocka,b, Vanessa Ferrisa,b, Reuben Sharmac,1, Jack Sunterc, Mick Baileyb, Mark Carringtonc, and Wendy Gibsona,2 aSchool of Biological Sciences, University of Bristol, Bristol BS8 1UG, United Kingdom; bDepartment of Clinical Veterinary Science, University of Bristol, Bristol BS40 7DU, United Kingdom; and cDepartment of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom Edited by Francisco J. Ayala, University of California, Irvine, CA, and approved January 27, 2011 (received for review December 23, 2010) Elucidating the mechanism of genetic exchange is fundamental for genetic exchange in T. brucei involves mixing of mitochondrial understanding how genes for such traits as virulence, disease (kinetoplast) and nuclear genomes, because hybrid progeny have phenotype, and drug resistance are transferred between pathogen hybrid kinetoplast DNA (kDNA) networks with mini-circles de- strains. Genetic exchange occurs in the parasitic protists Trypano- rived from both parents (14, 15). Plausible models for the gen- soma brucei, T. cruzi, and Leishmania major, but the precise cellular eration of hybrid kDNA networks are limited by the complex mechanisms are unknown, because the process has not been ob- structure and highly ordered replication of this concatenated served directly. Here we exploit the identification of homologs of mass of small DNA circles (16). Finally, trypanosomes belong to meiotic genes in the T. brucei genome and demonstrate that three the Euglenozoa, a deep branch within the excavate eukaryote su- functionally distinct, meiosis-specific proteins are expressed in the pergroup (17, 18). The production of four haploid gametes and nucleus of a single specific cell type, defining a previously unde- subsequent fusion to reform the diploid occurring in trypanosomes scribed developmental stage occurring within the tsetse flysalivary would strongly suggest the presence of a typical meiosis in the last gland.