Differentiation and division of Trypanosoma brucei in the mammalian bloodstream. A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Science. 1998 Kevin Tyler School of Biological Sciences i Division of Biochemistry ii Declaration No portion of this work has been submitted in support of an application for another degree or qualification of this or any other university or institute of learning. The following notes on copyright and the intellectual property rights: 1) Copyright in text of this thesis rests with the Author. Copies (by any process) either in full, or of extracts may be made only in accordance with instructions given by the Author and lodged in the John Rylands University Library of Manchester. Details may be obtained from the Librarian. This page must form part of any such copies made. Further copies (by any process) of copies made in accordance with such instructions may not be made without the permission (in writing) of the Author. 2) The ownership of any intellectual property rights which may be described in this thesis is vested in the University of Manchester, subject to any prior agreement to the contrary, and may not be made available for use by third parties without the written permission of the University, which will prescribe the terms and conditions of any such agreement. Further information on the conditions under which disclosures and exploitation may take place is available from the Head of Department. ii ACKNOWLEDGEMENTS Thanks must go first and foremost to Keif Matthews and Keith Gull, who initiated this project, for their exemplary supervision throughout. In addition, Keif provided many of the materials used, and assisted with some of the experiments. I am also indebted to my colleagues in the Gull lab who have all helped with this project. In particular, Klaus Ersfeld helped to adapt his in situ hybridization protocol to bloodstream forms; Rob Docherty collaborated in the differential display work; Trevor Sherwin contributed electron micrographs used in some of the studies; Christiane Hertz helped with and continued the work on in vitro culture; Aspasia Ploubidou worked with me in figuring out basal body positioning; and Val Scott taught me to run protein gels and make T-vector. Philippe Bastin and Derrick Robinson deserve a general mention for being fantastically helpful to the project. Philippe also kept me in biscuits during the write-up. Finally, my profound thanks to Lisa Butler for making sure there was somebody to drink with on a Friday night. Outside the lab, Paul Higgs collaborated with moving my simple algebraic models into the realms of calculus and computing. Also, many people kindly gave me antibodies to use for this project. Thanks in particular to George Hill and Minu Chaudhuri who let me have monoclonals to TAO before they published them. Luise Krauth-Siegel and Ralph Schoneck contributed DHLADH antibody; David Engman, mtp70 and hsp60 antibodies; Debbie Smith, lhsp70 antibody; Andrew Balber and Marla Brickman, the CB1 antibody. Thanks also to my surgeon Richard Cobb, who got me back to the project. Finally, thanks to my iii parents, sister, friends and relatives for life outside of science, and most especially to my wife, Allyson. iv ABSTRACT In the bloodstream of mammalian hosts, the African trypanosome Trypanosoma brucei, undergoes a life cycle stage differentiation from a long slender form to a short stumpy form. The slender form is proliferative and meets its energy requirements solely by glycolysis. The stumpy form is non-proliferative and partially preadapted for survival in the midgut of its vector, the tsetse-fly. This preadaption includes mitochondrial enlargement and activation of enzymes associated with Krebs cycle. Thus, three major events characterize the slender-to-stumpy differentiation: • Exit from the proliferative cell cycle. • Morphogenesis. • Partial mitochondrial biogenesis. This project sought, to provide markers associated with each of these events and, to use these markers to discriminate between theoretical models of the slender-to-stumpy differentiation. In this way, I aimed to determine the manner in which differentiation occurs, with respect to the co-ordination of its three major events. In the course of the project, organellar segregation and outgrowth of the new flagellum were characterized as markers of the slender form cell cycle; this facilitated the discrimination of cells committed to cell division. Flagellar length was found to be a good morphological marker of the slender-to-stumpy differentiation and expression of dihydrolipoamide dehydrogenase, hsp60 and mhsp70 were established as markers of mitochondrial biogenesis. When cells committed to division were investigated, evidence was found of heterogeneity between slender populations and differentiating populations. In differentiating populations many cells which were committed to division showed v increased expression of the mitochondrial marker mhsp70 and possessed a shortened new flagellum. This was taken as evidence that the slender-to-stumpy differentiation incorporates a round of cell division; a differentiation-division which is distinct from previous proliferative divisions. vi ABBREVIATIONS 5’ UTR 5’ untranslated sequence 5’SL 5’ splice leader sequence 3’UTR 3’ untranslated sequence BrdU 5-bromo-2-deoxyuridine DAPI 4,6-diamidino-2-phenylindole df degrees of freedom DHLADH dihydrolipoamide dehydrogenase dH20 (double) distilled water DIG digoxygenin EATRO East African Trypanosomiasis Research Organization E.M. electron microscope ES expression site ESAG expression site associated gene EST expressed sequence tag FCS foetal calf serum FITC fluoroscein isothiocyanate gRNA guide RNAs GUP Glasgow University Protozoology GUTat Glasgow University Trypanozoon antigenic type hsp heat shock protein J β-D-glucosyl-hydroxymethyluracil kDNA kinetoplast DNA MAP microtubule associated protein mhsp mitochondrial heat shock protein MOPS 3-(N-Mrpholino) propanesulphonic acid MS mean square MTOC microtubule organising centre NIH National Institute of Health PARP procyclic acidic repetitive protein PBS phosphate buffered saline vii PCR polymerase chain reaction PFR paraflagellar rod PSG Puck’s saline glucose RAPD randomly amplified polymorphic DNA RFLP restriction fragment length polymorphism slender form long slender form SS sum of the squares s standard deviation s2 variance S.E. standard error SDS-PAGE polyacrylamide gel electrophoresis using buffers containing sodium dodecyl sulphate stumpy form short stumpy form TAO Trypanosome alternative oxidase Tris Tris(hydoxymethyl)aminomethane TRITC tetramethylrhodamine isothiocyanate UTR untranslated region VAT variable antigenic type VSG variant-specific surface glycoprotein WHO World Health Organization (w/w) weight per weight (w/v) weight per volume viii CONTENTS CHAPTER 1 GENERAL INTRODUCTION 1 1.1 1.2 Trypanosoma brucei 2 1.3 Phylogeny and evolution 3 1.4 Life cycle of T.brucei 7 1.5 Trypanosome cell structure 9 1.5.1 The mitochondrion 11 1.5.2 The kinetoplast 12 1.6 Editing of mitochondrial pre-mRNAs 15 1.7 Glycosomes 18 1.8 The trypanosome nucleus 21 1.9 Trans-splicing of nuclear encode pre-mRNAs 22 1.10 Antigenic variation 25 1.11 The trypanosome cell cycle 32 1.12 Differentiation 34 1.12.1 Differentiation in trypanosomes 37 1.12.2 The stumpy-to-procyclic differentiation 38 1.13 The slender-to-stumpy differentiation 40 The aims of the project 43 ix CHAPTER 2 MATERIALS AND METHODS 45 2.1 Materials 46 2.1.1 The suppliers of consumables 46 2.1.2 Trypanosomes 46 2.1.3 Animals 47 2.1.4 Bacteria 47 2.1.5 Antibodies 48 2.1.6 Solutions and buffers 49 2.1.7 Bacterial culture media 53 2.1.8 Trypanosome culture media 54 2.2 Methods 56 2.2.1 Growth of trypanosomes in rodents 56 2.2.2 Cryopreservation of bloodstream trypanosomes 57 2.2.3 Purification of bloodstream trypanosomes from blood 57 2.2.4 Culture of procyclic trypanosomes 58 2.2.5 Culture of bloodstream trypanosomes 59 2.2.6 Bloodstream form to procyclic form differentiation 59 2.2.7 Preparation of air dried blood smears 60 2.2.8 Preparation of “wet fixed” slides for 60 immunofluorescence and in situ hybridization 2.2.9 Indirect immunofluorescence assay 61 2.2.10 NAD diaphorase assay 62 2.2.11 Preparation of protein samples 63 2.2.12 SDS-PAGE 63 2.2.13 Western blotting 64 2.2.14 Agarose gel electrophoresis of DNA 66 2.2.15 Restriction digestion of DNA 66 x 2.2.16 Polymerase chain reaction 67 2.2.17 Differential display of amplified cDNA fragments 67 2.2.18 Construction of a T-vector 68 2.2.19 Preparation of competent Escherichia coli 69 2.2.20 Purification and cloning of DNA fragments 69 from agarose gels 2.2.21 Ligation and cloning of DNA fragments 70 2.2.22 Small-scale plasmid preparation 71 2.2.23 Large-scale plasmid preparation 71 2.2.24 Preparation of total RNA from trypanosomes 72 2.2.25 Generation of riboprobes 73 2.2.26 Northern blotting 74 2.2.27 In situ hybridization 75 2.2.28 The dideoxy chain termination method 77 for DNA sequencing 2.2.29 Preparation of trypanosome cytoskeletons and 79 transmission electron microscopy xi CHAPTER 3 GROWTH, DIVISION AND DEVELOPMENT 80 OF BLOODSTREAM FORM TRYPANOSOMES 3.1 Introduction 81 3.1.1 Glossary 81 3.1.2 Maintenance of trypanosome parasitaemias 84 3.1.3 Trypanosome morphology during bloodstream form 86 differentiation and cell division 3.1.4 In vitro culture and differentiation of bloodstream forms 87 3.2 Results 91 3.2.1 Trypanosome growth in rodents 91 3.2.2 Bloodstream form trypanosome cytology