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Plasmodium Berghei SEXUAL AND SPOROGONIC DEVELOPMENT OF PLASMODIUM BERGHEI Anne Louise Dearsly A thesis submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy of the University of London and Diploma of Imperial College Imperial College Department of Pure and Applied Biology Prince Consort Road London October 1990 THE SEXUAL AND SPOROGONIC DEVELOPMENT OF PLASMODIUM BERGHEI. Plasmodium berghei infections were studied in vivo and in vitro and the production of gametocytes and ookinetes following 3, 8 and 14 sequential blood passages monitored. This allowed the definition of optimal conditions for the collection of parasite material for the study of the gametocyte and ookinete stages. The study also led to the identification of reliable indicators of ookinete production. A method was developed using mitomycin Cto obtain enriched/purified gametocyte populations. Giemsa stained smears of infected mouse blood showed an immediate drop in the asexual parasitaemia while the immediate numbers of gametocytes remained unaffected. Treated gametocytes went on to produce ookinetes in culture that were infective to mosquitoes and readily purified by Nycodenz centrifugation. Ookinete cultures were treated with metabolic inhibitors of DNA, RNA and protein synthesis and the effect on the morphological development was studied at both the light and electron microscope level. The effect of the inhibitors on protein synthesis was also studied by radiolabelling in the presence of the drugs and the differences between asexual and sexual stage protein synthesis determined. The synthesis of the 21kD transmission blocking protein was followed from the early asexual trophozoite to the mature ookinete. The time of the initial synthesis and expression on the surface of the parasite was established. Attempts to identify the trigger were made and indicated that synthesis is induced by gametogenesis and not fertilization. Glucosamine/mannose labelling of the protein and preliminary detergent extraction with triton X-114 were carried out to further characterise the protein. 2 Contents Page Acknowledgements ............................................................................. 13 Abbreviations ............................................................................................... 14 Chapter 1 General Introduction 1.1 Malaria and its control ................................................ 16 1.2 The Parasite ............................................................................ 16 1.3 The Plasmodial Life Cycle ..................................................... 16 1.3.1 Gametocyte Density................................................. 23 1.3.2 Blood Factors...................................................... 23 1.3.3 Host Immune Factors............................................. 24 1.4 Antimalarial Vaccines ........................................................... 26 1.5 Plasmodium berghei .............................................................. 27 1.6 The Biochemistry of Plasmodium ....................................... 27 1.7 Overview and Aims ..................................................... 30 Chapter 2 The Sexual Development of Plasmodium berghei in vivo and in vitro 2.1 Introduction ......................................................................................... 33 2.2 Materials and Methods ........................................................................ 36 2.2.1 In vivo Infection ................................................................... 36 2.2.2 Red blood cell count ................................................................ 36 2.2.3 Parasitaemia ............................................................................ 36 2.2.4 Exflagellation .......................................................................... 37 2.2.5 Culture Medium .................................................................... 37 2.2.5.1 Complete Ookinete Culture Medium ....................... 37 2.2.5.2 Methionine-free RPMI 1640 ..................................... 37 2.2.5.3 Serum-free Ookinete Medium ................................. 37 2.2.5.4 Gametocyte Culture Medium .................................... 38 2.2.5.5 Serum-free Gametocyte Medium ........................ 38 2.2.6 Ookinete Production .............................................................. 38 2.3 Results.................................................................................................... 39 2.3.1 Comparison of Asexual and Mixed Clones of P.berghei ...... 39 2.3.2 Asexual Parasitaemia ............................................................. 39 2.3.3 Red Blood Cell Count ............................................................ 39 3 Page 2.3.4 Sexual Parasitaemia ....................................................... 43 2.3.5 Production of Gametocytes During the Course of an Infection .................................................................................. 43 2.3.6 The Effect of Blood Passage on Gametocyte Production 37 and Activity .................................................................... 43 2.3.7 Ookinete Production .............................................................. 53 2.3.8 Sexual Stage Parasites as Indicators of Ookinete Production .............................................................................. 56 2.3.9 The Effect of Red Blood Cell Count on Ookinete Formation in vitro ................................................................ 56 2.3.10 The Effect of Asexual Parasites on Ookinete Formation in vitro ................................................................................... 58 2.4 Discussion ..................................................................................... 59 2.4.1 Conclusions ....................................................................... 67 Chapter 3 The Enrichment of Plasmodium berghei Gametocytes in vivo and ookinetes in vitro 3.1 Introduction ......................................................................................... 69 3.2 Materials and Methods ....................................................................... 70 3.2.1 Parasites ............................................................................ 70 3.2.2 Gametocyte Production ......................................................... 70 3.2.3 The Effect of the Duration of Mitomycin C Treatment ...... 70 3.2.4 Ookinete Culture ................................................................... 71 3.2.5 Ookinete Purification .................................................... 71 3.2.6 Mosquito Feeds ............................................................. 72 3.2.7 Radiolabelling of the Nucleic Acids ..................................... 72 3.3 Results ................................................................................................... 74 3.3.1 Mitomycin C treatment of P.berghei Parasites ................... 74 3.3.1.1 Effect of Time ............................................................. 74 3.3.1.2 Effect of Mitomycin C Concentration ........................ 74 3.3.2 Further Purification of Treated Parasites ...................... 79 3.3.3 Viability of Mitomycin C Treated Gametocytes .................. 79 3.3.4 Effect of Mitomycin C on the Incorporation of 32p into Nucleic Acids .......................................................................... 79 3.4 Discussion .................................................................................... 84 3.4.1 Conclusions ...................................................................... 87 4 Page Chapter 4 The Effect of Metabolic Inhibitors on the Development of Plasmodium berzhei Gametocvtes and Ookinetes 4.1 Introduction ........................................................................................ 89 4.2 Materials and Methods ....................................................................... 93 4.2.1 Micro Drug Culture Technique ......................................... 93 4.2.2 Electron Microscopy ............................................................ 95 4.2.3 35S-Methionine Labelling of Ookinete Cultures .............. 96 4.2.4 Analysis of Protein Samples ..................................... 96 4.2.5 SDS-PAGE ..................................................................... 99 4.2.6 Coomassie Blue Staining of Gels .............................. 100 4.2.7 Drying and Autoradiography of Gels ................................ 100 4.2.8 Densitometry of Autoradiographs .................................... 101 4.3 Results ................................................................................................ 102 4.3.1 Total Protein Synthesis .............................................. 102 4.3.2 Exflagellation ....................................................................... 102 4.3.3 Ookinete Development ...................................................... 112 4.3.3.1 Electron Microscopy of Untreated Control Cultures ................................................................... 112 4.3.3.2 DNA Synthesis Inhibitors ..................................... 112 4.3.3.2.1 Light Microscopy ..................................... 112 4.3.3.2.2 Electron microscopy ................................ 112 4.3.3.3 RNA Synthesis Inhibitors ..................................... 113 4.3.3.3.1 Light Microscopy ..................................... 113 4.3.3.3.2 Electron Microscopy ................................ 113 4.3.3.4 Protein Synthesis Inhibitors ................................... 122 4.3.3.4.1 Light
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