UNIVERSITY OF CALIFORNIA, SAN DIEGO Plasmodium Sexual Development and the Role of Plasmepsin X in Plasmodium falciparum transmission to Anopheles gambiae A Dissertation submitted in partial satisfaction of the Requirements for the Degree of Doctor of Philosophy in Biomedical Sciences by Viengngeun Bounkeua Committee in charge: Professor Joseph M. Vinetz, Chair Professor James R. Feramisco Professor Frances D. Gillin Professor Victor Nizet Professor Sharon L. Reed Professor Elizabeth A. Winzeler 2010 © Viengngeun Bounkeua, 2010 All rights reserved. The Dissertation of Viengngeun Bounkeua is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2010 iii DEDICATION In recognition of all those who have believed in me and helped me to reach this day: - my parents, Kingngeun Bounkeua and Dara Strong, who taught me to be compassionate, dedicated and hardworking through their own example - all of my family, especially my sister, Kay Bounkeua who lent countless hours of early-morning and late-night support, and my step-father, Jeffrey Strong - my research mentors: Dr. Jeffrey Griffith from the University of New Mexico; Phil Rodacy, Pamela Walker and Susan Bender from Sandia National Laboratories; Dr. Michael Lenardo from the National Institutes of Health, and Dr. Joseph Vinetz, my thesis advisor - my thesis committee, for their enthusiastic and thoughtful scientific input - members of the Vinetz lab, for their teachings and for creating an engaging learning environment for the past 4 years, especially Dr. Fengwu Li and Dr. Michael Matthias, Kenny Pettersen, Paula Maguina, Dr. Christian Ganoza and Dr. Shira Abeles - my graduate colleagues, for their technical help, moral encouragement and friendship, especially Neekesh Dharia, Jennifer Aron and Joshua Theisen - my dear friends, who loved and supported me during this time: Emily Mathews, Sherry He, Ilya Gertsman, Caleb Stokes, and Tara Herrmann iv TABLE OF CONTENTS Signature Page ............................................................................................................... iii Dedication ...................................................................................................................... iv Table of Contents ........................................................................................................... v List of Abbreviations .................................................................................................... vii List of Figures ............................................................................................................... xii List of Tables ............................................................................................................... xiv Acknowledgements ...................................................................................................... xv Vita .............................................................................................................................. xvi Abstract of the Dissertation ......................................................................................... xix Chapter 1 Introduction .................................................................................................... 1 Works cited ........................................................................................................... 9 Chapter 2 Biological features of in vitro-generated Plasmodium falciparum sexual stage parasites ............................................................................................................... 12 Abstract ............................................................................................................... 13 Introduction. ........................................................................................................ 13 Methods ............................................................................................................... 14 Results and Discussion. ....................................................................................... 20 Conclusion ........................................................................................................... 37 Works cited ......................................................................................................... 41 Chapter 3 Ex vivo-cultivation of Plasmodium vivax ookinetes .................................... 46 Abstract ............................................................................................................... 47 Introduction. ........................................................................................................ 47 Methods ............................................................................................................... 48 Results and Discussion. ....................................................................................... 52 Conclusion ........................................................................................................... 61 Works cited ......................................................................................................... 64 Chapter 4 The role of Plasmepsin X in Plasmodium transmission to mosquitos ......... 67 Abstract ............................................................................................................... 68 Introduction. ........................................................................................................ 68 Methods ............................................................................................................... 70 v Results and Discussion. ....................................................................................... 79 Conclusion ......................................................................................................... 101 Works cited ....................................................................................................... 105 Chapter 5 Proteomic analysis of Plasmodium falciparum sexual stage parasites and Plasmodium vivax macrogametes ........................................................................ 112 Abstract ............................................................................................................. 113 Introduction. ...................................................................................................... 113 Methods ............................................................................................................. 114 Results and Discussion. ..................................................................................... 120 Conclusion ......................................................................................................... 141 Works cited ....................................................................................................... 163 vi LIST OF ABBREVIATIONS 2DGE – two-dimensional electrophoresis 6Fam – 6-carboxyfluorescein BHQ – black hole quencher bp – base pairs cap – central apical pore cry – crystalloid CTRP – circumsporozoite and TRAP related protein DAPI – 4’,6-diamidino-2-phenylindole DHFR – dihydrofolate reductase DIG – digoxigenin DMEM – Dulbecco’s modified eagle’s media DNA – deoxyribonucleic acid DOZI – development of zygote inhibited DTT – dithiothreitol dv – digestive vacuole e – erythrocyte EDTA – ethylenediaminetetraacetic acid EGTA – ethylene glycol tetraacetic acid ELISA – enzyme-linked immuno-sorbant assay EM – electron microscopy FCS – fetal calf serum FITC – fluorescein isothiocyanate vii FKBP – forkhead binding protein gc – gametocyte GEMS – gene-enrichment motif searching gm – macrogamete GO – gene ontology GS – gamete exflagellation/emergence solution h – hemozoin HEPES – 4-(2-hydroxyethyl)-1-piperazineethanesulufonic acid HS – heat inactivated human serum IACUC – Institutional Animal Care and Use Committee IFA – immunofluorescence assay IgG – immunoglobulin subtype G IgM – immunoglobulin subtype M IMC – inner membrane complex IP – immunoprecipitation IPTG – isopropyl β-D-1-thiogalactopyranoside kDa – kilodalton KO – knockout l – liter LB-amp – laurel broth with ampicillin LCCL – Limulus factor C, cochlear protein Coch-5b2, and late gestation lung protein Lgl1 viii Leukostat – modified Wright stain used to visualize Plasmodium parasites by light microscopy m – microneme M – molar, moles of reagent / liter of solution mAb – monoclonal antibody MALDI-TOF – matrix-assisted laser desorption/ionization time-of-flight MCB – master cell bank µF – microfarad µg – microgram ml – milliliter µm – micrometer mM – millimolar MS – mass spectrometry mt – mitochondria MudPIT – multidimensional protein identification technology n – nucleus N – normal, gram equivalents of H+ ions / liter of solution NF – Nijmegen Falciparum isolate nm – nanometer o – ookinete OPI – ontology-based pattern identification PAGE – polyacrylamide gel electrophoresis PBS – phosphate buffered saline ix pCAM-BSD – genetic integration construct containing blasticidin-S-deaminase PCR – polymerase chain reaction PFA – paraformaldehyde Pfs16 – Plasmodium falciparum surface protein 16 Pfs25 – Plasmodium falciparum surface protein 25 pI – isoelectric point pm – plasma membrane PM – plasmepsin pr – polar ring pRBC – packed red blood cells PRISMA – Projectos en informatica medicina salud y agricultura Puf – Pumilio in Drosophila melanogaster and fem-3 binding factor in Caenorhabditis elegans Pvg377 – Plasmodium vivax gamete protein 377 Pvs230 – Plasmodium vivax surface protein 230