THE FUNCTIONAL CHARACTERIZATION OF APICOMPLEXAN TYPE II FATTY ACID SYNTHESIS IN TOXOPLASMA GONDII by JOLLY MAZUMDAR (Under the Direction of Boris Striepen) ABSTRACT Apicomplexan parasites cause important human diseases including malaria and AIDS associated opportunistic infections. Effectiveness of current drug treatments are challenged by side effects and wide spread resistance. The discovery of the apicoplast, an organelle derived from a prokaryote, and the metabolic pathways within, presents novel drug targets unique to the parasite. Apicoplast localized Type II fatty acid synthesis (FASII) is one such pathway. The remarkable divergence of apicoplast FASII from human FASI makes it a potential drug target. But the biological functions of this pathway are currently unknown. Moreover, some apicomplexans including Toxoplasma gondii, encodes an additional FAS I pathway. In the presence of potentially redundant mechanisms, the functional significance of apicoplast FASII remains elusive. The research presented here focuses on the elucidation of apicoplast FASII functions in the apicomplexan, T. gondii. Using a novel two marker approach, we engineered a TgFASII mutant, by the conditional knock-out of acyl carrier protein (ACP), a central FASII component. FASII knock down significantly reduced the growth and viability of parasites in cultured cells. FASII mutants formed smaller plaques, and were unable to establish disease in a mouse model, indicating an essential requirement of FASII for the growth and pathogenesis of T. gondii. Biochemical functions, extensively characterized by protein analysis, immunofluorescence assays, metabolic labeling and fluorescent transgene expression, indicate a role of FASII in the production of lipoic acid, an essential cofactor for the parasite’s sole pyruvate dehydrogenase complex (PDH). We also show a role of FASII in maintenance of the apicoplast. FASII knock down produces drastic effects on apicoplast morphology, resulting in organelle loss. Consistent with previous reports suggesting robust scavenge of fatty acids from the host cell by T.gondii, loss of FASII did not affect bulk fatty acid biosynthesis. In conclusion, we have generated a genetic model for the rigorous analysis of TgFASII functions. We show an essential requirement of apicoplast FASII for the maintenance of the apicoplast and enzymes within, including PDH. Most importantly, the critical nature of apicoplast FASII for the growth and pathogenesis of parasites validate this pathway as a viable drug target. INDEX WORDS: Apicoplast; plastid; fatty acid synthesis (FAS); Type I and Type II FAS; conditional gene knock-out THE FUNCTIONAL CHARACTERIZATION OF APICOMPLEXAN TYPE II FATTY ACID SYNTHESIS IN TOXOPLASMA GONDII by JOLLY MAZUMDAR B.S., University of Bombay, India, 1996 M.S., Barkatullah University, India, 1999 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2006 © 2006 Jolly Mazumdar All Rights Reserved THE FUNCTIONAL CHARACTERIZATION OF APICOMPLEXAN TYPE II FATTY ACID SYNTHESIS IN TOXOPLASMA GONDII by JOLLY MAZUMDAR Major Professor: Boris Striepen Committee: Marcus Fechheimer Jessica Kissinger Kojo Mensa-Wilmot Rick Tarleton Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2006 DEDICATION To my parents. Thank you for your ever lasting love and support. Also to my grandparents (Dadu and Arektama) and to the loving memories of Dadu and Thamma. iv ACKNOWLEDGEMENTS At this very special juncture of my scientific career, I am indebted to many people for their support and guidance. First, I will like to express my sincere gratitude to Dr. Boris Striepen, my major professor and my mentor. His guidance, training, constant encouragement and the countless enlightening conversations has not only helped me achieve this goal, but will also help me as a scientist in years to come. I would also like to thank the members of my committee, Dr. Marcus Fechheimer, Dr. Jessica Kissinger, Dr. Kojo Mensa-Wilmot and Dr. Rick Tarleton for their time and invaluable input into my research. I will like to thank all the past and present members of the Striepen lab. I will especially like to acknowledge Shipra Vaishnava, Chitra Thadhani and Marc-Jan Gubbels, for being good friends and excellent colleagues. I also express my gratitude to Dr. Guan Zhu, Dr. Dominique Soldati, Dr. Geoff McFadden and Dr. Paul Englund for sharing reagents and offering advice. I am indebted to Dr. Christopher Hunter and Dr. Emma Wilson for offering collaborative help. I will also like to acknowledge Ms. Julie Nelson, whose technical help resulted in most of the data presented in Chapter 3. Last but not the least, I will like to express my undying love and gratitude to my mother, father, Joy and Shilpi, CK uncle, aunty and to my close family members, for a lifetime of support, encouragement, and education. A special thanks to Thimmaiah Chendrimada for his wonderful scientific insight and critical reading of the thesis. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.............................................................................................................v LIST OF TABLES......................................................................................................................... ix LIST OF FIGURES .........................................................................................................................x CHAPTER 1 INTRODUCTION .........................................................................................................1 2 THE APICOPLAST; A RELICT CHLOROPLAST IN APICOMPLEXAN PARASITES (REVIEW OF THE LITERATURE)..................................................4 2.1 ENDOSYMBIOTIC ORIGIN OF PLASTIDS..........................................................5 2.2 APICOPLAST DIVISION: A NOVEL MECHANISM..........................................10 2.3 APICOPLAST PROTEIN TARGETING: A NOVEL MACHINERY...................16 2.4 APICOPLAST FUNCTIONS..................................................................................24 2.5 THE APICOPLAST PRESENTS NOVEL DRUG TARGETS ..............................32 2.6 CONCLUSIONS......................................................................................................35 3 A NOVEL SELECTION FOR THE EFFICIENT DETECTION OF ALLELIC REPLACEMENTS IN TOXOPAPLASMA GONDII.............................50 3.1 INTRODUCTION ..................................................................................................52 3.2 MATERIALS AND METHODS............................................................................55 vi 3.3 RESULTS ...............................................................................................................60 3.4 DISCUSSION.........................................................................................................64 4 APICOPLAST FATTY ACID SYNTHESIS IS ESSENTIAL FOR ORGANELLE BIOGENESIS AND PARASITE SURVIVAL IN TOXOPLASMA GONDII.........................................................................................73 4.1 INTRODUCTION ..................................................................................................75 4.2 MATERIALS AND METHODS............................................................................77 4.3 RESULTS ...............................................................................................................81 4.4 DISCUSSION.........................................................................................................87 5 CONCLUSIONS AND FUTURE DIRECTIONS.....................................................104 5.1 CONCLUSIONS....................................................................................................105 5.2 FUTURE DIRECTIONS .......................................................................................106 APPENDIX A: REACTIONS CATALYZED BY FAS II ENZYMES.....................................118 APPENDIX B: FAS II INHIBITOR (THIOLACTOMYCIN) GENERATES APICOPLAST MORPHOLOGY DEFECTS .......................................................120 APPENDIX C: VECTORS FOR TARGETING Tg FAS I PATHWAY...................................122 REFERENCES ...........................................................................................................................124 vii LIST OF TABLES Page Table 1: ToxoDB identification of T. gondii FAS II enzymes ......................................................47 Table 2: Identification of apicoplast genes involved in carbon and energy generation .....................................................................................................49 Table 3: Diversity of apicomplexan lipid biosynthesis................................................................115 viii LIST OF FIGURES Page Figure 2.1: The secondary endosymbiotic origin of the apicoplast...............................................37 Figure 2.2: Schematic representation of the “Chromalveolate tree of life”...................................39 Figure 2.3A: The FtsZ ring is the hallmark of bacterial cell division............................................41 Figure 2.3B: Model illustrating the molecular link between apicoplast and parasite cell division...........................................................................................41 Figure 2.4: Apicoplast protein import............................................................................................43 Figure