ABSTRACT Title of Dissertation: MOLECULAR EVOLUTIONARY
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ABSTRACT Title of Dissertation: MOLECULAR EVOLUTIONARY STUDIES ON TRYPANOSOMA CRUZI, THE AGENT OF CHAGAS DISEASE Carlos A. Flores-López, Doctor of Philosophy, 2013 Dissertation directed by: Professor Carlos A. Machado Department of Biology The use of DNA sequences to address diverse evolutionary questions has increased steadily with the growing availability of genome sequence data. In this study, I make use of DNA sequence data to describe several evolutionary aspects of the protozoan parasite responsible for Chagas disease, Trypanosoma cruzi. Chagas is estimated to infect 7.7 million people and cause the deaths of approximately ten thousand people every year in Latin America. Just like many other parasitic diseases, Chagas does not have a vaccine or an effective drug treatment. In this body of work, I specifically: (1) describe the evolutionary history of the major strains of the parasite through the use of phylogenetic analyses of 32 loci and demonstrate that the parasite’s original classification into two major evolutionary lineages does not reflect the evolutionary history of the parasite, (2) demonstrate that there is strong evidence for just one major recent hybridization event during the history of T. cruzi divergence and not two as previously suggested, (3) show that all major extant T. cruzi lineages diverged recently (less than 3 million years ago), well before the arrival of humans in the Americas, (4) describe a new T. cruzi lineage that appears to have diverged in North America (“TcNA”), (5) show that a significantly larger fraction of protein-coding genes have experienced positive selection in T. cruzi than in Leishmania spp., a pattern likely due to the greater versatility of T. cruzi in its host range, cell tropism and cell invasion mechanisms, and (6) illustrate a recent major expansion of a few surface protein families in T. cruzi that seem to be linked to the evolution of the parasite’s ability to invade multiple cell tissues and multiple host species. These results demonstrate the applicability and power of molecular evolutionary analyses for understanding parasitic diseases. MOLECULAR EVOLUTIONARY STUDIES ON TRYPANOSOMA CRUZI, THE AGENT OF CHAGAS DISEASE by Carlos A. Flores-López Thesis submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2013 Advisory Committee: Professor Carlos A. Machado, Chair Professor Michael P. Cummings Professor Charles Delwiche Professor Najib El-Sayed Professor Thomas D. Kocher © Copyright by Carlos A. Flores-López 2013 DEDICATION Esta tesis está dedicada a la persona que me inspiró en los momentos más difíciles a siempre mirar hacia delante. A mi hijo Ulises. ii ACKNOWLEDGMENTS I wouldn’t have been able to complete my PhD education without the wonderful support I have received from my parents Margarita López-Martínez and Carlos F. Flores-Luna. Their leadership by example, love and support since I was kid has always kept me motivated to pursue my goals. If I hadn't looked up to them I would have never been introduced to the many treasures that are connected with a life in science. I would also like to thank my advisor Dr. Carlos A. Machado for all the years I spent under his wing as his student. His support in all the definitions of the word (both work related and personal) has meant so much to me. This work wouldn’t have been possible without his help. I would also like to thank each member of my committee: Dr. Charles Delwiche, Dr. Michael Cummings, Dr. Thomas Kocher and Dr. Najib El-Sayed for the guidance, advice and support I received throughout my years at Maryland. This work would not of been possible without their crucial input. The many members of the Machado lab throughout the years that have been so supportive throughout all these years and who I owe so much, I say thank you. Lastly I would like to thank all my friends and colleagues that have supported me throughout the years (some a long time ago when this adventure began at the University of Arizona, some more recently when the adventure moved to Maryland, and some all the iii way back from México). Without their support and friendship in the stressful and hard times that come along with a PhD and the time spent away from my family and country for so many years, it is undisputable I would not been able to accomplish this work. Alejandra Velázquez, Gabi Flores, Javier and Liza Montenegro, Shane Bryan, Brian Dalton, Laura Rascón, Gladys Erives, Matt Palmer, Kawther Abdilleh, Kevin Nyberg, Bibek Yumman, Guillerhme Targino-Valente, Chris Arias, Matt Palmer, Miguel Carneiro, Rafael Tannus, Gonzálo de León-Girón, Alejandro González, Alonso Gutierrez, Ricardo Cruz, Ricardo Valencia, Sebastian Gómez, Enrique Gómez, Leonardo Carvalho and many others. Chapter 2. I thank 4 reviewers for their comments, and Michel Tibayrenc and Christian Barnabé for kindly providing T. cruzi DNA Chapter 3. I would like to thank all the collaborators of this project: Mitchell E., Reisenman C.E., and Williamson P. for their insightful comments on the manuscript. I would also like to thank Dr. Christian Barnabé for kindly donating T. cruzi reference strains (TcI-VI), to Teresa Gregory and William Savary for help collecting insects. Chapter 4. I would like to thank Maximilian J. Telford for help on the translation/alignment analysis, and Kawther Abdilleh and Guilherme Targino Valente for constructive discussions. iv TABLE OF CONTENTS Dedication ………………………………………………………………………………. ii Acknowledgments ……………………………………………………………………… iii Table of Contents ……………………………………………………………………….. v List of Tables ……………………………………………………………………………vii List of Figures ……………………………………………………………………………ix Chapter 1: Introduction to Dissertation ………………………………………………… 1 Chapter 2: Analyses of 32 loci clarify phylogenetic relationships among Trypanosoma cruzi lineages and support a single hybridization prior to human contact ……………… 5 Abstract ……………………………………………………………………………….. 6 Introduction …………………………………………………………………………… 7 Materials and Methods ……………………………………………………………….. 10 Results ………………………………………………………………………………... 16 Discussion …………………………………………………………………………… 20 Conclusion …………………………………………………………………………… 28 Tables ………………………………………………………………………………… 30 Figures ………………………………………………………………………………... 38 Chapter 3: Description of a new Trypanosoma cruzi lineage from the United States reveals an introduction into North America during the Pleistocene …………………….41 Abstract ………………………………………………………………………………..42 Introduction ……………………………………………………………………………42 Materials and Methods ……………………………………………………………….. 46 Results …………………………………………………………………………………49 Discussion ……………………………………………………………………………..52 Conclusions ……………………………………………………………………………55 Tables ………………………………………………………………………………….56 Figures …………………………………………………………………………………60 Chapter 4: Positive selection has played a larger role in the evolution of Trypanosoma cruzi proteins than in the evolution of Leishmania spp. proteins ……………………….64 Abstract ………………………………………………………………………………. 65 Introduction ……………………………………………………………………………65 Materials and Methods ……………………………………………………………….. 69 Results …………………………………………………………………………………74 Discussion ……………………………………………………………………………..78 Tables ………………………………………………………………………………….83 Figures …………………………………………………………………………………86 v Chapter 5: Genomic changes associated with the evolution of multicellular invasion ability and adaptation to multiple hosts in a pathogen: Protein family expansions in the parasitic lifestyle of Trypanosoma cruzi ……………………………………………….. 91 Abstract ………………………………………………………………………………. 92 Introduction ……………………………………………………………………………92 Materials and Methods ………………………………………………………………...94 Results …………………………………………………………………………………96 Discussion ……………………………………………………………………………..99 Figures ………………………………………………………………………………..105 Chapter 6. Overview and Conclusions to Dissertation ………………………………...108 Overview ……………………………………………………………………………..108 Future directions ……………………………………………………………………..111 Conclusion …………………………………………………………………………...113 Appendices ……………………………………………………………………………..114 Bibliography …………………………………………………………………………...220 vi LIST OF TABLES Table 2-1. The main Trypanosoma cruzi strains used in this study ................................. 30 Table 2-2. List of loci included in this study ................................................................... 31 Table 2-3. Results from the selection, distance and midpoint rooting analyses ……….. 34 Table 2-4. Bayesian estimates of divergence time (in mya) for different T. cruzi lineages …………………………………………………………………………………………. 37 Table 3-1. Information for isolated samples from Texas and Arizona ........................... 56 Table 3-2 Bayesian estimates of divergence (time in millions of years) for the main T. cruzi lineages and TcNA ……………………………………………………………… 59 Table 4-1. The number of proteins under positive selection in T. cruzi and Leishmania spp ……..……..……..……..……..……..……..……..……..……..……..……..…….. 83 Table 4-2. Functional over representation of genes showing evidence of positive selection ………….……..……..……..……..……..……..……..……..……..……..……..…….. 84 Table 4-3. Statistically overrepresented hypothetical protein clusters under positive selection in T. cruzi and Leishmania spp. …………………………………………….. 85 Table S2-1. Additional Trypanosoma cruzi strains used for some of the loci ……… 114 Table S2-2. Amplified nuclear loci and PCR primers ……………………………… 115 Table S2-3. Results of the Shimodaira-Hasegawa tests ……………………………… 117 Table S2-4. LRT of Molecular clock on genes that had a homolog in T. brucei