Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2016 Tracking the In Vivo Dynamics of Antigenic Variation in the African Trypanosome Monica R. Mugnier Follow this and additional works at: http://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons Recommended Citation Mugnier, Monica R., "Tracking the In Vivo Dynamics of Antigenic Variation in the African Trypanosome" (2016). Student Theses and Dissertations. Paper 317. This Thesis is brought to you for free and open access by Digital Commons @ RU. It has been accepted for inclusion in Student Theses and Dissertations by an authorized administrator of Digital Commons @ RU. For more information, please contact [email protected]. TRACKING THE IN VIVO DYNAMICS OF ANTIGENIC VARIATION IN THE AFRICAN TRYPANOSOME A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy by Monica R. Mugnier June 2016 © Copyright by Monica R. Mugnier 2016 TRACKING THE IN VIVO DYNAMICS OF ANTIGENIC VARIATION IN THE AFRICAN TRYPANOSOME Monica Mugnier, Ph.D. The Rockefeller University 2016 Trypanosoma brucei, a causative agent of African sleeping sickness in humans and nagana in animals, constantly changes its dense variant surface glycoprotein (VSG) coat to avoid elimination by the immune system of its mammalian host, using an extensive repertoire of dedicated genes. Although this process, referred to as antigenic variation, is the major mechanism of pathogenesis for T. brucei, the dynamics of VSG expression in T. brucei during an infection are poorly understood. In this thesis, I describe the development of VSG-seq, a method for quantitatively examining the diversity of expressed VSGs in any population of trypanosomes. Using VSG-seq, I monitored VSG expression dynamics in vivo during both acute and chronic mouse infections. My experiments revealed unexpected diversity within parasite populations, and the expression of as much as one-third of the functional genomic VSG repertoire after only one month of infection. In addition to suggesting that the host-pathogen interaction in T. brucei infection is substantially more dynamic and nuanced than previously expected, this observed diversity highlighted the importance of the mechanisms by which T. brucei diversifies its genome-encoded VSG repertoire. During infection, the parasite can form mosaic VSGs, novel variants that arise through recombination events within the parasite genome during infection. Though these novel variants had been identified previously, little was known about the mechanisms by which they form. VSG-seq facilitated the identification of mosaic VSGs during the infection, which allowed me to track their formation over time. My results provide the first temporal data on the formation of these variants and suggest that mosaic VSGs likely form at sites of VSG transcription. VSG-seq, which is based on the de novo assembly of VSGs, obviates the requirement for a reference genome for the analysis of expressed VSG populations. This allows the method to be used for the high-resolution study of VSG expression in any strain of T. brucei, whether in the lab or in the field. To this end, I have applied VSG-seq to samples grown in vitro, parasites isolated from natural infections, and extravascular parasites occupying various tissues in vivo. These extensions of the method reveal new aspects of T. brucei biology and demonstrate the potential of high-throughput approaches for studying antigenic variation, both in trypanosomes and in any pathogen that uses antigenic variation as a means of immune evasion. For my Dad. iii ACKNOWLEDGEMENTS I would like to thank my advisor, Nina Papavasiliou, for her enthusiasm and support throughout this process. Her belief in my abilities as a scientist gave me the confidence to pursue things, both scientifically and professionally, that I may not have been brave enough to try otherwise. I owe so much to her for her encouragement. I have been lucky enough to have a second advisor in George Cross, whom I thank for his incredible support, encyclopedic knowledge of VSG-related papers, and many genome assemblies. His critical approach to science has taught me so much. I thank my committee members, Kirk Deitsch and Michel Nussenzweig, for their time and thoughtful advice, along with Christian Tschudi for serving as the external examiner at my thesis defense. I am very grateful to my collaborators who have contributed to the work presented in this thesis: Keith Matthews, Al Ivens, Isabel Roditi, Kapila Gunasekera, Stijn Deborggraeve, Veerle Lejon, Luisa Figueiredo, and Filipa Ferreira. I would also like to thank the students who have contributed to this work and taught me a thing or two about being a mentor, Chris Patacsil, Jake Scott, and Tom Hart. Of course, my time in graduate school would not have been so much fun if it weren’t for all of the members of the Papavasiliou Lab. Thanks especially to Claire Hamilton, Eric Fritz, and Brad Rosenberg, for teaching me about sequencing and being my first friends in the lab, to Danae Schulz and Maryam Zaringhalam for the wine-fueled practice talks and trips to Baker St., and to Jason Pinger, Catherine Boothroyd, Galadriel Hovel-Miner, and Hee-Sook Kim for camaraderie in the world of trypanosomes. iv Thanks to Tom, Donovan, Kavi, Kate, Roman, and Ben for being my crew at Rockefeller. I am so lucky to have met all of you. My best friend, Melissa, deserves credit for cultivating my love for science. I can’t help but think our days in elementary school planning the Mugnier-Slane Animal Hospital have something to do with our eventual careers in biology. I thank my father, Joe Mugnier, for everything. A certain poem by Billy Collins comes to mind when I think about attempting to express my gratitude to him… Finally, I must thank my roommates and family, Bailey, The Stallion, and Patrick, for their walks, head-butts, dinners, and love. v TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................ iv TABLE OF CONTENTS ................................................................................................ vi LIST OF FIGURES ....................................................................................................... viii LIST OF TABLES ........................................................................................................... ix CHAPTER 1. Introduction ............................................................................................. 1 1.1 Trypanosoma brucei ................................................................................................. 1 1.2 The variant surface glycoprotein and antigenic variation ......................................... 4 1.3 VSG expression ......................................................................................................... 6 1.4 VSG switching ........................................................................................................... 8 1.5 The genomic VSG repertoire ..................................................................................... 9 1.7 Mosaic VSGs ........................................................................................................... 11 1.7 Mechanisms of mosaic formation ........................................................................... 12 1.8 Order in VSG switching .......................................................................................... 13 1.9 Dynamics of antigenic variation ............................................................................. 14 1.10 Antigenic variation in vivo: Rates of switching .................................................... 15 1.11 Antigenic variation in vivo: Diversity ................................................................... 16 1.12 The B cell response to T. brucei ........................................................................... 17 1.13 Antibody-parasite interactions .............................................................................. 19 1.14 Mosaic VSGs and antibody cross-reactivity ......................................................... 20 1.15 The T cell response to T. brucei and T cell recognition of VSG .......................... 21 1.16 Outstanding questions ........................................................................................... 22 CHAPTER 2. Development of a broadly applicable method for quantifying VSG expression ......................................................................................................................... 23 2.1 Introduction ............................................................................................................. 23 2.2 Optimization of VSG-seq library preparation strategy ........................................... 24 2.2.1 Enrichment for VSG sequences ........................................................................ 28 2.2.2 Read coverage across VSG genes .................................................................... 28 2.3 Optimizing VSG-seq analysis: mappability ............................................................ 31 2.4 Characterizing VSG-seq ......................................................................................... 33 2.4.1 Assembly of VSGs and relationship to number of input cells ......................... 36 2.4.2 Limits of quantification of VSG expression ....................................................