UNIVERSITY OF CALIFORNIA, IRVINE Unraveling host and parasite pathways in human innate immunity to Toxoplasma gondii DISSERTATION submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biological Sciences by Lanny Gov Dissertation Committee: Assistant Professor Melissa B. Lodoen, Chair Professor Andrea J. Tenner Professor Craig M. Walsh 2014 Chapter 2 © 2013 American Society for Microbiology All other materials © 2014 Lanny Gov DEDICATION To My mother: my hero and my champion; Jonathan Farnworth: my better half; My loving and supportive family: brothers George and James, and sister-in-law Kelly; My dearest friends: Eugenia Hu, Quen Vong, Tracy Tang, and Erika Villaruel Thank you for so many years of love, laughter, and encouragement, and for helping me believe I could accomplish anything. “Promise me you’ll always remember: You’re braver than you believe, and stronger than you seem, and smarter than you think.” –A.A. Milne, Winnie-the-Pooh “You don’t have to know everything, but you should try to.” -Nori Ueno, on preparation for my Advancement exam ii TABLE OF CONTENTS Page LIST OF FIGURES iv LIST OF TABLES v ACKNOWLEDGMENTS vi CURRICULUM VITAE ix ABSTRACT OF THE DISSERTATION xii CHAPTER ONE: Introduction to Toxoplasma gondii Immunity 1 Toxoplasma gondii Biology 2 T. gondii Clinical Disease 6 T. gondii Invasion of Host Cells 9 Three Modes of T. gondii Intracellular Entry 12 Immunity to T. gondii 13 Toll-like Receptor Sensing of T. gondii 15 Nod-like Receptor Sensing of T. gondii 17 Modulation of Host Cell Pathways by T. gondii Effectors 19 Monocytes in Immunity to T. gondii 26 CHAPTER TWO: Human Innate Immunity to Toxoplasma gondii is 31 Mediated by Host Caspase-1 and ASC and Parasite GRA15 Introduction 32 Results 35 Discussion 50 Materials and Methods 54 Acknowledgments 61 CHAPTER THREE: Toxoplasma gondii Preferentially and Actively 62 Invades Classical and Intermediate Human Monocytes Introduction 63 Results 66 Discussion 80 Materials and Methods 83 Acknowledgements 87 CHAPTER FOUR: Concluding Remarks 88 REFERENCES 97 iii LIST OF FIGURES Page Figure 1.1 Strain-specific modulation of host immunity by T. gondii ROP18 24 Figure 2.1 IL-1β and IL-18 synthesis in primary human monocytes and THP-1 cells in 36 response to T. gondii infection Figure 2.2 T. gondii induction of IL-1β in shASC cells 38 Figure 2.3 Role of caspase-1 in parasite-mediated IL-1β release 41 Figure 2.4 Effects of blocking parasite invasion on IL-1β production 43 Figure 2.5 Strain specificity of T. gondii-induced IL-1β in human monocytes 46 Figure 2.6 Role of GRA15 in T. gondii-mediated IL-1β induction in human monocytes 48 Figure 3.1 Among human PBMC, T. gondii is preferentially associated with monocytes 67 Figure 3.2 Among elutriated human monocytes, T. gondii is preferentially associated 69 with classical and intermediate monocytes Figure 3.3 A majority of intracellular T. gondii are GRA7+ 71 Figure 3.4 Classical and intermediate monocytes are more permissive to parasite 75 survival Figure 3.5 Intracellular IL-1β expression levels in the three monocyte subsets 77 iv LIST OF TABLES Page Table 1.1 T. gondii virulence factors and their strain-specific effects on the host cell 21 Table 1.2 Heterogeneity among monocytes 28 v ACKNOWLEDGMENTS Among the most valuable things I have gained in the past five years of graduate school is all the new relationships I have been fortunate enough to have forged. The multitude of mentors, friends, and mentees I have worked with here have undeniably shaped not only my scientific career, but have enriched my life in countless ways. First and foremost, I would like to express my deepest gratitude and sincere appreciation for my thesis advisor, Dr. Melissa Lodoen. Over the years, Melissa has become much more than just “my boss.” She is an outstanding scientist and the embodiment of a great mentor. Melissa’s door was always open, and she answered any and every question I had with patience, enthusiasm, and kindness. Her silver lining optimism is one trait I value dearly and will sorely miss. She always found a bright side when experiments failed, and this was especially meaningful when whole projects failed, something I was not a stranger to. Melissa has helped shape me into the scientist and person that I am today, and for that, I am incredibly fortunate. Dr. Naomi Morrissette has informally been a second advisor to me and she’s the person I look for when I can’t find Melissa. Naomi, thank you for all the moral support and intellectual contributions that you’ve given to me throughout the years. Your smiling face and nods of approval has gotten me through many talks, especially as an early stage graduate student. Your sense of humor and warmth has greatly enriched my time here. The Lodoen Lab has been a wonderful “home away from home” to me over the years and I would like to thank them all for uniquely shaping my graduate experience. In particular, my lab hubby, Pedro Morgado, was here with me from the beginning. Together, we were Melissa’s first graduate students, and we overcame many challenges together. I could turn to Pedro vi whenever things were rough, both at work and in my personal life. Pedro, thank you for your love, support, countless hugs, and even more coffee dates. Nori Ueno, I only wish you had joined our lab sooner. You brought with you a drive for science and perfection that has undoubtedly rubbed off on me. You have been a wonderful mentor, friend, confidant, and colleague, especially this last year. It has been a bumpy year, filled with long days and late night time-points, but it has been fun with you by my side (even when we go on our adventures to the trashcans). Last but not least of the Lodoen Lab, I would like to thank my mentee, Alborz Karimzadeh. Alborz worked with me as a volunteer after graduating and contributed vastly to the IL-1β story told here. Alborz brought a drive to our project that few undergrads do, and quickly became an independent worker. Contrary to how a mentor-mentee relationship is supposed to work, he lifted my spirits when experiments were not working! Alborz, your optimism and tenacity will serve you well as you embark on your own Ph. D. journey. I was lucky to have had the opportunity to train you and am proud of all you have and will do. To my friends, old and new, thank you for being there for me. I was fortunate enough to have crossed paths and built lasting friendships with Elizabeth Clarke, Roxanna Ochoa, Heidi Contreras, Michael Hernandez, Thomas Lechuga, Kelley Fracchia, Vivian Prindle, Sharmila Mallya, and Milton Greenberg here at UCI. No one quite understands the trials and tribulations of graduate school like a fellow graduate student does. Words cannot express how grateful I am for the lunch and coffee dates, vent sessions, and laughter that we shared. I would also like to thank all my old friends who thought I was crazy for pursuing a graduate degree after spending my whole life in school, but supported me anyways: Eugenia Hu, Quen Vong, Erika Villaruel, and Tracy Tang. You girls are my rock and I would have quit long ago if it weren’t for you. Eugenia, vii thank you for always finding time for me and for making things better. Quen, even though you are far away and our crazy schedules make it difficult to catch each other, I know you have been looking out for me since sixth grade. Erika and Tracy, you girls keep me sane and more importantly, young. To all my girls, thank you for your unconditional love and the many ways you express it; you are family to me. I would not be where I am today without my family. As the youngest child, I was extremely lucky to have not one, but two, older brothers to look up to. George and James, along with my new sister Kelly, have given me unconditional love and support as I navigate the rough waters that is grad school. Thank you for always listening to my project updates (well, for George, mostly listening to me), sharing in my accomplishments, and encouraging me to overcome my setbacks. Jon, my better half, the year I met you was indeed a hard one, and I did not quite come to realize how lucky I was at the time. Thank you for standing by me through some of my most challenging moments, and time and time again, believing in me and helping me believe in myself. I now realize how lucky I am. I am deeply grateful to have you in my life, and cannot thank you enough for all the love, hugs, and countless massages you have given me. Last, but not least, this dissertation is dedicated to my mother. My whole life, I have seen my mom work so hard and sacrifice so much so that I could have a better life. She persevered against all odds and raised three Bruins along the way! Her resilience has been a constant inspiration to me, especially when life gets tough. From an early age, she instilled in me the value of an education and has steadfastly supported me every step of the way. All that I have accomplished and all that I will is because of her. Mom, I love you and I couldn’t have done this without you. viii CURRICULUM VITAE Lanny Gov EDUCATION UNIVERSITY OF CALIFORNIA, IRVINE, Irvine, CA 2009-2014 Ph.D.