EVASION OF ADAPTIVE IMMUNE DEFENSES BY THE LETHAL CHYTRID FUNGUS BATRACHOCHYTRIUM DENDROBATIDIS By Jeffrey Scott Fites Jr. Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biological Sciences May, 2014 Nashville, TN Approved: Katherine Friedman, Ph. D., chair Clint Carter, Ph. D. Julián Hillyer, Ph. D. D. Borden Lacy, Ph. D. Louise Rollins-Smith, Ph. D., thesis advisor DEDICATION To my family, My loving wife Meg, My newborn son Peter, My parents Jeff and Robin, And my sister Kateri. ii ACKNOWLEGMENTS I have many people to acknowledge for the achievements I have made in graduate school both personal and academic. I could not have accomplished a paper in Science or completed my Ph. D. thesis work without their assistance and support. I practiced karate for eight years from elementary school through high school. At every promotion to a higher rank, my karate instructor would remind all the students that they did not make achievements alone and that they must “put all their ducks in a row” to thank all the people responsible helping us along our way. He would say that we should start by thanking the oldest living members of our families, our parents, grandparents, and great-grandparents because, “if it wasn’t for our great-grandparents, our grandparents wouldn’t be here; and if it wasn’t for our grandparents, our parents wouldn’t be here; and if it wasn’t for our parents, we wouldn’t be here.” In such fashion, I will begin by thanking my oldest living relatives, my grandparents, Jack and Pauline Fites. When I get a chance to call up my grandparents I often discuss my research or scientific research in general with my granddad. My grandparents are by no means scientists, but my granddad would always discuss research and graduates school with great interest. When I told them that I had published a paper in Science, they did not really understand what that meant, but they were both excited and proud when I told them. My parents, Jeff and Robin, have been a great support system for my sister and me throughout all of our years of schooling, now about 42 years for the both of us combined. They have given my sister and me great education opportunities that most kids never have. My dad’s favorite past-time has always been travel, and he would spend months researching interesting iii locations around the world to find affordable rates to show my sister and me the world from a young age. Travel was the best educational experience I had growing up. An appreciation for nature came from the pristine waters of the Antarctic, the thick canopy of the Amazon rainforests, and the majestic Himalaya. An understanding of history flourished in the temples of Luxor, the streets of Pompeii, and the plazas of Teotihuacán. An openness to the excitement of the world came floating over Victoria Falls in a microlight, meeting Shaolin monks, and experiencing the Christkindlmarkt. I must not forget to thank my wife, Meg. For all of the times when lab got in the way and for all the weekends I spent writing or working on data, I thank you for your patience and understanding. It has been rough these last few months with the pregnancy, and I hope this thesis did not keep me from supporting you and the baby. I also must thank you for loving me and for moving to Tennessee. I know that science will be a part of my life for the rest of my life, and I appreciate that you understand that. Finally, I must thank you the most for bringing little Peter into the world and for pushing on through one of the most difficult pregnancies ever. To little Peter, although you came early and greatly complicated my dissertation defense, you are a wonderful addition to my life. Your early appearance has greatly diverted my anxiety about my defense; I have probably stressed out less about my defense than any other graduate student in the history of Ph. D dissertations. Peter, your presence has made me so happy these last few weeks; I look forward to watching you grow. Kateri, thank you for being a great sister. It was fun growing up with you. You have become a strong, intelligent, and beautiful young woman. You are going to be a great biomedical engineer. I wish you and Ben the best in Seattle. iv I would not have even made it to graduate school if it had not been for all of the countless teachers and mentors I had along the way. To all the teachers of St. Susanna, Cardinal Ritter, and the University of Evansville, I thank you. I would especially like to recognize Linda Bloomer, Kathy Chapin, Jean Reed, Vicky Branchini, Jill Wynalda, Mary Pat O’Connor, Linda Mann, Michael Cullen, Pat Acrobawi, Joyce Stamm, Mark Davis, Cris Hoechwender, and Phil Morgan. Thank you especially to the research mentors I had as an undergraduate, Jae-Ho Lee, Wen-Chen Xiong, Dale Edwards, and Brian Ernsting. The research experiences I had as an undergraduate sparked my development as a scientist and prepared me well for graduate school. In completion of my Ph. D. dissertation thesis, I owe the most gratitude to my research advisor Louise Rollins-Smith. Near the end of my first year at Vanderbilt, I came to Louise and told her that I wanted to pursue this project to investigate the inhibition of lymphocytes by Batrachochytrium dendrobatidis. Despite all of the funding issues, Louise found a way to keep me and my project funded. Also, it was a long, difficult struggle to publish our paper in Science. I was very anxious to publish a first author publication; especially because I had no first author publication at the beginning of my fifth year in graduate school. Thank you Louise for continuing to push to publish in a high impact journal; all of the stress and hard work paid off in the end. I also would like to thank Louise for being available and working to prepare me into the scientist I am today. I hope that I can continue to represent you well in my future research endeavors. Along with Louise, I have received much support and assistance from all of the members of the Rollins-Smith lab. Laura Reinert, our lab manager, deserves recognition for all that she does in keeping the Rollins-Smith lab running. Laura, it was a pleasure working with you. Jeremy Ramsey, a previous graduate student in the Rollins-Smith lab, initiated the research v which eventually evolved into my Ph. D thesis. Jeremy, you gave a great start to this amazing story about chytridiomycosis and were a great mentor to me during my early years in the lab. Whitney (Gammill) Holden, you have been a great colleague to have in the lab, and it has been wonderful collaborating with you. I would also like to acknowledge Jim Pask, another former graduate student in the Rollins-Smith lab. Rotation students and undergraduates who have contributed to tangential projects were Diana Contreras, Tim Chappell, Heather Wells, Julia Coleman, and Jack Lee. I would have not had the great success without all of collaborators I had through my thesis work. Kyra Oswald-Richter completed the experiments determining the effects of B. dendrobatidis supernatants on human CD4+ T cells. Sarah Parker Collier and the Aune lab provided me with mouse splenocytes and aided in designing the experiments I completed with mouse lymphocytes. Danica Sutherland in the lab of Terrence Dermody worked with me to test my factors on two cell lines with which she had experience: CHO and HeLa cells. Sophia Gayek completed several experiments present in the Science manuscript during her rotation in the Rollins-Smith lab. Michelle Reyzer completed MALDI/MS to analyze the components of supernatants. Thomas Umile, a post-doc in the lab of Kevin Minbiole at Villanova, has been working to understand the small molecule metabolites produced by B. dendrobatidis. Thomas has identified some of the small molecule components of B. dendrobatidis supernatants and is continuing to identify others. Alex Roberts, a post-doc in the lab of Lee Berger at James Cook University in Townsville, Australia, has been working to isolate the large molecular factors. Pavlina Ivanova in the lab of Alex Brown at Vanderbilt completed the analysis to determine if lipids might be responsible for inhibition of lymphocytes. John Galgiani, the director of the Valley Fever Center for Excellence at the University of Arizona also provided the nikkomycin Z vi drug which was essential for key components of my research. Thank you all for your assistance and open collaboration. Thank you also to my committee who has helped me in my success. Kathy Friedman, my chair, has aided me in many things including obtaining a Dissertation Enhancement Grant from the graduate school. Julián Hillyer provided me with great advice and the facilities of his lab so that I could complete necessary experiments for the Science manuscript. Borden Lacy has given great advice about how to proceed along my project. Clint Carter stayed on my committee even after retiring and was an important component of my committee having served on Jeremy Ramsey’s committee. I have received much assistance in completing my work at Vanderbilt by other labs and core facilities. I have had to rely on the generosity of these labs and cores to allow me to use the equipment necessary to obtain the necessary data. I used a scintillation counter in the lab of Luc Van Kaer to complete many of the proliferation assays throughout my time at Vanderbilt.