Clemson University TigerPrints All Dissertations Dissertations May 2021 Glycosome Heterogeneity, Import Complex, and Metabolism in Trypanosoma brucei Christina L. Wilkinson Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Recommended Citation Wilkinson, Christina L., "Glycosome Heterogeneity, Import Complex, and Metabolism in Trypanosoma brucei" (2021). All Dissertations. 2817. https://tigerprints.clemson.edu/all_dissertations/2817 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected]. GLYCOSOME HETEROGENEITY, IMPORT COMPLEX, AND METABOLISM IN TRYPANOSOMA BRUCEI A Dissertation Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Genetics by Christina Wilkinson May 2021 Accepted by: Meredith Morris, Committee Chair Kimberly Paul Julia Frugoli Zhicheng Dou i ABSTRACT The kinetoplastid parasite Trypanosoma brucei is responsible for both human African trypanosomiasis (HAT) and the wasting disease nagana found in cattle. Unique to kinetoplastids are the specialized peroxisomes, named glycosomes, which compartmentalize the first several steps of glycolysis and gluconeogenesis, nucleotide sugar biosynthesis, and many other metabolic processes. There are many studies surrounding the heterogeneity and complexity of glycosomes as well as how these organelles proliferate and import their proteins. Here, I first explored new methods to analyze glycosome heterogeneity by flow cytometry. The advancement of flow cytometry has yielded methods that enable the identification of vesicles between 30-1000 nm in diameter. I adapted these techniques for the identification of glycosome populations by flow cytometry and the isolation of distinct populations via organelle sorting. With this technology, I detected populations of glycosomes (200 nm) harboring different amounts of a fluorescent glycosome protein reporter. I used a cell sorter to sort glycosomes based on their eYFP expression. T. brucei is unique in that they have two Pex13s, Pex13.1 and Pex13.2, whereas other eukaryotes have a single Pex13, and together, Pex13.1, Pex13.2, and Pex14 comprise the glycosome import complex. Phosphoproteomics reveal that Pex13.2 is phosphorylated and here I analyzed the role of this phosphorylation on both import complex formation and glycosomal protein import. Using Pex13.2 phosphorylation mutants revealed that the phosphorylation state of Pex13.2 does not impact import complex binding or the translocation of matrix proteins. However, Pex13.2 phosphorylation may affect import complex size. Additionally, I examined the activity and protein expression of gluconeogenic enzyme fructose 1,6- bisphosphatase (FBPase) between cell strains. FBPase is localized to the glycosome ii and recent metabolic labeling experiments revealed the gluconeogenesis is active in both stages of T. brucei. Here, I showed that FBPase is regulated in an unexpected manner. Under low glucose conditions the enzyme activity is undetectable, but in high glucose conditions activity levels are high. Also, I revealed that this activity pattern is both density dependent and strain dependent. Because glycosomes are essential and parasite specific, understanding their biology is critical for development of therapeutics. iii DEDICATION I dedicate this to my parents, Karen and Ron Wilkinson, my brother Gregory, and my partner Brian LeFevre. Without your love and unending support I would not be the person I am today or have made it this far. iv ACKNOWLEDGMENTS The work presented here has been made possible by all my amazing colleagues and friends over the last several years. I want to especially thank Dr. Meredith Morris, who has been a wonderful and compassionate mentor. She took me into her lab and has made a huge impact on my growth as a scientist and person. I also want to thank Dr. Jim Morris, who has always been there for me for advice and support. The Morris lab is not only a place a work, but the people have become family and I feel truly lucky to have worked with them every day. Over the years so many people have influenced me and helped me through the best and worst of times. I want to truly thank Dr. Logan Crowe, Emily Knight, and Jessica Tetterton, who have all contributed directly to my work, as well as Dr. Jimmy Suryadi, Dr. Evan Qiu, Jessica Jones, and Jillian Milanes. Thank you all so much for making my time at Clemson so special and help shape me into the person I am today. Your friendship means everything to me and I am incredibly fortunate to have been surrounded by such amazing people, including all of EPIC, for the support. Additionally, I would like to thank my committee members for always being available for questions and advice: Dr. Kim Paul, Dr. Julia Frugoli, and Dr. Zhicheng Dou. Thank you to the Clemson Light Imaging Facility, Dr. Terri Bruce and Rhonda Powell, for all of your help with microscopy and teaching me how to use the different pieces of equipment. I also want to thank Julie Nelson at the University of Georgia for helping me with the technical aspects of cell sorting and allowing us to use her equipment to sort glycosomes. Finally, I want to thank Dr. Jordan Wesel, Rodney Colon- Reyes, and Heather Walters for all of the support and friendship in science and everyday life. v TABLE OF CONTENTS Page TITLE PAGE .................................................................................................................i ABSTRACT .................................................................................................................. ii DEDICATION .............................................................................................................. iv ACKNOWLEDGMENTS ...............................................................................................v LIST OF TABLES ........................................................................................................ ix LIST OF FIGURES .......................................................................................................x CHAPTER I. Literature Review ....................................................................................... 1 Human African Trypanosomiasis .......................................................... 1 Lifecycle of T. brucei ............................................................................ 4 Peroxisomes and protein import ........................................................... 8 Biogenesis of peroxisomes ................................................................ 10 Peroxisome to glycosome evolution ................................................... 11 Glycosomes and protein trafficking..................................................... 14 Kinetoplastids have two unique Pex13s ............................................. 19 Peroxin phosphorylation ..................................................................... 21 Glycosome metabolic processes ........................................................ 23 Peroxisome and glycosome heterogeneity ......................................... 26 Summary............................................................................................ 30 References ......................................................................................... 32 II. New Methods to Analyze Glycosome Heterogeneity in Trypanosoma brucei ...................................................................................................... 52 vi TABLE OF CONTENTS (Continued) Page Abstract .............................................................................................. 53 Introduction ........................................................................................ 54 Materials and methods ....................................................................... 57 Results ............................................................................................... 61 Discussion .......................................................................................... 70 Acknowledgements ............................................................................ 74 References ......................................................................................... 75 III. Discerning the Role of Pex13.2 Phosphorylation on Glycosome Protein Import and Biogenesis in the Protozoan Parasite T. brucei ...................... 81 Abstract .............................................................................................. 82 Introduction ........................................................................................ 83 Materials and methods ....................................................................... 87 Results ............................................................................................... 91 Discussion ........................................................................................ 101 Acknowledgements .......................................................................... 103 References ....................................................................................... 104 IV. Density and Strain Dependent Regulation of Fructose 1,6-bisphosphatase in the Protozoan Parasite Trypanosoma brucei ......................................... 111 Abstract ...........................................................................................