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Genetic and Biochemical Insights Into the Mycobacterial Prrab System As a Regulator Of Genetic and Biochemical Insights into the Mycobacterial PrrAB System as a Regulator of Respiration and Central Metabolism by Jason Dean Maarsingh A Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Approved April 2019 by the Graduate Supervisory Committee: Shelley E. Haydel, Chair Heather Bean Todd Sandrin Kenneth Roland ARIZONA STATE UNIVERSITY May 2019 ABSTRACT Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the 10th leading cause of death, worldwide. The prevalence of drug-resistant clinical isolates and the paucity of newly-approved antituberculosis drugs impedes the successful eradication of Mtb. Bacteria commonly use two-component systems (TCS) to sense their environment and genetically modulate adaptive responses. The prrAB TCS is essential in Mtb, thus representing an auspicious drug target; however, the inability to generate an Mtb ΔprrAB mutant complicates investigating how this TCS contributes to pathogenesis. Mycobacterium smegmatis, a commonly used M. tuberculosis genetic surrogate was used here. This work shows that prrAB is not essential in M. smegmatis. During ammonium stress, the ΔprrAB mutant excessively accumulates triacylglycerol lipids, a phenotype associated with M. tuberculosis dormancy and chronic infection. Additionally, triacylglycerol biosynthetic genes were induced in the ΔprrAB mutant relative to the wild-type and complementation strains during ammonium stress. Next, RNA-seq was used to define the M. smegmatis PrrAB regulon. PrrAB regulates genes participating in respiration, metabolism, redox balance, and oxidative phosphorylation. The M. smegmatis ΔprrAB mutant is compromised for growth under hypoxia, is hypersensitive to cyanide, and fails to induce high-affinity respiratory genes during hypoxia. Furthermore, PrrAB positively regulates the hypoxia-responsive dosR TCS response regulator, potentially explaining the hypoxia-mediated growth defects in the ΔprrAB mutant. Despite inducing genes encoding the F1F0 ATP synthase, the ΔprrAB mutant accumulates significantly less ATP during aerobic, exponential growth compared to the wild-type and complementation strains. Finally, the M. smegmatis ΔprrAB mutant exhibited growth impairment in media containing gluconeogenic carbon sources. M. tuberculosis mutants unable to utilize these substrates fail to establish chronic infection, suggesting that PrrAB may regulate Mtb central carbon metabolism in response to chronic infection. In conclusion, 1) prrAB is not universally essential in mycobacteria; 2) M. smegmatis PrrAB regulates genetic responsiveness to nutrient and oxygen stress; and 3) PrrAB may provide feed-forward control of the DosRS TCS and dormancy phenotypes. The data i generated in these studies provide insight into the mycobacterial PrrAB TCS transcriptional regulon, PrrAB essentiality in Mtb, and how PrrAB may mediate stresses encountered by Mtb during the transition to chronic infection. ii DEDICATION To all the people who have helped shape the person I am. Whether they were positive or negative influences, I wouldn’t be who I am without them. Special dedication to my family, because there’s nothing more important than family and I’m one of the lucky ones to have incredibly supportive relatives. iii ACKNOWLEDGMENTS First and foremost, I need to acknowledge my father, Ron, whose constant support has been the single-most influential contribution toward my success in grad school and life in general. Through all my ups-and-downs, including too many years of uncertainty in my life’s direction, he never gave up on me. There is no stronger person in my life and if I can become half the man he is, I’m confidant I’ll be ok. I love you dad! To my mother, who passed away from liver cancer when I was two years old. Though I never had the chance to grow up with this wonderful woman, her spirit permeates into every good thing I do. I don’t fully believe or reject the existence of an afterlife, but I feel she is with me during my trials and tribulations. I’m trying to make you proud, mama. Special thanks to my aunt Martha and uncle Stewart, who became my second parents after mom died. I wouldn’t have achieved my successes without you. Martha, you filled mom’s shoes better than anyone could have, and I know she’s thanking you for doing so. Stewart, I will always be grateful for the lessons and morals you imparted to me during my youth, now, and in the future. Additionally, to my cousins (better yet, brothers) Rusty (Max), Cody, and Yari. Thanks for being the siblings I never had as an only child. We’ve done some stupid things in the past, but I wouldn’t change a thing. Though our lives take us in different directions, for better or worse, I would be in a much worse place without you. To Dr. Maneesha Muralinath (ASU mama), my first mentor at ASU who believed in me and helped me get my scientific “sea legs”. To Dr. Valerie Stout, who’s ever warming smile and advice provided me with a sense of calm and reassurance. To my committee, Dr. Heather Bean, Dr. Kenneth Roland, and Dr. Todd Sandrin: thank you for your advice and encouragement during my graduate school tenure. It’s been a pleasure having your guidance and support throughout my graduate education. Finally, to Dr. Shelley Haydel, my committee chair. You gave me the opportunity to work in a formal lab setting. I know I haven’t always been your star student, but you showed ever- lasting patience and a refusal to give up on me. I wouldn’t be the scientist I am today without your mentorship and guidance. Thanks for giving me the once-in-a-lifetime chance to make something iv out of myself. I will do everything I can to make you proud in my future career and scientific endeavors. To my girlfriend, Sam. You are my muse and gave me a reason not to quit during my last few years of grad school. Thanks for dealing with all my late nights and constant weekends working in the lab. I’m glad we had the chance to tackle the challenges of grad school together. I love you and will always be your support, friend, and partner. Finally, to all the Haydel lab members of the present and past. Thanks to Dr. Caitlin Otto for the honest input regarding details of grad school. To Ryan Lamarca, my lab brother. We started our programs together and I couldn’t have had a better lab mate. Your work ethic and intelligence will take you far! Rachell Barret, my first lab sister, whom I had the pleasure to mentor during my first year. I’ll never forget our rides to-and-from the ASU West campus! I wish you and Nick a long, blissful marriage. To Michelle Stevens, the super smarty pants undergraduate and my second lab sister. It was an absolute pleasure seeing someone as young and talented as you mature in the lab. Thanks to my second lab sister, Rebecca Melo. I know you and Bryan are going to do well in California and best of luck during grad school! I’m excited to see lil’ Grayson’s pics when he’s born! To everyone else (because there are too many to list), thanks for working hard to support Shelley’s research goals. I know I can be intense sometimes, but please realize that I always had the lab’s best interest in mind. Continue the proud Haydel lab tradition and take care of it well! v TABLE OF CONTENTS Page LIST OF TABLES ................................................................................................................................. viii LIST OF FIGURES ................................................................................................................................. ix LIST OF ABBREVIATIONS ................................................................................................................. xiii CHAPTER 1 INTRODUCTION ................................................................................................................ 1 Mycobacterium tuberculosis and its Global Implications ............................................... 1 Two-Component Regulatory Systems............................................................................ 2 The PrrAB TCS of Mycobacteria ................................................................................ 4 Host-Pathogen Interactions and the Mtb PrrAB TCS ................................................. 8 The Mtb PrrAB TCS as a Potential Therapeutic Target ........................................... 11 M. smegmatis as a Genetic Model for Mtb ............................................................... 12 Nitrogen Metabolism in Mycobacteria ...................................................................... 14 Mycobacterial Lipids ................................................................................................. 19 Respiration and Oxidative Phosphorylation in Mycobacteria ................................... 22 Mycobacterial Dormancy and the DosRS Two-Component Regulatory System ..... 27 Carbon Metabolism in Mycobacteria ........................................................................ 28 2 MYCOBACTERIUM SMEGMATIS PRRAB TWO-COMPONENT SYSTEM INFLUENCES TRIACYLGLYCEROL ACCUMULATION DURING AMMONIUM STRESS ............................................................................................................................. 38 Publication Note ........................................................................................................ 38 Abstract ..................................................................................................................... 38 Introduction ..............................................................................................................
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