MECHANISMS OF STAPHYLOCOCCUS AUREUS PERSISTENCE AND ERADICATION OF CHRONIC STAPHYLOCOCCAL INFECTIONS by Rebecca Yee A dissertation submitted to the Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland December, 2018 ABSTRACT Bacteria can exist in different phenotypic states depending on environmental conditions. Under stressed conditions, such as antibiotic exposure, bacteria can develop into persister cells that allow them to stay dormant until the stress is removed, when they can revert back to a growing state. The interconversion of non-growing persister cells and actively growing cells is the underlying basis of relapsing and chronic persistent infections. Eradication and better treatment of chronic, persistent infections caused by Staphylococcus aureus requires a multi- faceted approach, including a deeper understanding of how the bacteria persist under stressed conditions, regulate its cell death pathways, and development of novel drug therapies. Persisters were first discovered in the 1940s in a staphylococcal culture in which penicillin failed to kill a small subpopulation of the cells. Despite the discovery many decades ago, the specific mechanisms of Staphylococcus aureus persistence is largely unknown. Recently renewed interest has emerged due to the rise of chronic infections caused by pathogens such as M. tuberculosis, B. burgdorferi, S. aureus, P. aeruginosa, and E. coli. Treatments for chronic infections are lacking and antibiotic resistance is becoming a bigger issue. The goal of our research is to define the mechanisms involved in S. aureus persistence and cell death to improve our knowledge of genes and molecular pathways that can be used as targets for drugs to eradicate chronic infections. Using several high-throughput genetic screens, we identified and confirmed several core regulators of bacterial persistence and cell death upon exposure to bactericidal antibiotics and environmental stresses such as heat and acid stress. Currently, drug combinations approved for clinical usage do not target persister cells. One exception is the treatment regimen for tuberculosis that includes pyrazinamide, an anti- persister drug that targets unconventional drug targets such as proteins involved in energy ii metabolism and trans-translation. Upon the addition of pyrazinamide, which kill persister bacteria to the combination of rifampin and isoniazid which kill growing bacteria, the duration of tuberculosis treatment was shortened from 9-12 months to 6 months. Here, using the treatment for tuberculosis as an example that demonstrates the powerful activity of an anti-persister drug such as pyrazinamide, we screened for drugs with high activity against growing and non-growing forms of S. aureus to formulate drug combinations that can effectively kill the heterogeneous population of bacteria in biofilms. To test this approach in a clinically-relevant animal model, we established a chronic, skin infection mouse model of S. aureus to validate the improved efficacy of drug combinations in clearing persistent skin infections than currently-approved regimens used in the clinics. Our findings reveal that drug combinations consisting of drugs targeting both actively growing bacteria and persister cells, such as the combination of meropenem + daptomycin + clinafloxacin, can eradicate biofilms more effectively than doxycycline + rifampin, a combination used currently on patients. Our in vivo mouse model studies further validate the efficacy of such drug combinations in clearing the bacteria in the lesions, reducing the pathology, and completely healing the chronic lesions formed on the skin. These findings have important implications for treating other persistent bacterial infections. Advisor: Dr. Ying Zhang Thesis Committee: Dr. Jie Xiao Dr. J. Marie Hardwick Dr. Meghan Davis Dr. Nicole Parrish Alternates: Dr. Valeria Culotta Dr. Gary Ketner iii ACKNOWLEDGEMENTS I would first like to express my gratitude to my advisor, Dr. Ying Zhang, for his support and encouragement throughout the years. I gained independence, a wide range of experience in bacteriology research, and confidence as a scientist. Thank you for listening to me when I expressed interest to pursue research on S. aureus and allowing me to conduct research not limited to the genetic basis of S. aureus persistence but also treatment and even diagnostics. I am extremely grateful for your support of my ultimate career goal in becoming a clinical microbiologist and for your creative research ideas that were applicable to the clinical field. Thank you for giving me the freedom to explore and grow. It has been an amazing time working with the past and present members of the Zhang lab. Thank you all for keeping the lab exciting and lively at all times. I especially have to thank Dr. Wanliang Shi for training me during my rotation in the lab and providing a positive experience that drew me back to joining the lab. I would like to thank my thesis committee members (both past and present), Dr. Jie Xiao, Dr. J. Marie Hardwick, Dr. Meghan Davis, Dr. Nicole Parrish, and Dr. Arturo Casadevall for providing constructive feedback and also having conversations about my career goals along the way. In particular, I would like to thank both Dr. Hardwick and Dr. Parrish who have been with me since the beginning of my PhD career, from my preliminary oral exam to my final oral exam. You both watched me grow and I appreciated all our conversations ranging from personal to professional career advice. The members of the Hardwick lab (Zach, Jason, Heather, and Madhura) were also instrumental in helping me execute some of my studies. I also need to thank my past exam committee members, Dr. David Sullivan, Dr. Winnie Tang, and Dr. Randy Bryant, who still periodically check up on me about the progress that I have made. I thank you all for the continuous support throughout the years. iv Special thanks go to my classmates and colleagues at Johns Hopkins University. To my collaborators, Naina and Andreina, we came to know each other very well after some unsuccessful but then successful experiments together. I am happy to be able to learn a lot from you both but more importantly, we are friends and not simply colleagues. From MMI, a special big thanks to Snow, Phil, Jane, Yuting, Jasmine, and Gail. Thank you all for your friendship, encouragement, and our random conversations after Forum or Seminar. It's great to have a life outside of lab! A bigger thanks to my roommate, Jessie, for being a great roommate, scientist, friend and confidant as we both navigated our PhD journey together. And, the biggest thanks go to my partner, Eric. Thanks for being a great listener and constant supply of humor, optimism, laughter, food, and bubble tea. I always knew you all would have my back and made sure I was not falling through anywhere. I will miss you all but I am confident that we will all do great things in the future and we will cross paths once again. Lastly, I express much thanks to my family members. Thank you for letting me pursue my dreams and despite the long duration spent in education, you only encouraged me to push further with your unconditional love and support. I thank my parents, especially my father who was a teacher in his past career, for instilling the importance of education and discipline at a young age. By forcing me to memorize my multiplication tables and perfect my cursive before any of my peers did taught me how a strong work ethic during times of struggle will lead to many successes, such as a PhD. Thank you to my sister Jessica for being around to pick up the slack when I could not be home. I'm glad we have each other during the good and bad times. v TABLE OF CONTENTS Abstract ........................................................................................................................................... ii Acknowledgements ........................................................................................................................ iv Table of Contents ........................................................................................................................... vi List of Figures .............................................................................................................................. viii List of Tables ...................................................................................................................................x Chapter 1: Introduction ....................................................................................................................1 Staphylococcus aureus ...............................................................................................................2 Antibiotic Resistant S. aureus .....................................................................................................3 Mechanisms of Antibiotic Resistance ..........................................................................................4 Drug Resistance Mechanisms in S. aureus ..................................................................................5 Detection of Antibiotic Resistance in Bacteria ............................................................................6 The Phenomenon of Antibiotic Persistence .................................................................................7 Persisters and Persistent Infections ..............................................................................................8 Biofilms