Investigation of Peptidyl-prolyl cis/trans isomerases in the virulence of Staphylococcus aureus A Dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Rebecca A. Keogh August 2020 © 2020 Rebecca A. Keogh. All Rights Reserved. 2 This Dissertation titled Investigation of Peptidyl-prolyl cis/trans isomerases in the virulence of Staphylococcus aureus by REBECCA A. KEOGH has been approved for the Department of Biological Sciences and the College of Arts and Sciences by Ronan K. Carroll Assistant Professor of Biological Sciences Florenz Plassmann Dean, College of Arts and Sciences 3 ABSTRACT REBECCA A. KEOGH, Doctorate of Philosophy, August 2020, Biological Sciences Investigation of peptidyl-prolyl cis/trans isomerases in the virulence of Staphylococcus aureus Director of Dissertation: Ronan K. Carroll Staphylococcus aureus is a leading cause of both hospital and community- associated infections that can manifest in a wide range of diseases. These diseases range in severity from minor skin and soft tissue infections to life-threatening sepsis, endocarditis and meningitis. Of rising concern is the prevalence of antibiotic resistant S. aureus strains in the population, and the lack of new antibiotics being developed to treat them. A greater understanding of the ability of S. aureus to cause infection is crucial to better inform treatments and combat these antibiotic resistant superbugs. The ability of S. aureus to cause such diverse infections can be attributed to the arsenal of virulence factors produced by the bacterium that work to both evade the human immune system and assist in pathogenesis. Many of these virulence factors have redundant or overlapping functions, and consequently, vaccines and antibiotics designed to target one or a small number of virulence factors have had limited success in combatting infection. An alternative approach to targeting individual factors has been to identify global regulators or systems that affect the expression of numerous virulence factors or virulence factor activity in the cell. Peptidyl-prolyl cis/trans isomerases (PPIases) are a family of enzymes that have been shown to regulate virulence factor activity and assist in protein secretion in 4 numerous bacteria. In addition, the deletion of certain PPIases has resulted in an attenuation of virulence, demonstrating their importance in infection. The objective of this dissertation was to characterize three PPIase proteins: PrsA, PpiB and trigger factor (TF) encoded by S. aureus and determine if they contribute to virulence factor production, regulation and disease. PrsA is a membrane-bound lipoprotein that has been shown to assist in protein secretion and contribute to virulence in the pathogens Listeria monocytogenes and Streptococcus pyogenes. In S. aureus, PrsA was found to be involved in oxacillin resistance and to have PPIase activity, but little was known about the ability of the protein to contribute to infection. The data in Chapter 3 reveal that PrsA affects the activity of the major hemolytic alpha-toxin (Hla) in S. aureus, and that it interacts with multiple membrane-anchored proteins that contribute to antibiotic resistance. Despite these findings, Chapter 3 also demonstrates that PrsA is dispensable for virulence in a murine sepsis model of infection, a murine abscess model of infection, and in macrophage and nasal epithelial cell infection. PpiB was characterized as a predicted cytoplasmic protein encoding a putative PPIase domain in S. aureus. Initial work by our group confirmed the cellular location and PPIase activity of PpiB and showed that a ppiB mutant had reduced hemolytic and nuclease activity. Chapter 2 of this dissertation identifies a role for PpiB in virulence, and shows that the PPIase activity of PpiB does not contribute to disease. Further characterization of how PpiB contributes to virulence is outlined in Chapter 3, where PpiB is revealed to affect the activity of numerous virulence factors including the alpha 5 phenol-soluble modulins (αPSMs) and Hla. This project identified the first known PPIase to contribute to S. aureus infection, and identified specific virulence factors regulated by PpiB. TF is the best characterized bacterial PPIase that was first identified in Escherichia coli for its ability to bind to the ribosome and assist in the chaperoning of nascent polypeptides. Numerous studies on Gram-positive pathogens have identified a role for TF in virulence-associated processes. Despite its apparent role in virulence in multiple pathogens, no work was done on S. aureus TF prior to this dissertation. In Chapter 4, we identify a role for TF in biofilm formation and virulence in S. aureus. The work presented in this dissertation reveals that S. aureus PPIases act through distinct mechanisms to regulate virulence factors and in some cases, contribute to infection. Identification of PPIases as novel regulators of virulence strengthens our knowledge of S. aureus virulence factor regulation and can be used to better inform future treatments. 6 DEDICATION To my parents, Joanie and Paul Keogh. This dissertation would not have been possible without your endless love, support, encouragement, and supplied wine. 7 ACKNOWLEDGMENTS Ronan, there are so many lessons you taught me over the last four years, too many to include here so I will highlight a few that have been particularly impactful. First, I remember interviewing with you and thinking wow, this person doesn’t just want a student, they want me. That feeling of importance is something you have continued to foster throughout my graduate career, and every talk we had along the way helped me fight my crippling imposter syndrome. You assured me I belonged in this community and that was possibly the most important lesson of all. Second, you taught me how to be a great mentor and therefore inspired me to continue a career in academia. You gave me advice to better myself, and you asked me for my advice in return. You made me feel like my voice mattered, and that I was a colleague as well as a mentee. I strive to be that mentor, one who grows along with their students. Third, you taught me how not to do retro-orbital infections. Next, to the Carroll lab past and present, thank you for accepting me at my best and my worst. For helping me finish the celebratory champagne and blocks of cheese and pretending my eyes weren’t puffy after I cried behind the freezer. Each of you are what made work so special to me. A special thank you to my partner in crime, Rachel Zapf. I couldn’t have asked for a better person to have there with me every step of the way. You were always there to listen, giving incredible advice both in science and in life. I can only hope I gave you the same. To my other scientific mentors. Clay Caswell, I wouldn’t be in science if you didn’t take a chance on me at Virginia Tech. You taught me what it meant to be a 8 researcher and laid the foundation for my career and my confidence. Jimmy Budnick and Lauren Sheehan, I am so grateful for your mentorship in the lab and in navigating my career. Also, for the pizza rolls. To my committee, Dr. Erin Murphy, Dr. Donald Holzschu and Dr. Sarah Wyatt. This dissertation would not have been possible without your advice and suggestions for improvement. Thank you for believing in me. To my family and friends. Graduate school is hard, and I often doubted myself along the way. You never doubted me. Mom and dad, thank you for being my best friends and biggest cheerleaders. In your eyes I can do whatever I set my mind to, and your unwavering support is what gets me through the hardest of times. My fellow grad- students, you made these four years incredible. I appreciate each and every one of your friendships which I will take with me well-beyond Athens. Finally, a thank you to my funding sources, without whom I would not have a career or a dissertation. Those sources include the National Institute of Allergy and Infectious Disease, the Bill and Melinda Gates Foundation, the Ohio University Student Enhancement Award, the College of Arts and Sciences Graduate Student Research Fund, and the Ohio University Donald Clippinger Fellowship. 9 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3 Dedication ........................................................................................................................... 6 Acknowledgments............................................................................................................... 7 List of Tables .................................................................................................................... 12 List of Figures ................................................................................................................... 13 Chapter 1: Peptidyl-prolyl cis/trans isomerases (PPIases) in Staphylococcus aureus ..... 15 Staphylococcus aureus ................................................................................................ 15 Disease Prevalence and Presentation .................................................................... 15 Virulence Factors and Protein Secretion............................................................... 16 PPIases ........................................................................................................................ 19 Function and Activity ..........................................................................................