The Type II Secretion System in Acinetobacter baumannii: Its Role in Pathogenesis and Translational Implications By Ursula Bernice Waack A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Microbiology and Immunology) in the University of Michigan 2017 Doctoral Committee: Associate Professor Maria B. Sandkvist, Chair Assistant Professor Nicole Koropatkin Associate Professor Lyle S. Simmons Professor Michele S. Swanson Ursula B. Waack [email protected] ORCID: 0000-0003-4517-6790 © Ursula B. Waack 2017 Dedication For: Eric, Rose, Mom, Dad, Hannah, Louis, and Emma. ii Acknowledgements As with any great endeavor, I did not make it here on my own. I was supported by family, friends, colleagues, and mentors. I would like to thank Maria Sandkvist for being my graduate mentor. I have learned a lot from you and you have made me a better scientist. I hope to make you proud as I continue my career. My labmates made my graduate career bearable. Shilpa Gadwal and Chelsea Rule, you have helped me through the ups and downs of graduate school. Thank you for being there for me and preventing me from giving up. A special thanks to Nathan Porter, Chelsea Rule, and Elizabeth Rettedal for reading through my thesis and giving me feedback. Your time and thoughts were greatly appreciated. Finally, I would like to thank my family who have supported me since I was a child. I know you are all extremely proud of me and I could not have done it without you. Thank you to my wonderfully supportive husband, Eric. You have given me an incredible amount of freedom, support, and love. iii Contents Dedication ........................................................................................................................ii Acknowledgements ......................................................................................................... iii List of Tables ...................................................................................................................vi List of Figures ................................................................................................................. vii Abstract ......................................................................................................................... viii Chapter I: ........................................................................................................................ 1 Introduction ..................................................................................................................... 1 Acinetobacter baumannii Infection ............................................................................... 1 Antibiotic Resistance .................................................................................................... 2 Mechanisms of Pathogenesis ...................................................................................... 4 Type II Secretion System ............................................................................................. 7 Host Response to Bacterial Infections ....................................................................... 12 Coagulation Pathway ................................................................................................. 13 Clotting and Pathogenesis ......................................................................................... 16 Factor XII ................................................................................................................... 17 Scope of this Study .................................................................................................... 18 Chapter II: ..................................................................................................................... 20 Acinetobacter baumannii is Dependent on the Type II Secretion System and Its Substrate LipA for Lipid Utilization and in vivo Fitness .................................................. 20 Abstract ...................................................................................................................... 20 Importance ................................................................................................................. 21 Introduction ................................................................................................................ 21 Materials and Methods ............................................................................................... 24 Results ....................................................................................................................... 28 Discussion.................................................................................................................. 36 Chapter III: .................................................................................................................... 42 iv The Adamalysin-like Protease CpaA Secreted by Acinetobacter baumannii Promotes in vivo Colonization and Inactivation of Coagulation Factor XII ......................................... 42 Abstract ...................................................................................................................... 42 Introduction ................................................................................................................ 43 Materials and Methods ............................................................................................... 47 Results ....................................................................................................................... 51 Discussion.................................................................................................................. 59 Chapter IV: .................................................................................................................... 63 Targeting the Type II Secretion System: The Development, Optimization, and Validation of a High-Throughput Screen to Identify Small Molecule Inhibitors ............................... 63 Abstract ...................................................................................................................... 63 Introduction ................................................................................................................ 64 Methods ..................................................................................................................... 68 Results ....................................................................................................................... 71 Discussion.................................................................................................................. 87 Chapter V: ..................................................................................................................... 92 Discussion ..................................................................................................................... 92 Discovery of the T2SS in A. baumannii ...................................................................... 92 The T2SS, CpaA, and fXII.......................................................................................... 95 Targeting the T2SS .................................................................................................. 100 Concluding Remarks ................................................................................................ 102 References .................................................................................................................. 103 v List of Tables Table 1.1. Examples of T2S system substrates. .......................................................................................... 12 Table 2.1. Plasmids and Bacterial Strains ................................................................................................... 40 Table 2.2. Primers used for plasmid construction ...................................................................................... 41 Table 3.1. Strains and plasmids used in this study. .................................................................................... 62 Table 3.2. Primers used for plasmid construction. ..................................................................................... 62 Table 4.1. Optimization of assay conditions for development of a High-Throughput Screen. .................. 78 Table 4.2. IC50 values of compounds identified in primary screen and tested for concentration dependent inhibition. .................................................................................................................................................... 82 Table 4.3. List of Bacterial Strains and Plasmids used in this study. ........................................................... 91 vi List of Figures Figure 1.1. A schematic representation of the T2S System in A. baumannii .............................................. 10 Figure 1.2. A simplified coagulation cascade .............................................................................................. 14 Figure 2.1. T2S genes in A. baumannii. ....................................................................................................... 28 Figure 2.2. A. baumannii growth on lipid agar and LB. ............................................................................... 30 Figure 2.3. Sequence alignment of LipA. .................................................................................................... 31 Figure 2.4. Extracellular A. baumannii lipase activity. ...............................................................................