UNDERSTANDING THE REGULATORY DYNAMIC OF THE TWO-COMPONENT SYSTEMS, PLRSR AND BVGAS, DURING BORDETELLA COLONIZATION OF THE MAMMALIAN RESPIRATORY TRACT Mary Ashley Sobran A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Microbiology and Immunology in the School of Medicine. Chapel Hill 2019 Approved by: Peggy A. Cotter Rita Tamayo Robert B. Bourret Miriam Braunstein Brian Conlon © 2019 Mary Ashley Sobran ALL RIGHTS RESERVED ii ABSTRACT Mary Ashley Sobran: Understanding the Regulatory Dynamic of the Two-Component Systems, PlrSR and BvgAS, During Bordetella Colonization of the Mammalian Respiratory Tract (Under the direction of Peggy A. Cotter) Pertussis is a severe respiratory disease caused by the bacterium Bordetella pertussis. Despite high vaccination coverage, the number of pertussis cases has rebounded in recent years. To combat this disease, more efficacious vaccines and improved knowledge of Bordetella virulence is critical. Bacteria often use two-component systems (TCSs) to coordinate essential cellular processes in response to the diverse environments they encounter. In B. pertussis and the closely related subspecies B. bronchiseptica, the TCS, BvgAS, controls the expression of almost all known virulence factor-encoding genes and is considered the main virulence regulatory system. Recently, another TCS, PlrSR, was identified that is also required for Bordetella infection within the lower respiratory tract (LRT). However, why PlrSR is important for colonization is unknown. Using engineered strains of B. bronchiseptica and genetic reporters, we demonstrated that PlrS is required for the maintenance of BvgAS activity in the lungs of mice, indicating that PlrSR, along with BvgAS, coordinates virulence specifically in the LRT. Moreover, our data indicate that PlrSR may regulate genes that are required for persistence in the LRT independent of BvgAS. Importantly, these genes and their corresponding proteins may serve as new vaccine components or therapeutic targets. We have also shown that the Per-Arnt- Sim (PAS) domain of BvgS is required for BvgS inactivation in the absence of PlrS, indicating that the PAS domain is important for the PlrSR-BvgAS connection and functions as an independent signaling domain. Based on published data and our findings, we hypothesize that iii PlrSR controls the expression of high-affinity cytochrome oxidases (HACOs) that are required for bacterial respiration and maintenance of BvgAS activity in the LRT. We have demonstrated that two of four HACOs contribute to LRT colonization. However, BvgAS activity is unaffected by the loss of these HACOs and it is unknown if PlrSR regulates the expression of any HACO encoding genes. Together, this work provides a more detailed understanding of the coordinated regulation imposed by PlrSR and BvgAS to promote Bordetella survival within the mammalian host. iv To my family. Thank you for all your support along the way. v TABLE OF CONTENTS LIST OF TABLES ......................................................................................................................... xi LIST OF FIGURES ...................................................................................................................... xii LIST OF ABBREVIATIONS ...................................................................................................... xiv CHAPTER 1. INTRODUCTION ................................................................................................... 1 Introduction ......................................................................................................................... 1 Current pertussis vaccines fail to protect individuals from Bordetella pertussis colonization and pertussis disease ................................................................ 1 Despite high rates of vaccination, pertussis remains a burden in the United States and throughout the world ...................................................................... 2 The progression of pertussis disease is most severe in young patients and can last for many months ...................................................................................... 2 Bordetella pertussis produces many essential virulence factors during human infection ........................................................................................................... 3 The complex, two-component phosphorelay system, BvgAS, is essential for Bordetella colonization of the mammalian host and is considered the master regulator of virulence ............................................................... 4 Although the importance of BvgAS for Bordetella virulence has been well established, the contributions of other two-component systems to bacterial survival and virulence in the mammalian host is less understood .................................................................................................................... 6 PlrSR is a newly identified putative two-component system required for B. bronchiseptica colonization of the lower respiratory tract in rats ..................... 7 Research Objective ............................................................................................................. 7 REFERENCES ............................................................................................................................... 9 CHAPTER 2. THE BORDETELLA PLRSR REGULATORY SYSTEM CONTROLS BVGAS ACTIVITY AND VIRULENCE IN THE LOWER RESPIRATORY TRACT ............................................................................................................. 15 vi Introduction ....................................................................................................................... 15 Results ............................................................................................................................... 18 PlrS is required for enhanced BvgAS-dependent virulence-associated phenotypes in response to elevated CO2 concentrations ............................................ 18 PlrS likely affects BvgAS-dependent phenotypes via PlrR ....................................... 18 B. bronchiseptica lacking plrS modulate to the Bvg– phase within the lower respiratory tract ................................................................................................ 20 B. bronchiseptica lacking plrS fail to activate BvgAS within the lower respiratory tract .......................................................................................................... 22 PlrS is required for B. bronchiseptica persistence in the lower respiratory tract, independent of its effects on BvgAS activity ................................. 23 PlrS is required for survival and persistence of B. pertussis in the lower respiratory tract ................................................................................................ 24 Discussion ......................................................................................................................... 25 Materials and Methods ...................................................................................................... 29 Growth media and bacterial strains ........................................................................... 29 Construction of cloning plasmids and bacterial strains ............................................. 30 In vitro adherence assays ........................................................................................... 31 Macrophage cytotoxicity assays ................................................................................ 31 Bacterial adherence to J774 cells and in response to being cultured in media with decreased pH ........................................................................................... 32 Evaluation of B. bronchiseptica bvgAS transcription in vitro ................................... 32 Evaluation of B. bronchiseptica promoter activity in vitro ....................................... 33 Bacterial colonization of the mouse respiratory tract ................................................ 33 Evaluation of B. bronchiseptica phenotypic phase transition in vivo ........................ 34 Ethics statement ......................................................................................................... 34 Statistical analysis ...................................................................................................... 35 Figures and Tables ............................................................................................................ 36 vii REFERENCES ............................................................................................................................. 47 CHAPTER 3. THE BVGS PAS DOMAIN: AN INDEPENDENT SENSORY PERCEPTION MODULE IN THE BORDETELLA BRONCHISEPTICA BVGAS PHOSPHORELAY ....................................................................................................................... 53 Introduction ....................................................................................................................... 53 Results ............................................................................................................................... 57 Mutants used in this study ......................................................................................... 57 Contribution of the PAS domain in regulating BvgS activity in vitro