Identification and Characterization of Candidate Treponema pallidum Vascular Adhesins by Heterologous Expression in the Lyme Disease Pathogen by Wei-Chien (Andrew) Kao A thesis submitted in conformity with the requirements for the degree of Master of Science Faculty of Dentistry University of Toronto © Copyright by Wei-Chien (Andrew) Kao 2016 Identification and characterization of candidate Treponema pallidum vascular adhesins by heterologous expression in the Lyme disease pathogen Wei-Chien (Andrew) Kao Master of Science Faculty of Dentistry University of Toronto 2016 Abstract This thesis reports novel techniques to study the candidate adhesin of unculturable syphilis pathogen Treponema pallidum in the Lyme disease pathogen Borrelia burgdorferi. The Treponema pallidum laminin-binding adhesin, Tp0751, was heterologously expressed and surface localized in a non-infectious, less-adherent B. burgdorferi strain which has the attenuated ability to interact with cultured mammalian cells in vitro and vascular endothelial cells in vivo. By using the flow chamber techniques and intravital microscopy approach, I demonstrated that Tp0751 was able to restore borrelial-endothelial interactions in vitro, and was able to confer vascular adhesion in vivo. This study has illustrated that gain-of-function B. burgdorferi strain can serve as a machinery to study the activity of treponemal outer surface proteins. The analysis of the adhesive role of Treponema pallidum will reveal its mechanisms of dissemination and tissue colonization during syphilis infection. ii Table of contents List of diagrams ............................................................................................................................. vi List of figures and tables ............................................................................................................... vii List of abbreviations .................................................................................................................... viii Chapter 1. Introduction and literature review ................................................................................. 1 1.1 Pathogenic spirochetes ..................................................................................................... 1 1.1.1 Borrelia burgdorferi ................................................................................................. 2 1.1.2 Treponema pallidum ............................................................................................... 14 1.2 Production of outer membrane lipoproteins in B. burgdorferi and T. pallidum ............ 19 1.2.1 Secretion and localization of lipoproteins in other gram-negative bacteria ........... 20 1.2.2 Current knowledge of lipoproteins secretion and localization in B. burgdorferi and T. pallidum .............................................................................................................. 21 1.3 Strategy utilized to characterize B. burgdorferi vascular adhesins ................................ 22 1.3.1 In vivo phage display to identify potential adhesins ............................................... 22 1.3.2 Generation of gain-of-function and loss-of function strains to measure the cell- and ligand-binding activity promoted by the adhesins .................................................. 23 1.3.3 Flow-chamber imaging to determine bacterial-endothelial interaction under the environment with fluid shear stress ........................................................................ 23 1.3.4 Imaging techniques including intravital microscapy (IVM) and whole body imaging to observe spirochetal colonization and dissemination in vivo ................. 24 1.3.5 Short-term intravenous infection model to measure vascular interaction promoted by the adhesins ........................................................................................................ 25 1.4 Rationale, objective and hypothesis of M.Sc. studies .................................................... 26 Chapter 2. Manuscript - Heterologous expression of the Treponema pallidum vascular adhesin Tp0751 in the Lyme disease pathogen Borrelia burgdorferi ....................................................... 27 2.1 Abstract .......................................................................................................................... 27 2.2 Introduction .................................................................................................................... 27 2.3 Materials and methods ................................................................................................... 30 2.4 Results ............................................................................................................................ 43 2.5 Discussion ...................................................................................................................... 51 2.6 Figures and tables ........................................................................................................... 56 iii 2.7 Supplemental movies ..................................................................................................... 85 Chapter 3. Thesis discussion ......................................................................................................... 86 References ..................................................................................................................................... 90 iv Original contribution to science I completed all experimental work and writing associated with this thesis except: Chapter 2 (Manuscript): Introduction, figures, legends, first half of results drafted/written with/by Helena Pětrošová and rewritten by me Experiments and analysis for following figures were conducted by others: o Fig. 1A: Collaborator Rebecca Hof (Cameron lab) generated gain-of-function constructs, and I transformed them into Borrelia o Fig. 1B-C: FACS analysis of protein expression and localization performed by collaborator Yi-Pin Lin (Leong lab) o Fig. 2A: Collaborator Karen Lithgow (Cameron lab) performed static adhesion assays o Fig. 6A: Intravital microscopy was performed with assistance of collaborator Pablo Rojas (Moter lab) o Fig. 6B: Co-author Yang (Shirley) Zhang (Moriarty lab) performed qPCR to measure bacterial burden in blood o Fig. S1: Collaborator Charmaine Wetherell (Cameron lab) performed Tp0751 PCR screening o Fig. S2: Co-author Rhodaba Ebady (Moriarty lab) performed all experiments for GCB1586 (RevB), tracking and analysis for this figure v List of diagrams Page Diagram 1. The natural infectious cycle of Borrelia burgdorferi 2 Diagram 2. The course of Borrelia burgdorferi infection and Lyme disease in humans 3 vi List of figures and tables Page Figure 1. Surface production of Tp0751 in transformed B. burgdorferi strains 56 Figure 2. Interaction of Tp0751-expressing strains with human endothelia under static and 58 physiological shear stress conditions Figure 3. Physical properties of B. burgdorferi interactions mediated by Tp0751 60 Figure 4. Effect of Tp0751 on tether-dependent and tether-independent interactions 62 Figure 5. Effect of increasing shear stress on physical properties of Tp0751-dependent 64 interactions Figure 6. Interaction of Tp0751-expressing strains with vasculature in vivo 66 Figure S1. PCR screening of Tp0751-expressing strains, showing the presence of Tp0751 68 insert, pTM259 and pTM61 plasmids Figure S2. Effect of FLAG-tag in bacterial interactions under physiological shear force 70 Figure S3. Dissociation kinetics of tether-dependent and tether-independent interactions 72 under physiological fluid shear force Figure S4. B. burgdorferi interaction trajectories of tether-dependent interactions 74 Figure S5. B. burgdorferi interaction trajectories of tether-independent interactions 76 Figure S6. Secondary structure prediction of BBK32 and Tp0751 78 Table S1. B. burgdorferi strains used in this study 80 Table S2. Constructs used in this study 81 Table S3. Primers used in this study 82 Table S4. Bacterial length of individual B. burgdorferi strains 84 vii List of abbreviations BSK II Barbour-Stoenner-Kelly II CRASP Complement regulator-acquiring surface protein DB Dextrose and BSA ECM Extracellular matrix ELISA Enzyme-linked immunosorbent assay FACS Fluorescence-activated cell sorting Fb Force imposed on the bonds FBS Fetal bovine serum Fn Fibronectin GAG Glycosaminoglycan GFP Green fluorescent protein HBSC HEPES buffer with sodium chloride and calcium chloride HIV Human immunodeficiency virus HUVEC Human umbilical vein endothelial cell ICAM-1 Intercellular adhesion molecule 1 iNKT cell Invariant natural killer T cell IVM Intravital microscopy Koff Dissociation rate constant Lol pathway Lipoprotein outer membrane localization pathway MFI Mean fluorescence index pFn Plasma fibronectin qPCR Quantitative PCR Sec pathway Secretory pathway SRP Signal recognition particle TAT pathway Twin-arginine translocation pathway VCAM-1 Vascular cell adhesion molecule 1 viii Chapter 1. Introduction and literature review 1.1 Pathogenic spirochetes Spirochetes belong to a phylum of a large group of morphologically unique bacteria which are widespread in aquatic environments and in animals (Paster and Dewhirst, 2000). They possess unique spiral shapes with length and diameter ranging from 5 to 250μm and 0.1 to 0.5μm, respectively (Pavia, 1994). Unlike other bacteria, spirochetes utilize periplasmic flagella located in the periplasm to promote their motility (Wolgemuth, 2015). During early infection, spirochetes