Investigating the Role of Pallilysin in the Dissemination of the Syphilis Spirochete Treponema pallidum by Yavor Denchev Bachelor of Science, University of Victoria, 2011 A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE in the Department of Biochemistry and Microbiology Yavor Denchev, 2014 University of Victoria All rights reserved. This thesis may not be reproduced in whole or in part, by photocopy or other means, without the permission of the author. ii Supervisory Committee Investigating the Role of Pallilysin in the Dissemination of the Syphilis Spirochete Treponema pallidum by Yavor Denchev Bachelor of Science, University of Victoria, 2011 Supervisory Committee Dr. Caroline Cameron, Department of Biochemistry and Microbiology Supervisor Dr. Terry Pearson, Department of Biochemistry and Microbiology Departmental Member Dr. John Taylor, Department of Biology Outside Member iii Abstract Supervisory Committee Dr. Caroline Cameron, Department of Biochemistry and Microbiology Supervisor Dr. Terry Pearson, Department of Biochemistry and Microbiology Departmental Member Dr. John Taylor, Department of Biology Outside Member Syphilis is a global public health concern with 36.4 million cases worldwide and 11 million new infections per year. It is a chronic multistage disease caused by the spirochete bacterium Treponema pallidum and is transmitted by sexual contact, direct contact with lesions or vertically from an infected mother to her fetus. T. pallidum is a highly invasive pathogen that rapidly penetrates tight junctions of endothelial cells and disseminates rapidly via the bloodstream to establish widespread infection. Previous investigations conducted in our laboratory identified the surface-exposed adhesin, pallilysin, as a metalloprotease that degrades the host components laminin (major component of the basement membrane lining blood vessels) and fibrinogen (primary component of the coagulation cascade), as well as fibrin clots (function to entrap bacteria and prevent disseminated infection). Furthermore, pallilysin expressed on the surface of the non-invasive spirochete Treponema phagedenis conferred upon this bacterium the ability to degrade fibrin clots. It was hypothesized that pallilysin is integral to the process of T. pallidum dissemination, and interference with its functioning will prevent spread throughout the host and establishment of chronic infection. To test this hypothesis, a two- pronged approach was undertaken during my thesis research. Bioinformatics analyses were used to trace the evolutionary history of pallilysin in an attempt to gain further insight into its role in the pathogenesis of T. pallidum. The iv sequence conservation of pallilysin was analyzed in the context of its homologues. The bioinformatics analyses revealed homologues in three spirochete genera, namely Treponema, Spirochaeta, and Borrelia, presented in decreasing order of the degree of sequence conservation. The HEXXH motif, part of the active site of the pallilysin metalloprotease, was fully conserved only in T. pallidum and T. paraluiscuniculi, both of which are systemic pathogens. However, the flanking sequences showed a high degree of conservation, especially in the Treponema and Spirochaeta genera. The minimum laminin-binding region of pallilysin identified previously was partially conserved among the treponema and spirochaeta homologues with the highest degree of conservation observed with the homologues from T. paraluiscuniculi and T. phagedenis, as well as among the homologues from the human oral pathogens. In vitro dissemination studies were performed to investigate the dissemination capacity of T. phagedenis heterologously expressing pallilysin. Human Umbilical Vein Endothelial Cells were seeded and grown to confluence on permeable inserts coated with growth factor-reduced Matrigel to create an artificial endothelial barrier. Wild type T. phagedenis, and T. phagedenis transformed either with the pallilysin open reading frame or its empty shuttle vector, were incubated with the barriers under anaerobic conditions. Dissemination across the barrier was assessed as percent traversal by both dark-field microscopic counts of treponemes and real-time quantitative PCR of genomic DNA extracted from the treponemes. The results were inconclusive. However, a traversal trend suggested heterologous expression of pallilysin may facilitate traversal of T. phagedenis across the artificial endothelial barrier. v This study presented the first step towards elucidating the role of pallilysin in endothelial monolayer traversal and provided supporting evidence for the role of pallilysin in the widespread dissemination of T. pallidum in vivo. vi Table of Contents Supervisory Committee ...................................................................................................... ii Abstract .............................................................................................................................. iii Table of Contents ............................................................................................................... vi List of Tables ................................................................................................................... viii List of Figures .................................................................................................................... ix List of Abbreviations .......................................................................................................... x Acknowledgments............................................................................................................. xii Dedication ........................................................................................................................ xiii Chapter 1: Introduction ....................................................................................................... 1 1.1 Syphilis ..................................................................................................................... 1 1.1.1 Epidemiology ..................................................................................................... 1 1.1.2 Syphilis Pathology and Course of Infection ...................................................... 4 1.2 Treponema pallidum subspecies pallidum ................................................................ 7 1.2.1 Taxonomy .......................................................................................................... 7 1.2.2 Biology of T. pallidum ..................................................................................... 10 1.2.2 In vivo and in vitro growth of T. pallidum ................................................... 12 1.2.2 Treponema pallidum cell envelope ultrastructure........................................ 13 1.2.2 Treponema pallidum outer membrane proteins ........................................... 15 1.2.2 Identification of Treponema pallidum outer membrane proteins ................ 17 1.3 Dissemination Capacity of T. pallidum .................................................................. 19 1.3.1 Dissemination Capacity of T. pallidum ........................................................... 23 1.3.1.1 Basement Membrane and Endothelial Cell Behaviour ............................. 25 1.4 Pallilysin, a Treponema pallidum Surface-Exposed Adhesin and Protease ........... 26 1.4.1 Pallilysin, a Treponema pallidum Surface-Exposed Adhesin and Protease .... 29 1.5 Research Hypotheses and Objectives ..................................................................... 31 1.6 Experimental Approach .......................................................................................... 33 Chapter 2: Materials and methods .................................................................................... 35 2.1.1 Bacterial Strain and Growth Conditions .............................................................. 35 2.1.2 Umbilical Vein Endothelial Cell Line and Monolayer Growth Conditions ........ 36 2.2 Artificial Endothelial Barrier .................................................................................. 37 2.3 Measurement of HUVEC Barrier Function by TEER ............................................ 38 2.4 Dissemination across the Artificial Endothelial Barrier ......................................... 39 2.4.1 Assay Medium ................................................................................................. 39 2.4.2 Treponemal manipulations............................................................................... 40 2.4.3 Experimental Design ........................................................................................ 40 2.4.4 Incubation of treponemes with the HUVECs .................................................. 32 2.5 Dissemination across the Artificial Endothelial Barrier ......................................... 42 2.5.1 Dark-field Microscopy ..................................................................................... 42 2.5.2 Genomic DNA extraction ................................................................................ 43 2.5.3 Real-Time Quantitative PCR ........................................................................... 44 2.6 Confirmation of Strain Identity ............................................................................... 46 2.7 Statistical Analysis .................................................................................................. 47 2.8 In vitro pallilysin