TprK Variation and Syphilis Pathogenesis Tara Brinck Reid A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2014 Reading Committee: Sheila Lukehart, Chair Patricia Totten Wesley Van Voorhis Program Authorized to Offer Degree: Pathobiology i ©Copyright 2014 Tara Brinck Reid ii University of Washington Abstract TprK Variation and Syphilis Pathogenesis Tara Brinck Reid Chair of the Supervisory Committee: Professor Sheila Lukehart Departments of Medicine and Global Health Treponema pallidum subsp. pallidum is the etiologic agent of syphilis, a multistage disease that poses a global health problem. Despite the availability of curative therapy and ongoing public health efforts to eradicate syphilis, the disease is re-emerging in developed countries and remains prevalent in other locations. Calculating the basic reproductive number (R o) of syphilis allows for the estimation of the number of people infected by a single infectious person. R o is proportional to the duration of infectiousness, efficiency of transmission, and the number of new susceptible contacts. An infectious disease is expected to increase prevalence in a population when R o>1, and will die out if R o< 1. It is known that antibodies directed to exposed surface antigens are critical for opsonophagocytosis and clearance of this pathogen from lesions. TprK is a T. pallidum antigen that undergoes antigenic variation during infection. Evidence shows that antibodies are directed to the variable regions of the protein and TprK sequence changes abrogate antibody binding, thus allowing immune escape. This dissertation describes TprK‘s effect on two of the three parameters used to calculate R o. The first is the role of TprK variation in duration of infectiousness, exemplified by the association found between TprK variants and secondary lesions. Length of the infectious period is increased by facilitating development of the second stage of syphilis in the face of developing opsonic antibody to TprK. The second iii component of Ro impacted by TprK variation is the number of new susceptible contacts an infected individual encounters. TprK variation contributes to T. pallidum’s ability to reinfect or superinfect individuals that have been previously exposed by altering opsonic antibody epitopes such that newly infecting treponemes escape the established immune response. In summary, antigenic variation of TprK is associated with the ability of the spirochete to re- infect previously exposed individuals, and increase the duration of infectiousness by facilitating persistence into the second stage of syphilis. Antigenic variation of the outer membrane protein, TprK, accounts for a larger estimated R o value for syphilis, and thus may be associated with its centuries of success as a devastating disease. iv Table of Contents List of Figures ..................................................................................................................... vi List of Tables ..................................................................................................................... vii I. Background and Introduction .......................................................................... 1 Historical Perspectives ............................................................................ 1 Epidemiology ........................................................................................... 4 Natural History......................................................................................... 7 Attachment and Invasion ........................................................................13 Immunology ............................................................................................16 Diagnostic Tools .....................................................................................19 Treatment ...............................................................................................22 Biology of the Etiologic Agent .................................................................24 Animal Models ........................................................................................25 Vaccines.................................................................................................28 Outer Membrane Proteins ......................................................................29 Treponema pallidum repeat Protein K (TprK) .........................................32 Antigenic Variation and Pathogenesis ....................................................34 Figures ...................................................................................................37 II. Antigenic Variation of TprK Facilitates Development of Secondary Syphilis . 41 Abstract ..................................................................................................41 Introduction ............................................................................................42 Methods .................................................................................................44 Results ...................................................................................................50 Discussion ..............................................................................................55 Tables and Figures .................................................................................61 III. Repeated Syphilis Infection is Facilitated by Antigenic Variation of TprK ...... 73 Abstract ..................................................................................................73 Introduction ............................................................................................74 Methods .................................................................................................77 Results ...................................................................................................83 Discussion ..............................................................................................89 Tables and Figures .................................................................................95 IV. Conclusions and Future Directions ............................................................. 106 Figures ................................................................................................. 117 References ...................................................................................................................... 118 v List of Figures Figure I-1. Syphilis natural history ............................................................................................37 Figure I-2. Prevalence of strain types in Seattle .......................................................................38 Figure I-3. Model of TprK variation by gene conversion ...........................................................39 Figure I-4. Predicted structure of TprK .....................................................................................40 Figure II-1. Examples of TprK V7 sequences ...........................................................................69 Figure II-2. Phylogenetic analysis of tprK DNA sequences from secondary lesions .................70 Figure II-3. Proportions of lesions arising from TprK variants ...................................................71 Figure II-4. Analysis of antibody reactivity in secondary syphilis ...............................................72 Figure III-1. Comparison of strains studied ............................................................................. 101 Figure III-2. Alignment of majority TprK sequences ................................................................ 102 Figure III-3. Nichols Houston E challenge sites ...................................................................... 103 Figure III-4. Pre-challenge antibody reactivity......................................................................... 105 Figure IV-1. Serologic reactivity to TprC ................................................................................. 117 vi List of Tables Table II-1. Oligonucleotides used in Chapter II .........................................................................61 Table II-2. Oligopeptides used in Chapter II .............................................................................63 Table II-3. GenBank accession numbers ..................................................................................65 Table III-1. Oligonucleotides used in Chapter III .......................................................................95 Table III-2. Oligopeptides used in Chapter III ...........................................................................97 Table III-3. Development of homologous immunity after 90-day initial infection ........................99 Table III-4. Isolates with a full repertoire of 50 donor sites (DS) are able to superinfect .......... 100 vii Acknowledgements Throughout the process of obtaining this degree, I have leaned heavily on the UW Syphilis research group and am deeply grateful for their continued patience, support, and enthusiasm. I could never have hoped for a more friendly and supportive environment to grow as a person and as a scientist. Additionally, I would like to thank my doctoral committee for their patient mentoring over these years. To my father, I am grateful for instilling in me a sense of adventure in learning and excitement in undertaking new and varied projects. I have found this outlook on, and approach to, learning to be critical