University of North Dakota UND Scholarly Commons Theses and Dissertations Theses, Dissertations, and Senior Projects January 2018 Characterization Of Infectious Isolates Of Borrelia Burgdorferi In North Dakota, Bb0399 And Bbb28 In B. Burgdorferi, And Borrelia Miyamotoi In Vitro And In Vivo Brandee Lynn Stone Follow this and additional works at: https://commons.und.edu/theses Recommended Citation Stone, Brandee Lynn, "Characterization Of Infectious Isolates Of Borrelia Burgdorferi In North Dakota, Bb0399 And Bbb28 In B. Burgdorferi, And Borrelia Miyamotoi In Vitro And In Vivo" (2018). Theses and Dissertations. 2356. https://commons.und.edu/theses/2356 This Dissertation is brought to you for free and open access by the Theses, Dissertations, and Senior Projects at UND Scholarly Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UND Scholarly Commons. For more information, please contact [email protected]. CHARACTERIZATION OF INFECTIOUS ISOLATES OF BORRELIA BURGDORFERI IN NORTH DAKOTA, BB0399 AND BBB28 IN B. BURGDORFERI, AND BORRELIA MIYAMOTOI IN VITRO AND IN VIVO by Brandee Lynn Stone Masters of Science, California State University, Chico, 2011 A Dissertation Submitted to the Graduate Faculty of the University of North Dakota In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Immunology Grand Forks, North Dakota May 2018 i Copyright 2018 Brandee Lynn Stone ii PERMISSION Title: CHARACTERIZATION OF INFECTIOUS ISOLATES OF BORRELIA BURGDORFERI IN NORTH DAKOTA, BB0399 AND BBB28 IN B. BURGDORFERI, AND BORRELIA MIYAMOTOI IN VITRO AND IN VIVO Department: Microbiology and Immunology Degree: Doctor of Philosophy In presenting this dissertation in partial fulfillment of the requirements for a graduate degree from the University of North Dakota, I agree that the library of this University shall make it freely available for inspection. I further agree that permission for extensive copying for scholarly purposes may be granted by the professor who supervised my dissertation work, or in his absence, by the Chairperson of the department or the dean of the School of Graduate Studies. It is understood that any copying or publication or other use of this dissertation or part thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of North Dakota in any scholarly use which may be made of any material in my dissertation. Brandee Lynn Stone May, 2018 iviv iv TABLE OF CONTENTS LIST OF TABLES .................................................................................. xi LIST OF FIGURES .................................................................................xii ACKNOWLEDGEMENTS ........................................................................ xiv ABSTRACT ......................................................................................... xvi CHAPTER 1. AN INTRODUCTION TO BORRELIA BURGDORFERI AND BORRELIA MIYAMOTOI ................................................. 1 Borrelia .................................................................... 1 The Unusual Nature of B. burgdorferi S.L. Genetics ............................... 5 Relapsing Fever Borrelia Genetics is also Unusual in Nature ................................... 7 Vectors and Associated Borrelia spp ............................. 8 Life Cycle of Ixodes and Enzootic Cycle of B. burgdorferi s. ............................................... 8 Life Cycle of Ornithodoros and Enzootic Cycle of Tick-Borne Relapsing Fever (TBRF) Borrelia ...................................... 11 Habitats and Behavior of Ixodes and Ornithodoros ............................................ 13 Borrelia spp. Suitably Adapted to the Differences in Vector ....................................... 14 Tick-Borne Spirochete Diseases ................................. 16 Lyme Disease ................................................. 16 vv v Tick-Borne Relapsing Fever .............................. 19 Hard Tick-Borne Relapsing Fever or Borrelia miyamotoi Disease .............................. 19 General Immune Responses and How Borrelia Deal with Them ........................... 23 Osps and VlsE of Lyme Borrelia ........................ 24 Vmps of Relapsing Fever Borrelia ...................... 28 Mechanisms of Immune Evasion by B. miyamotoi ................................................. 28 Problem .................................................................. 29 2. THE WESTERN PROGRESSION OF LYME DISEASE: INFECTIOUS AND NON-CLONAL BORRELIA BURGDORFERI SENSU LATO POPULATIONS IN GRAND FORKS COUNTY, NORTH DAKOTA ............................................................... 31 Introduction ............................................................ 31 Materials and Methods .............................................. 35 Animal Care and Use ....................................... 35 Sample Collection and Culturing Spirochetes ...... 35 Amplification and Sequencing of ospA, ospC, flaB, 016S, 16S-ile tRNA IGS, and p66 ..... 36 NCBI Nuccore and BLAST Database Searches ..... 38 Alignments and Phylogeny ............................... 38 OspC Typing .................................................. 39 Multilocus Sequence Typing (MLST) .................. 40 Infectivity of B. burgdorferi M3 ......................... 41 Enzyme-Linked Immunosorbent Assay (ELISA) ................................................ 42 DNA Extraction ............................................... 42 vivi vi Quantitative PCR (qPCR) ................................. 42 Detection of B. burgdorferi DNA in Nymphal I. scapularis ................................................... 43 GenBank Accession Numbers ........................... 44 Results ................................................................... 44 Sequence and Phylogeny Confirm Spirochetes are B. burgdorferi and Represent Non-Clonal Populations .................................................... 44 The Eastern North Dakota Populations are Most Closely Related to B. burgdorferi Found in the Upper Midwest ............................. 51 Needle-Injected B. burgdorferi M3 Infects and Survives in BALB/c Mice and is Acquired by Larval I. scapularis During Feeding ................... 55 B. burgdorferi M3 Survives the I. scapularis Larval Molt and is Subsequently Transmitted to Naïve BALB/c Mice During a Blood Meal ......... 56 Discussion .............................................................. 57 Acknowledgements .................................................. 60 3. IN VITRO AND IN VIVO CHARACTERIZATION OF TWO ANKYRIN PROTEINS, BB0399 AND BBB28,IN BORRELIA BURGDORFERI ................................................................. 61 Introduction ............................................................ 61 Ankyrin Proteins ...................................................... 62 AnkA ............................................................. 62 AnkB ............................................................. 63 Oxidative Stress ...................................................... 63 Oxidative Stress and B. burgdorferi .................. 64 B. burgdorferi Ankyrin Proteins .................................. 67 B. burgdorferi bbb28 ....................................... 67 viivii vii B. burgdorferi bb0399 ..................................... 69 Objective ................................................................ 70 Materials and Methods .............................................. 71 Cloning ......................................................... 71 Expression of Recombinant BB0399 and BBB28 .................................................... 74 Growth Curves ............................................... 75 Purification of Recombinant Proteins ................. 76 Dot Blots ....................................................... 77 Production of Polyclonal Anti-BBB28 Antibodies ..................................................... 78 Oxidative Stress ............................................. 79 Results and Discussion ............................................. 81 In silico Analysis of BB0399 and BBB28 ............. 81 Cloning and Recombinant Protein Expression ..... 83 Growth Curves ............................................... 90 Codon Usage .................................................. 99 Recombinant Protein Properties ........................ 99 Vector and Expression E. coli Choice ............... 100 Induction Parameters .................................... 100 Oxidative Stress ........................................... 100 Conclusions........................................................... 104 4. IN VITRO CHARACTERIZATION OF BORRELIA MIYAMOTOI AND THE EFFECT OF AGE ON BORRELIA MIYAMOTOI INFECTION IN WILD-TYPE C3H/HEN MICE ......................... 105 Introduction .......................................................... 105 viiiviii viii An Overview of the Complement System ......... 108 Inhibition of the Mammalian Complement System by Borrelia and Ixodes ....................... 111 Factor H, Factor H-Like Protein 1 (FHL-1), and Factor I ................................................. 112 Factor H-Binding Proteins and CRASPs ............ 113 C4b-Binding Protein ...................................... 114 FHBP, C4bp, and Borrelia Niche ...................... 114 CD59-Like Protein ......................................... 116 Complement Inhibition by Ixodes and Ornithodoros Salivary