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Characterization of the Unique Flagellar Hook Structure of the Spirochetes Borrelia Burgdorferi and Treponema Denticola

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Characterization of the Unique Flagellar Hook Structure of the Spirochetes Borrelia Burgdorferi and Treponema Denticola Graduate Theses, Dissertations, and Problem Reports 2013 Characterization of the Unique Flagellar Hook Structure of the Spirochetes Borrelia burgdorferi and Treponema denticola Kelly Ann Heintzelman Miller West Virginia University Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Miller, Kelly Ann Heintzelman, "Characterization of the Unique Flagellar Hook Structure of the Spirochetes Borrelia burgdorferi and Treponema denticola" (2013). Graduate Theses, Dissertations, and Problem Reports. 4983. https://researchrepository.wvu.edu/etd/4983 This Dissertation is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Dissertation in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Dissertation has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. Characterization of the Unique Flagellar Hook Structure of the Spirochetes Borrelia burgdorferi and Treponema denticola Kelly Ann Heintzelman Miller Dissertation submitted to the School of Medicine at West Virginia University in partial fullfillment of the requirements for the degree of Doctor of Philosophy in Immunology and Microbial Pathogenesis Nyles W. Charon, Ph.D., Chair Michael R. Miller, Ph.D. Thomas Elliott, Ph.D. Slawomir Lukomski, Ph.D. Rita Rio, Ph.D. Department of Microbial Pathogenesis and Immunology Morgantown, WV 2013 Keywords: flagella, hook, Borrelia burgdorferi, Treponema denticola, mass spectrometry, peptide cross-linking, spirochete ABSTRACT Characterization of the Unique Flagellar Hook Structure of the Spirochetes Borrelia burgdorferi and Treponema denticola Kelly Ann Heintzelman Miller The long-term goal of this project is to better understand Borrelia burgdorferi and Treponema denticola motility as it relates to pathogenesis. The structure of the spirochete periplasmic flagella is unique when compared to externally located flagella. One unusual characteristic is the flagellar hook, which serves as a universal joint coupling rotation of the membrane bound motor complex to the flagellar filament. The hook is composed of approximately 120 FlgE monomers, and in bacteria with externally located flagella, these structures readily disassociate into monomers (~50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa. We hypothesize that this high molecular weight complex (HMWC) is the result of covalent cross-linking of FlgE proteins. Cross-linking of bacterial flagella proteins has not been examined previously. Our goals are to thoroughly characterize the cross-linking in the B.burgdorferi and T. denticola hook proteins. We anticipate that once we have this information, new drugs could be developed to inhibit this cross-linking, which we anticipate will severely affect the spirochetes' motility and virulence capability. Thus, the proposed project’s long-term goal is centered on developing new drug treatments for Lyme disease as well as other spirochetal diseases, including syphilis. We found that the HMWC of both B. burgdorferi and T. denticola are stable to a variety of denaturants and is not an artifact of boiling. Previously, western blot analysis was the sole indication that spirochetes cross- link their flagellar hook proteins. By making several modifications to the published procedure for isolating periplasmic flagella, I was able to obtain enough hook protein of both B. burgdorferi and T. denticola for mass spectrometry analysis. The mass spectrometry analysis confirmed that the spirochete HMWC is composed of FlgE, identified FlgE as the sole protein in these complexes, and that in specific regions the mass spectrometry patterns are different for the monomer and HMWC. We observed that T. denticola FlgE over-expressed in Escherichia coli forms a stable HMWC in vitro, and used the in vitro HMWC to study the effects of both chemical treatments and point mutations on HMWC formation. Finally, I propose a mechanism of cross-linking that is consistent with the mass spectrometry, biochemical, and mutational analyses. We hypothesize that FlgE cross-linking strengthens the hook structure for optimal spirochete motility. Acknowledgements I wish to dedicate this work to my snuggle buddy Kodiak Lee. You are the joy that keeps me going over day through the highs and lows. You have taught me patience and make me laugh when I'm about to cry. There are several people I would like to thank for support and guidance through this journey. Thank God. Without him, nothing is possible. My husband Eric for patience, listening to me vent, and comforting me when stressed. My parents for their support, time, and taking care of Kodiak. Milinda for being a rock and maintaining my sanity. Giving me support both inside and outside of lab. My mentors Nyles and Mike for taking on one more graduate student before retirement, letting me work on a really cool project, guiding me through this process, and for being good friends. My friends Amanda, Dacie, and Chrissy. For always being there, for good times, and helping me with the job search. Andy Cockburn for listening and good suggestions. You always looked at things from a different angle and gave fresh ideas. My committee: Thomas Elliott, Slawomir Lukomski, and Rita Rio for ideas, suggestions, looking over results with me, assisting in experimental design, and facilitating my scientific development. iii Collaborators: Chris Li (SUNY, Buffalo), Brian Crane (Cornell), Jun Liu (University of Texas), MD Motaleb (ECU), Justin Hettick (NIOSH), Melissa Caimano (University of Connecticut), Roger Hendrix and Bob Duda (University of Pittsburgh). Thank you for your contributions to this project. My dogs Lucky and Reba for stress relief and nice walks. iv Table of Contents Chapter 1: Introduction....................................................................................... 1 Medical Importance of T. denticola................................................................... 2 Medical Importance of B. burgdorferi............................................................... 3 Flagellar structure and motility.......................................................................... 4 Structure of periplasmic flagella and how spirochetes swim............................ 12 Motility as a virulence factor of spirochetes...................................................... 15 Covalent bonding of peptides in microorganisms............................................. 17 Methodology to detecting and deciphering cross-links..................................... 22 Indirect evidence of cross-linking in spirochete flagella.................................... 23 Research objectives......................................................................................... 25 References............................................................................................... 26 Chapter 2: Initial characterization of the FlgE high molecular weight complex of Borrelia burgdorferi......................................................................... 39 Abstract............................................................................................................ 40 Introduction....................................................................................................... 41 Materials and Methods..................................................................................... 43 Results and Discussion.................................................................................... 48 References............................................................................................... 59 Chapter 3: The biochemistry of the Treponema denticola FlgE high molecular weight complex.................................................................................. 68 Abstract............................................................................................................ 69 Introduction....................................................................................................... 70 Materials and Methods..................................................................................... 71 Results.............................................................................................................. 76 Discussion........................................................................................................ 98 References....................................................................................................... 104 Chapter 4: Discussion......................................................................................... 111 Appendix 122 The unique paradigm of spirochete motility and chemotaxis............................ 130 Visualizing the sequential assembly of bacterial flagella in Borrelia 154 burgdorferi by cryo-electron tomography.......................................................... Curriculum Vitae 176 v List of Figures Figure 1.1: Flagellar components of S. enterica...................................... 4 Figure 1.2: Proposed structure
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