Graduate Theses, Dissertations, and Problem Reports 2006 Measuring chemotaxis in Borrelia burgdorferi the Lyme disease spirochete Richard Gerrit Bakker West Virginia University Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Bakker, Richard Gerrit, "Measuring chemotaxis in Borrelia burgdorferi the Lyme disease spirochete" (2006). Graduate Theses, Dissertations, and Problem Reports. 2423. https://researchrepository.wvu.edu/etd/2423 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]. Measuring Chemotaxis in Borrelia burgdorferi the Lyme Disease Spirochete Richard Gerrit Bakker Dissertation submitted to the School of Medicine at West Virginia University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Microbiology and Immunology Nyles W. Charon Ph.D. (Chair) Melanie Fisher MD Daniel Flynn Ph.D. Michael Miller Ph.D. Rosana Schafer Ph.D. David Yelton Ph.D. Department of Microbiology Immunology and Cell Biology Morgantown, West Virginia 2004 Keywords: Borrelia burgdorferi, chemotaxis, flow cytometry, Lyme disease, CheA complementation Copyright 2004 Richard G. Bakker ABSTRACT Measuring Chemotaxis in Borrelia burgdorferi the Lyme Disease Spriochete Richard Gerrit Bakker Borrelia burgdorferi is the spirochete causative agent of Lyme disease. The chemotaxis and motility systems of these bacteria are far less well described than that of Escherichia coli or Salmonella enterica. This dissertation explores the role of the CheA proteins in the chemotactic response and describes the first defined attractants for B. burgdorferi. In order to test hypotheses, we developed or optimized three protocols. To characterize the motion of cells, two motion tracking systems were optimized. The Hobson BacTracker allowed for tracking cell motions in real time. This hardware/software chimera, while powerful for the specific application, utilizes a cumbersome interface. Therefore, the software package Volocity was adopted. While the tracking itself is somewhat slower, the interface greatly facilitates data collection, organization, and presentation, making it much faster. To assay chemotaxis with the capillary tube assay, one must enumerate cells. This was previously difficult because cell enumeration was slow, laborious, and ineffectual at low concentrations. We overcame these limitations by initially developing a protocol for enumerating cells by flow cytometry. Once this enumeration method was validated with direct comparisons to Petroff-Hausser counting chamber data, we were able to screen for attractants using a modified capillary tube assay. We found that B. burgdorferi mutants in cheA2 were non-chemotactic to defined attractants. Complementation of cheA2 restored the wild-type phenotype. Mutants in cheA1 failed to show any discernable phenotype. The modified capillary tube chemotaxis assay was used to screen for chemoattractants. To date serine, glycine, N-n-diacetyl-chitobiose, glucose, glutamate, putricine, spermidine, rabbit serum, and glucosamine, have been tested, the latter five had at least some chemoattractant ability. Finally, this is the first work to correlate the ability of the cells to reverse with chemotaxis. In conclusion, this work developed techniques to track the motion of B. burgdorferi and measure the chemotactic response with a high throughput assay. These tools are being used in a screen of compounds which has already found 5 specific compounds that act as chemoattractants. The techniques developed will be useful not only for B. burgdorferi, but will facilitate measuring the chemotactic response in other slow growing prokaryotic species. iv I wish to dedicate this work to: My Parents: Leslie and Geert Bakker and My Grandmother: Ellen Smith Thank you for all your support, without you none of this would have been possible. v I wish to acknowledge and thank a number of people for their time, help, and support over the course of my research. At West Virginia University: The Charon laboratory – for all the help and patience Dr. Nyles Charon Melanie Sal Dr. Chunhao Li Dr. Md. Motaleb Pang Jia My fellow graduate students – for technical help and harassment Lisa Rucks Matt Hirsh Jeff Miller Alicia Mathers Sarah Dodson The Flow Cytometric Core Facility Dr. Cynthia Cunningham – for help, technical and otherwise, as well as chocolate provisions At the University of California Los Angles: Dr. Renata Lux - for teaching me tissue penetration assays At the University of Minnesota: Dr. Stuart G. Goldstein – for making mathematics work for me My Committee: Melanie Fisher MD Daniel Flynn Ph.D. Michael Miller Ph.D. Rosana Schafer Ph.D. David Yelton Ph.D. At home: Mark A. Blake – for being you, and putting up with me vi Table of Contents Chapter 1 : Introduction ........................................................................................1 A. History of Lyme Disease and Classification of Borrelia species ......................2 B. Structure and Motility of B. burgdorferi and Escherichia coli. ...........................4 Spirochete Structure and Motility.......................................................................4 Escherichia coli Motility and Chemotaxis ..........................................................5 Similarities and Differences between E. coli and B. burgdorferi ........................9 C. Clinical manifestations of Lyme Borreliosis ....................................................10 Disease Staging ..............................................................................................10 Immune Response ..........................................................................................12 Serologic Diagnosis.........................................................................................13 European and Asian Manifestations of Disease..............................................14 Treatment and Vaccine ...................................................................................15 D. Chemotaxis and Motility as Virulence Factors................................................16 Chemotaxis and Motility as Virulence Factors in B. burgdorferi ......................17 Chemotaxis and Motility as Virulence Factors in Other Species .....................17 Chemotaxis and Motility as Virulence Factors in other Spirochete Species....19 E. Borrelia burgdorferi Life Cycle and Gene Expression.....................................20 Life Cycle and Vectors ....................................................................................20 Gene Expression.............................................................................................21 F. Genetic Manipulation of B. burgdorferi ...........................................................23 Chromosome Structure and Replication..........................................................24 Genetic Manipulation.......................................................................................24 Shuttle Vectors and Complementation............................................................25 Lateral Gene Transfer: Transposons and Phages...........................................25 G. The Structure and Function of Methyl Accepting Chemotaxis Proteins.........26 Mcp Structure and Function ............................................................................26 Mcp Clustering and Amplification ....................................................................27 Mcp Response to Repellants...........................................................................29 H. The Structure and Function of Chemotaxis Protein A (CheA) ........................30 CheA Activation...............................................................................................31 CheA in B. burgdorferi.....................................................................................32 vii I. Capillary Tube Assay and Flow Cytometric Enumeration of B. burgdorferi......33 The Capillary Tube Assay ...............................................................................33 Modifications of the Capillary Tube Assay.......................................................34 Enumeration of B. burgdorferi using Flow cytometry.......................................35 Flow Cytometric Enumeration of B. burgdorferi...............................................36 J. Cell Motility Tracking Procedures....................................................................38 Film Based Tracking........................................................................................38 Prokaryotic Translational Velocity ...................................................................41 Chapter 2: Asymmetrical flagellar rotation in Borrelia burgdorferi cheA mutants 43 Abstract ...........................................................................................................44