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Analysis of Virulence-Associated Petrobactin Reacquisition in Bacillus Anthracis

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Analysis of Virulence-Associated Petrobactin Reacquisition in Bacillus Anthracis Analysis of Virulence-Associated Petrobactin Reacquisition in Bacillus anthracis by Shandee Dawn Dixon A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Microbiology and Immunology) in The University of Michigan 2013 Doctoral Committee: Professor Philip C. Hanna, Chair Professor Carol A. Fierke Associate Professor Maria B. Sandkvist Professor David H. Sherman The Bacillus anthracis Petrobactin ATP-Binding Cassette (ABC) Import System ©Shandee D. Dixon 2013 DEDICATION For my Family ii PREFACE This written dissertation summarizes the labor and thought invested in my graduate studies to develop a better understanding of how Bacillus anthracis reacquires the vital siderophore petrobactin back into the cytoplasm after sequestering iron from its host. The identification of this system and the elucidation of the mechanisms involved in the reacquisition of iron-bound petrobactin will greatly improve our current knowledge of Bacillus anthracis pathogenesis. This body of work is divided into four chapters. Chapter 1 introduces the general importance of iron in microbial biology and aspects of iron acquisition in Bacillus anthracis. A n emphasis is placed on c urrent research surrounding the virulence- associated siderophore petrobactin and introduces the uptake mechanisms employed by bacteria to reacquire iron-bound siderophores. Additionally, Chapter 1 describes work I performed along with my colleagues during my first year in the Hanna laboratory which provides the first description of the only receptor protein required for the reacquisition of petrobactin. This research has been published as an article in the journal Molecular Microbiology (Carlson PE Jr, Dixon SD, Janes BK, Carr KA, Nusca TD, Anderson EC, Keene SE, Sherman DH, and Hanna PC 2008. “Genetic analysis of petrobactin transport in Bacillus anthracis.” Mol Microbiol. 2010 Feb;75(4):900-9). Chapter 2 describes work performed to identify the remaining components of the petrobactin import system. Mutants of candidate permease and ATPase genes were iii generated allowing for characterization of multiple petrobactin ATP-binding cassette (ABC)-import systems. The research put forth in Chapter 3 ha s been published in the journal of Molecular Microbiology (Dixon SD, Janes BK, Bourgis A, Carlson PE Jr, and Hanna PC. 2012. “Multiple ABC transporters are involved in the acquisition of petrobactin in Bacillus anthracis”. Mol Microbiol. 2012 Apr;84(2):370-82.) Findings in Chapter 2 directed my focus onto designing a biochemical assay to identify small molecule inhibitors of petrobactin ABC-import machinery. The identified ABC-transport proteins described in Chapter 2 are essential in cell viability, virulence and pathogenicity, and have conserved sequences across all bacterial species, making these proteins intriguing targets for therapeutics. Because active ATPases can be easily purified and only requires ATP as a substrate, these enzymes are prime candidates for a high-throughput inhibitor search. Chapter 3 describes my efforts to design and implement a high-throughput screen (HTS) at the University of Michigan Center for Chemical Genomics (CCG) to search for novel molecules capable of inhibiting the activity of these crucial ATPase proteins. By investigating methods to inhibit the activity of these ATPases, my overall goal was to devise a strategy to shut down siderophore import and thus the necessary act of iron acquisition by B. anthracis and other dangerous pathogens. As a closing section, Chapter 4 a ims to summarize the research presented in Chapters 2 and 3 as well as its possible implications in the fields of microbial iron- acquisition and the development of novel therapeutics against bacterial infection. Generally, Chapter 4 encompasses hypothetical and future avenues of experimentation that were made more apparent by the body of this dissertation; however, in some instances, preliminary data is presented. iv TABLE OF CONTENTS Dedication………………………………………………………………………………...ii Preface…………………………………….……………………………………………..iii List of Figures…………………………….………………………………………….......ix List of Tables…………………………….…………………………………………..….xii Abstract ………………………………………………………………………………..xiii Chapter 1………………………….………………………………………………….......1 Introduction………….……………………………………………………….......1 1.1 Introduction……….……………………………………………………….......1 1.2 Importance of Iron during Anthrax Infection...………….………….…….......2 1.3 Heme-iron Acquisition……………………………...…………………………4 1.4 Bacillus anthracis Siderophores………………………………………...…….8 1.5 Petrobactin Identification and Biosynthesis………………………………….11 1.6 Petrobactin is a “Stealth” Siderophore…...……………………….………….14 1.7 Iron Response Regulation…… ……………………………………………...16 1.8 Siderophore Recognition………………………………………….........…....18 1.9 ATP-Binding Cassette Transport Systems…...................................................21 ABC Transporter Structure……………..………………………………..25 ATPase Domains…………………………………….…………………..26 v ABC Transporter Assembly………………………………..……………29 ABC Transport and Petrobactin Utilization….…………….…………….30 1.10 References……..……………………………………………………...….....32 Chapter 2………………………….……………………………………………….........49 Multiple ABC Transporters are Involved in the Acquisition of Petrobactin by Bacillus anthracis……………………………………………………………….49 2.1 Introduction…………………………………………………………………..49 2.2 Results………………...….…………………………………………………..51 Identification of Two Individual Permeases Involved in Petrobactin Reacquisition……………...……………………………………………...51 The ∆fpuB∆fatCD Mutant is Attenuated for Virulence in a Murine Model of Inhalational Anthrax………………………...………………...............59 Identification of Three ATPase Components Involved in Petrobactin Transport……………………………...…….…………...……………….62 2.3 Discusion…………….…………………………………………………...…..66 2.4 Experimental Procedures………….………………………………………....71 Bacterial Growth and Sporulation Conditions…..……………………….71 Isolation of Mutants and Construction of Complementation Plasmids.…73 Measurement of Catechol Accumulation…………….…..………………74 Murine Virulence Assays………………………..……………………….75 2.5 Supplemental Material…………….................……………………………....76 vi 2.6 References………………………………………………………………........78 Chapter 3………………………….……………………………………………….........85 A High Throughput Screen for Inhibitors of the Bacillus anthracis Petrobactin ABC-Transporter ATPases ………………………………...…...85 3.1 Introduction……………...…………………………………………………..85 3.2 Results………………………………………………………………………..88 Heterologous Over-Expression and Purification of Three ATPases….…88 Colorimetric Determination of ATPase Activity…………….………….91 Optimization of Malachite Green Based ATPase Activity Assay………94 High-Throughput Screen for Small Molecule Inhibitors of FpuD Mediated ATP Hydrolysis………………………………………………………….96 Reconfirmation of Inhibitory Activity of Hit Compounds……………..103 In vitro Antimicrobial Activity of Top FpuD Inhibitors……….……….105 3.3 Conclusions and Future Directions.………………………………………...111 3.4 Experimental Procedures.…………………………………………………..116 Bacterial Strains and Plasmids……………….…………………………116 Cloning and Over-Expression of B. anthracis ABC transport ATPases and ATPase Mutants………………………………….……………………..116 Purification of B. anthracis ABC Transport ATPase-His and ATPase- E167Q-His Mutants……………………………………………….……117 ATPase Activity Assay…..……………………………………………..118 vii Small-Molecule Libraries………………………………………………120 High Throughput Screening Protocol and Determination of Z’……..…120 Dose Response Assays and Determination of AC50 for Inhibitors..……122 Secondary Screen Against Metal Chelating Compounds………………122 Cytotoxicity Assays and Determination of MIC Values for Inhibitors...123 3.7 References………………………………………………………………......123 Chapter 4………………………….………………………………………….……......128 Discussion of Research………………………………………………………..128 4.1 Summary……………………...…………………………………...………..128 4.2 Iron Release……..……..…………………...…..………….……..………...129 4.3 Petrobactin Efflux and Cycling………………………………………….….133 4.4 The Role of Bacillibactin……….…...…………...……………..………......138 4.5 Xenosiderophore Recognition……………………………………………...141 4.6 Targeting B. anthracis Iron Import; The Search for a New Therapeutic…..144 4.7 References……………………………...…………….…………...………...146 viii LIST OF FIGURES Figure 1-1 Known Mechanisms of Iron Acquisition from Heme or Bacillibactin in Bacillus anthracis …………………………………………………………...……………………..7 1-2 The Two Siderophores Produced by Bacillus anthracis..……………….......………..9 1-3 B. anthracis Virulence-Associated Siderophore Petrobactin Biosynthetic Operon and Molecular Structure ………………..……….………………………………...……..12 1-4 Growth of B. anthracis Mutant Strains in Iron Depleted Media and Attenuation of Virulence of the ΔfpuA Strain in a Murine Model of Infection ……………….……20 1-5 Iron Acquisition in Gram-positive Bacteria Using Siderophore Secretion and ABC- Transport…………..………………………………………………………...……….24 1-6 Gram-Positive ATP-Binding Cassette (ABC) Transporter Domains and Conserved motifs in the ATPase…………………………………………………………………28 2-1 Organization of Genes Investigated in this Work……………..………………..…...52 2-2 Growth Phenotypes of B. anthracis Mutant Strains......……………………...……...54 2-3 Petrobactin Supplementation does not Restore Growth of Transport Deficient Mutants in IDM ……………………………………………..……………………….55 2-4 Accumulation of Extracellular Catechols in Petrobactin Transport Deficient strains.57 2-5 Resistance of Petrobactin Transport Deficient Strains to Gallium Toxicity..……….58 2-6 Attenuation of Double Permease Mutant and ATPase Mutants in Murine Model of Inhalational Anthrax………………………..………………………………………..61 ix 2-7
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