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Iron Is an Essential Nutrient 4 Bacteria and Humans

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Iron Is an Essential Nutrient 4 Bacteria and Humans Abstract The bhuTUV and bhuO genes play vital roles in the ability of Brucella abortus to use heme as an iron source and are regulated in an iron-responsive manner by RirA and Irr by Jenifer F. Ojeda April, 2012 Dissertation Advisor: RM Roop II Department of Microbiology and Immunology Brucella abortus is a Gram negative intracellular pathogen that causes the zoonotic disease brucellosis. Antibiotic treatment for brucellosis in humans is prolonged and sometimes followed by relapses. Currently, the United States employs prevention of the illness in humans through cattle vaccinations, eliminating the bacterium in its natural host. Unfortunately, these vaccine strains cause the disease in humans, and Brucella research ultimately aims to identify new vaccine targets as well as alternative treatment options. Brucella abortus resides in the phagosomal compartment of the host macrophage where essential nutrients such as iron are limited. Most bacteria need iron, and within the macrophage, heme is a likely source of iron due to the breakdown of red blood cells by the host macrophage. Heme transporters in Gram negative bacteria are highly conserved, and include components for outer membrane, periplasmic, and cytoplasmic membrane transport. BhuA has been previously characterized as the outer membrane heme transporter of Brucella abortus and here we report that BhuT, BhuU, and BhuV (BhuTUV) are the periplasmic and cytoplasmic heme transport components and that they are required in order for Brucella abortus to transport heme as an iron source. Utilization of heme as an iron source requires the breakdown of heme into ferrous iron, carbon monoxide, and biliverdin by a heme oxygenase. BhuO has been identified as a heme oxygenase in Brucella abortus, and although there seems to be more than one heme oxygenase in Brucella, this study shows that BhuO is needed for the use of heme as an iron source under iron starvation conditions in vitro. Further, both bhuTUV and bhuO are regulated in an iron- responsive manner. The iron responsive regulator Irr directly represses bhuO, which shares an operon with rirA. Then the rhizobial iron regulator RirA in turn represses the bhuTUV operon. Together, these regulators help to maintain iron homeostasis within the bacterial cell, protecting it from damaging hydroxyl radicals produced by Fenton chemistry. The bhuTUV and bhuO genes play vital roles in the ability of Brucella abortus to use heme as an iron source and are regulated in an iron-responsive manner by RirA and Irr A Dissertation Submitted to the Graduate Faculty of the Brody School of Medicine at East Carolina University In Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy in the Department of Microbiology and Immunology By Jenifer F. Ojeda April 2012 © Jenifer F. Ojeda, 2012 The bhuTUV and bhuO genes play vital roles in the ability of Brucella abortus to use heme as an iron source and are regulated in an iron-responsive manner by RirA and Irr By Jenifer F. Ojeda APPROVED BY: DIRECTOR OF DISSERTATION_________________________________ Roy M. Roop II, Ph.D. COMMITTEE MEMBER________________________________________ Charles J. Smith, Ph.D. COMMITTEE MEMBER________________________________________ Everett C. Pesci, Ph.D. COMMITTEE MEMBER________________________________________ Cindy Putnam-Evans, Ph.D. COMMITTEE MEMBER________________________________________ Mark D. Mannie, Ph.D. CHAIR OF THE DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY _________________________________ Charles J. Smith, Ph.D. DEAN OF THE GRADUATE SCHOOL _________________________________ Paul J. Gemperline, Ph.D. Dedication This dissertation is dedicated to my husband Dan, who by all accounts earned this degree with me. He held the magic to obtaining the most obscure reference articles, the most visually appealing graphs, and had the wildest imagination when it came to scientific solutions. He rode the emotional turmoil of graduate school with me, and this work truly would not have been possible without his constant love and support. Acknowledgements First and foremost I offer my sincerest gratitude to my supervisor, Dr. Marty Roop, who has supported me thoughout my project with his patience and knowledge while still allowing me the room to work in my own way. I would like to thank him for taking the time to teach me how to fish (literally!) and for showing me that with the right attitude you can catch fish no matter how many pieces your fishing pole has broken into. One simply could not wish for a better or friendlier supervisor. I would also like to thank JT Paulley for taking the time to train me properly. I have missed the late night beer and cigar talks with JT, Eric, and Mike, who instilled in me a joy for arguing science. So many side projects began this way.... I would like to thank my committee for their patience and understanding during all of the curve balls life threw my way. I appreciate your guidance throughout this project. Lastly, I would like to thank the current members of the Roop Lab for the teamwork and fun that we have shared over the years. It is nice have such strong resources for help when technical challenges arise. Table of Contents CHAPTER 1 Literature Review.............................................................................................................................1 Brucella ....................................................................................................................................1 The causative agent of brucellosis .......................................................................................1 A stealthy intracellular pathogen .........................................................................................4 The bacterial necessity for iron ................................................................................................7 The chemistry of iron ..........................................................................................................7 Bacterial adaptations to the chemistry of iron .....................................................................8 Availability of iron within the host ........................................................................................13 Iron trafficking within the host ..........................................................................................13 Host iron sources during infection ....................................................................................16 Pathogenic bacteria acquire iron from the host ......................................................................17 Acquisition of ferric and ferrous iron from the host..........................................................17 Acquisition of heme from the host ....................................................................................19 References ......................................................................................................................................25 Figures............................................................................................................................................48 STATEMENT OF THE PROBLEM .........................................................................................56 CHAPTER 2 bhuTUV encodes proteins required for heme transport in Brucella abortus 2308 and is regulated by RirA ...........................................................................................................................59 Abstract ......................................................................................................................................59 Introduction ................................................................................................................................60 Materials and Methods ...............................................................................................................63 Results and Discussion ...............................................................................................................68 Acknowledgements ....................................................................................................................74 References ..................................................................................................................................75 Figures and Tables .....................................................................................................................81 CHAPTER 3 The bhuO gene encodes a heme oxygenase that contributes to the ability of Brucella abortus 2308 to use heme as an iron source and is regulated by Irr ...........................................................93 Abstract ......................................................................................................................................93 Introduction ................................................................................................................................94 Materials and Methods ...............................................................................................................95 Results and Discussion .............................................................................................................102 Acknowledgements ..................................................................................................................107 References ................................................................................................................................108 Figures and Tables ...................................................................................................................115
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