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THE CONTRIBUTION of ONE-CARBON METABOLISM to the PATHOGENESIS of FRANCISELLA TULARENSIS by Matthew Jude Brown B.S. in Biological

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THE CONTRIBUTION of ONE-CARBON METABOLISM to the PATHOGENESIS of FRANCISELLA TULARENSIS by Matthew Jude Brown B.S. in Biological THE CONTRIBUTION OF ONE-CARBON METABOLISM TO THE PATHOGENESIS OF FRANCISELLA TULARENSIS by Matthew Jude Brown B.S. in Biological Sciences – Molecular Genetics, University of Rochester, 2008 Submitted to the Graduate Faculty of the School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2013 UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Matthew Jude Brown It was defended on November 5, 2013 and approved by Dr. Bruce McClane, Ph.D. Professor, Department of Microbiology and Molecular Genetics Dr. Saleem Khan, Ph.D. Professor, Department of Microbiology and Molecular Genetics Dr. Robert Shanks, Ph.D. Associate Professor, Department of Microbiology and Molecular Genetics Dr. Carolyn Coyne, Ph.D. Associate Professor, Department of Microbiology and Molecular Genetics Dissertation Advisor: Dr. Gerard Nau, M.D., Ph.D. Assistant Professor, Department of Microbiology and Molecular Genetics ii Copyright © by Matthew Jude Brown 2013 iii THE CONTRIBUTION OF ONE-CARBON METABOLISM TO THE PATHOGENESIS OF FRANCISELLA TULARENSIS Matthew Jude Brown, PhD University of Pittsburgh, 2013 An emerging paradigm shift in bacterial pathogenesis has resulted in renewed interest in metabolism during infection. The acquisition and synthesis of metabolites by pathogens in the host represents a critical obstacle to successful colonization and infection. The value of bacterial metabolic pathways during infection remains poorly characterized for many pathogens and warrants further investigation. One aspect of this, the contribution of one-carbon metabolism to pathogenic fitness was assessed. This metabolic pathway primarily transfers single carbons and contributes to the synthesis of amino acids, DNA, and proteins. The donated carbon is typically derived from 5,10-methylenetetrahydrofolate, a central compound in one- carbon metabolism. Since the requirement of a metabolic pathway may vary among nutritionally diverse sites within a host, a systemic infection involving multiple niches would be an ideal model to assess the contribution of these pathways. Francisella tularensis is a gram-negative bacterium, a tier one bioterrorism agent, and the causative agent of a debilitating febrile illness known as tularemia. Since this organism is capable of establishing a systemic infection throughout the host, it provides an excellent opportunity to evaluate bacterial metabolism during infection. To evaluate this system in F. tularensis, mutagenesis of the two known sources of 5,10-methylenetetrahydrofolate, the glycine cleavage system and the serine hydroxymethyltransferase, was performed. Loss of either of these pathways resulted in serine auxotrophy, identifying one-carbon metabolism as the exclusive serine biosynthetic pathway of iv F. tularensis. Under standard assay conditions, neither the glycine cleavage system nor the serine hydroxymethyltransferase proved to be essential for intracellular replication in the virulent F. tularensis subsp. tularensis. Despite this result, both 5,10-methylenetetrahydrofolate producing pathways contributed to pathogenesis in a murine model of pneumonic tularemia. Further, these pathways contributed to pathogenic fitness of Francisella to varying degrees throughout the host. Critically, a significant reduction in bacteremia was associated with the loss of either system. These studies highlight differing nutritional environments of distinct sites in the host and confirm that these sites exert variable metabolic stresses on this invasive pathogen. This work identifies one-carbon metabolism as a key bacterial metabolic pathway employed by F. tularensis to overcome nutritional limitation during infection. v TABLE OF CONTENTS PREFACE .................................................................................................................................. XII 1.0 INTRODUCTION ........................................................................................................ 1 1.1 FRANCISELLA TULARENSIS .......................................................................... 1 1.1.1 The Discovery of Francisella tularensis ......................................................... 1 1.1.2 Taxonomy ......................................................................................................... 2 1.1.2.1 Francisella tularensis............................................................................. 3 1.1.2.2 Francisella philomiragia ....................................................................... 6 1.1.2.3 Francisella noatunesis ........................................................................... 6 1.1.2.4 Francisella halioticida ........................................................................... 7 1.1.2.5 Francisella hispaniensis ........................................................................ 7 1.1.2.6 Francisella guangzhouensis .................................................................. 8 1.1.3 Tularemia ......................................................................................................... 8 1.1.4 Bacteriology .................................................................................................... 14 1.1.5 Facultative Intracellular Life Cycle ............................................................. 15 1.1.6 Virulence Factors ........................................................................................... 18 1.1.7 Francisella Metabolism ................................................................................. 24 1.1.7.1 Amino Acids......................................................................................... 25 1.1.7.2 Purines and Pyrimidines .................................................................... 26 vi 1.1.7.3 Micronutrients – Elements and Vitamins ......................................... 28 1.2 ONE-CARBON METABOLISM ..................................................................... 30 1.2.1 5’10-methylenetetrahydrofolate (5’10-mTHF) ........................................... 32 1.2.2 Serine Hydroxymethyltransferase (SHMT) ................................................ 35 1.2.3 Glycine Cleavage System (GCS) .................................................................. 38 1.2.4 Role of one-carbon metabolism in F. tularensis .......................................... 42 1.3 STATEMENT OF THE PROBLEM ............................................................... 47 2.0 THE CONTRIBUTION OF THE GLYCINE CLEAVAGE SYSTEM TO THE PATHOGENESIS OF FRANCISELLA TULARENSIS .......................................................... 48 2.1 ABSTRACT........................................................................................................ 48 2.2 INTRODUCTION ............................................................................................. 49 2.3 MATERIALS AND METHODS ...................................................................... 51 2.3.1 Bacterial strains ............................................................................................. 51 2.3.2 Generation of deletion mutants, complements, and vector controls ......... 52 2.3.3 Growth kinetics in broth culture .................................................................. 54 2.3.4 Generation of primary macrophages and propagation of A549 cells ....... 54 2.3.5 Intracellular growth assays (standard)........................................................ 55 2.3.6 Intracellular growth assays (minimal media) ............................................. 56 Detection of TNF- ........................................................................................ 56 2.3.8 Mouse model of pneumonic tularemia ......................................................... 57 2.4 RESULTS ........................................................................................................... 57 2.4.1 The glycine cleavage system of F. tularensis is required for growth in serine limiting conditions ........................................................................................... 57 vii 2.4.2 gcvT homologs do not contribute to in vitro virulence phenotypes in standard tissue culture assays ................................................................................... 61 2.4.3 gcvT contributes to the pathogenesis of virulent Francisella tularensis in a murine model .............................................................................................................. 62 2.4.4 gcvT increases bacterial burden at sites of dissemination .......................... 64 2.4.5 gcvT contributes to intracellular replication in vitro in serine limiting conditions .................................................................................................................... 65 2.5 DISCUSSION ..................................................................................................... 67 3.0 THE CONTRIBUTION OF THE SERINE HYDROXYMETHYLTRANSFERASE TO THE PATHOGENESIS OF FRANCISELLA TULARENSIS .............................................................................................................................. 73 3.1 ABSTRACT........................................................................................................ 73 3.2 INTRODUCTION ............................................................................................. 74 3.3 MATERIALS AND METHODS ...................................................................... 77 3.3.1 Routine culture and source of bacterial strains
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