THE IRON-DEPENDENT CYANIDE AND HYDROGEN PEROXIDE CO-TOXICITY IN ESCHERICHIA COLI AND ITS CATASTROPHIC CONSEQUENCES FOR THE CHROMOSOME BY TULIP MAHASETH DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology in the Graduate College of the University of Illinois at Urbana-Champaign, 2015 Urbana, Illinois Doctoral Committee: Professor Andrei Kuzminov, Chair and Director of Research Professor John E. Cronan Professor Jeffrey F. Gardner Associate Professor Carin K. Vanderpool ABSTRACT 2+ Hydrogen peroxide (H2O2) can oxidize cytoplasmic ferrous ions (Fe ) to produce highly reactive hydroxyl radicals (•OH) via Fenton’s reaction that can damage various biomolecules causing oxidative stress. Even though at concentrations higher than 20 mM H2O2 by itself can efficiently kill micro-organisms, it is metabolically impossible for eukaryotic cells to generate H2O2, an uncharged molecule, in such large quantities inside the cell. We propose that potentiation of physiologically relevant amounts of H2O2 by various small molecules serves as a more feasible and safe mechanism to combat invading microbes. NO potentiation of H2O2 toxicity is a known bactericidal weapon employed by macrophages. In fact, in human neutrophils activated by bacterial infection, the myeloperoxidase enzyme catalyzes the formation of hydrogen cyanide (HCN) from serum thiocyanate (SCN-). In the past, researchers have reported that a combination of low millimolar doses of H2O2 and cyanide (CN), which are individually bacteriostatic, caused rapid synergistic killing in Escherichia coli. Our aim is to understand the immune cells antimicrobial responses by investigating the mechanism of CN potentiation of H2O2 toxicity and its chromosomal consequences. We have found that the ability of CN to recruit iron from intracellular depots such as ferritin contributes to its potentiation of H2O2 toxicity, whereas the major stationary phase intracellular iron depot protein, Dps, can sequester this iron, thereby quelling Fenton's reaction. Our work has also demonstrated that this synergistic toxicity is associated with catastrophic chromosomal fragmentation, which is blocked by iron chelators. Moreover, the CN + H2O2 treatment also resulted in destruction of ribosomal RNA, indicating that RNA is equally susceptible to the oxidative damage induced. The ii catastrophic chromosomal fragmentation induced by CN and H2O2 was found to be cell density-dependent, but replication- and translation-independent. We propose that disrupting intracellular iron trafficking to cause catastrophic chromosomal fragmentation is a common strategy employed by the immune system to kill invading microbes. We also showed that the base excision repair pathway plays the major role in preventing this catastrophic chromosomal fragmentation, which indicates that the double-strand breaks induced by CN + H2O2 are preceded by a significant number of base modifications, removed by base-excision repair via the abasic site intermediates. These lesions can be converted into double-strand breaks that are repairable via the recombinational repair system. On the other hand, termination of the CN + H2O2 treatment, which results in termination of the CN-induced block of respiration and ATP production, was found to trigger significant linear DNA degradation and disintegration of the nucleoid structure. We have shown that this disassembly of the nucleoid structure following the removal of CN and H2O2 is affected by the nucleoid-associated proteins and depends on ongoing transcription, thereby revealing the role of transcription in the nucleoid dynamics. Therefore, we conclude that cyanide potentiates hydrogen peroxide toxicity by recruiting iron from intracellular depots directly onto the DNA, where the DNA-iron complexes catalyze self-targeted Fenton's reaction leading to catastrophic DNA damage. This massive DNA damage is actively countered by base-excision repair mechanisms, but it eventually saturates the cellular DNA repair capacity and causes disintegration of the trancriptionally-active nucleoids. iii To my family and loved ones iv ACKNOWLEDGEMENTS This work would not have been possible without Dr. Andrei Kuzminov’s incredible guidance and support. Andrei is a brilliant scientist, from whom I have learned so much. I am extremely grateful to have the opportunity to work alongside him for the past several years. Thank you for always steering me in the right direction, in science and otherwise. I would like to thank Dr. James Imlay and all the members of my thesis committee – Dr. John Cronan, Dr. Jeffrey Gardner and Dr. Carin Vanderpool, for their advice and invaluable feedback throughout graduate school. Jim, an expert on hydrogen peroxide toxicity, has been especially patient and supportive of my work, which was, in fact, pioneered by him during his time as a graduate student at Berkeley. I am thankful to him for all the times he has listened to the several obstacles I faced with this project and provided his invaluable insights on them. I would also like to thank all the members of the Kuzminov Lab – Dr. Elena Kouzminova, Pritha Rao, Dr. Sharik Khan, Dr. Glen Cronan and Dr. Ka wai Kuong, for all their help and suggestions over the years, and for making the lab a fun and wonderful place to work. I am also thankful to Micah Hodosh for his love and companionship, and for seeing me through the ups and downs of graduate school. Last, but certainly not the least, I would like to thank my family – Dada, Mamma and Neil, for their love, support, encouragement and patience. Without them I would not be where I am today. v TABLE OF CONTENTS CHAPTER 1: INTRODUCTION...............................................................................1 1.1. BIOLOGICAL SIGNIFICANCE OF HYDROGEN PEROXIDE..................... 1 1.l.1. Relevance of H2O2 at Physiological Levels...........................................1 1.1.2. Biological Sources of H2O2 and Their Roles ........................................2 1.2. HYDROGEN PEROXIDE TOXICITY IN ESCHERICHIA COLI ................... 4 1.2.1. Oxidative Stress and Reactive Oxygen Species....................................4 1.2.2. Sources of ROS in the Cell ...................................................................5 1.2.3. Targets of H2O2.....................................................................................6 1.2.3.1. DNA damage caused by H2O2 ...............................................6 1.2.3.2. RNA damage caused by H2O2 ...............................................8 1.2.3.3. Protein oxidation..................................................................10 1.2.3.4. Lipid peroxidation................................................................11 1.3. IRON METABOLISM AND TOXICITY........................................................ 11 1.3.1. Iron-Containing Enzymes and Their Distribution in Metabolism ...................................................................................................11 1.3.2. The Iron Paradox.................................................................................12 1.3.3. Iron Scavenging in E. coli...................................................................13 1.3.4. The Iron Depots of E. coli...................................................................14 1.3.4.1. The DNA binding, iron detoxification and storage protein, Dps.......................................................................................14 1.3.4.2. Ferritin - The primary cellular iron storage protein.............16 1.3.5. Regulation of Iron Homeostasis by Fur ..............................................17 1.4. FREE RADICAL STRESS RESPONSES AND SCAVENGERS................... 19 1.4.1. The OxyR Response ...........................................................................19 1.4.1.1. H2O2 scavengers: catalases and peroxidases........................21 1.4.2. The SoxRS Response..........................................................................23 − 1.4.2.1. The O2 scavengers: SodA, SodB and SodC .......................24 1.5. MECHANISMS OF CHROMOSOMAL FRAGMENTATION AND REPAIR ....................................................................................................................27 vi 1.5.1. Mechanisms of Replication-Dependent Chromosomal Fragmentation ...............................................................................................28 1.5.2. Mechanisms of DNA Repair...............................................................31 1.5.2.1. Recombinational repair pathway .........................................31 1.5.2.2. Base excision repair pathway...............................................32 1.5.2.3. Nucleotide excision repair pathway.....................................32 1.6. POTENTIATED HYDROGEN PEROXIDE TOXICITY ............................... 33 1.6.1. The Concept of Potentiated Toxicity ..................................................33 1.6.2. Mechanism of NO + H2O2 Co-Toxicity..............................................36 1.6.3. Mechanism of Cysteine-Enhanced H2O2 Toxicity .............................37 1.6.4. The Cyanide + H2O2 Co-Toxicity.......................................................38 1.6.4.1. The potential of cyanide + H2O2 co-toxicity as a cytotoxic weapon in activated immune cells...................................40 1.7. SCOPE OF THIS THESIS................................................................................ 41 1.7.1. Cyanide Potentiates H2O2 Toxicity by Causing Catastrophic Chromosomal Fragmentation