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Title:The iron-dependent cyanide and hydrogen peroxide co-toxicity in Escherichia coli and its catastrophic consequences for the chromosome
Author(s):Mahaseth, Tulip
Director of Research:Kuzminov, Andrei
Doctoral Committee Chair(s):Kuzminov, Andrei
Doctoral Committee Member(s):Cronan, John E.; Gardner, Jeffrey F.; Vanderpool, Carin K.
Department / Program:Microbiology
Discipline:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):cyanide
potentiated hydrogen peroxide toxicity
catastrophic chromosomal fragmentation
iron metabolism
Fenton's reaction
immune system's anti-microbial mechanisms
nucleoid dynamics
Abstract:Hydrogen peroxide (H2O2) can oxidize cytoplasmic ferrous ions (Fe2+) 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 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.
Issue Date:2015-06-22
Type:Thesis
URI:http://hdl.handle.net/2142/88252
Rights Information:Copyright 2015 Tulip Mahaseth
Date Available in IDEALS:2015-09-29
Date Deposited:August 201


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