The nature of the synergistic lethality of nitric oxide and hydrogen peroxide in Escherichia coli and its effect on chromosome fragmentation

Immune cells kill invading microbes by producing reactive oxygen and nitrogen species, primarily hydrogen peroxide (H2O2) and nitric oxide (NO). However, their combined bactericidal action raises several issues. The nature of the synergy is unclear as is the lethal DNA lesion. To resolve this matter, we treated E. coli with various concentrations of H2O2-alone, NO-alone, and H2O2+NO and measured survival and chromosome stability. We found that the nature of the synergy was via potentiation and that NO potentiated H2O2 toxicity. H2O2 reacts with Fe(II) via Fenton reaction generating DNA damaging hydroxyl radicals. Indeed, higher H2O2 doses alone as well as the lower H2O2 doses in the presence of NO introduced double-strand breaks in the DNA leading to catastrophic chromosomal fragmentation. Heme-containing catalases that degrade H2O2 represent obvious targets of NO inhibition and we found that NO increased the half-life of H2O2 in cell cultures. Catalase-deficient mutants were killed equally by H2O2-alone or H2O2+NO treatments while also showing similar levels of chromosome fragmentation. The Fenton reaction, in addition to H2O2, requires Fe(II), which H2O2 instantly converts into Fenton-inert Fe(III). To make Fenton continuous a steady supply of reduced iron should also be present. Genetic analysis revealed that a potential contributor to this supply is Fe(II) released from FtnA by flavin reductase. Our observations also support the idea that NO binding of ubiquinol oxidases drives Fe(III) reduction by blocking respiration. We modelled the respiration block by employing a NADH dehydrogenase deficient mutant. We found that, like the catalase mutant, the dehydrogenase mutant was similarly sensitive to and fragmented its chromosome with H2O2-alone and H2O2+NO treatment. Moreover, the quadruple mutant lacking both catalases and dehydrogenases showed rapid killing by H2O2-alone, which NO immediately delayed instead of potentiated, indicating absence of further targets for NO potentiation. We conclude that NO bolsters both Fenton reactants, H2O2 and Fe(II) making hydroxyl-radical production and ensuing oxidative damage to the chromosome continuous. Finally, we investigated the mechanism behind the generation of the double-strand breaks (DSBs) using nicks as the starting point. We found that majority of the DSBs are replication independent. The breaks arose all over the chromosome including the unreplicated portion of the chromosome making them irreparable. We observed a DSB density higher than that expected simply from co-incidence of random nicks. The chromosomal fragmentation inflicted by H2O2+NO could be explained by nick clustering, indicative of persistent iron-DNA complexes.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2022

Author dc:creator

• Agashe, Pooja Sanjiv

Contributors dc:contributor

• Kuzminov, Andrei
• Imlay, James A
• Metcalf, William W
• Kehl-Fie, Thomas E

• oxidative damage
• oxidative stress
• catalase
• hydrogen peroxide
• nitric oxide
• iron metabolism
• ferritin
• respiration
• double-strand DNA breaks
• DNA damage
• chromosome fragmentation

Statement dc:rights

• Copyright 2022 Pooja Agashe

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/115662

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F115662