Oxidation of the Guanine Nucleotide Pool Underlies Cell Death by Bactericidal Antibiotics

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Science (Impact Factor: 31.48). 04/2012; 336(6079):315-9. DOI: 10.1126/science.1219192
Source: PubMed

ABSTRACT A detailed understanding of the mechanisms that underlie antibiotic killing is important for the derivation of new classes
of antibiotics and clinically useful adjuvants for current antimicrobial therapies. Our efforts to understand why DinB (DNA
polymerase IV) overproduction is cytotoxic to Escherichia coli led to the unexpected insight that oxidation of guanine to 8-oxo-guanine in the nucleotide pool underlies much of the cell
death caused by both DinB overproduction and bactericidal antibiotics. We propose a model in which the cytotoxicity of beta-lactams
and quinolones predominantly results from lethal double-strand DNA breaks caused by incomplete repair of closely spaced 8-oxo-deoxyguanosine
lesions, whereas the cytotoxicity of aminoglycosides might additionally result from mistranslation due to the incorporation
of 8-oxo-guanine into newly synthesized RNAs.

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    • "Norfloxacin is a synthetic antibacterial agent and a broad-spectrum antibiotic that is active against both gram-positive and gram-negative bacteria . Norfloxacin induces accumulation of ROS, highly destructive molecules that appear to oxidize DNA and lethal double-strand DNA breaks caused by incomplete repair can contribute to cell death in bacteria (Kohanski et al. 2007; Foti et al. 2012). The assay results showed that nano-Ag has antibacterial activity. "
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    ABSTRACT: Silver nanoparticles are known to have antimicrobial properties and have been used extensively in medicine, although the mechanism(s) of action have not yet been clearly established. In the present study, the findings suggest a novel mechanism for the antibacterial effect of silver nanoparticles on Escherichia coli, namely, the induction of a bacterial apoptosis-like response. We propose a possible mechanism for the bacterial apoptosis-like response that includes the following: accumulation of reactive oxygen species (ROS) (detected with H2DCFDA staining), increased intracellular calcium levels (detected with Fura-2 AM), phosphatidylserine exposure in the outer membrane (detected with Annexin V) which is the hallmarks of early apoptosis, disruption of the membrane potential [detected with DiBAC4(3)], activation of a bacterial caspase-like protein (detected by FITC-VAD-FMK staining) and DNA degradation (detected with TUNEL assay) which is the hallmarks of late apoptosis in bacterial cells treated with silver nanoparticles. We also performed RecA expression assay with western blotting and observed activation of SOS response to repair the damaged DNA. To summarize, silver nanoparticles are involved in the apoptosis-like response in E. coli and the novel mechanisms which were identified in this study, suggest that silver nanoparticles may be an effective antimicrobial agent with far lower propensity for inducing microbial resistance than antibiotics.
    BioMetals 08/2014; 27(6). DOI:10.1007/s10534-014-9782-z · 2.69 Impact Factor
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    • "Moreover, a recent work identified a significant decrease in mutation rates in MMR deficient, laboratory evolved E. coli lines associated with enhanced ROS detoxification (Turrientes et al. 2013). The weak effect of deactivated error-prone DNA repair in ÁdinBÁfur and ÁdinBÁumuDCÁfur mutants indicates that the majority of resistance mutations is formed by direct oxidation of DNA and not by incorporation of oxidized nucleotides through error-prone DNA polymerases (Foti et al. 2012). Indeed, we failed to find significant differences in the spectra of accumulated adaptive mutations between WT and Áfur populations (supplementary fig. "
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    ABSTRACT: Evolution of antibiotic resistance in microbes is frequently achieved by acquisition of spontaneous mutations during antimicrobial therapy. Here we demonstrate that inactivation of a central transcriptional regulator of iron homeostasis (Fur) facilitates laboratory evolution of ciprofloxacin resistance in Escherichia coli. To decipher the underlying molecular mechanisms, we first performed a global transcriptome analysis and demonstrated that the set of genes regulated by Fur change substantially in response to antibiotic treatment. We hypothesized that the impact of Fur on evolvability under antibiotic pressure is due to the elevated intracellular concentration of free iron and the consequent enhancement of oxidative damage-induced mutagenesis. In agreement with expectations, over-expression of iron storage proteins, inhibition of iron transport, or anaerobic conditions drastically suppressed the evolution of resistance, while inhibition of the SOS response-mediated mutagenesis had only minor effect in the fur deficient populations. Last, we provide evidence that a cell permeable iron chelator inhibits the evolution of resistance. In sum, our work revealed the central role of iron metabolism in the de novo evolution of antibiotic resistance, a pattern that could influence the development of novel antimicrobial strategies.
    Molecular Biology and Evolution 07/2014; 31(10). DOI:10.1093/molbev/msu223 · 14.31 Impact Factor
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    • "The fact that thiourea reduces mutation frequency is supportive of a hydroxyl radical–based mechanism of DNA damage as a result of the antibiotic treatment, as proposed for E. coli (Kohanski et al. 2007, 2010). In addition, the ability of thiourea to decrease the mutation rate in untreated cultures suggests that a significant amount of the observed background mutation frequency also resulted from hydroxyl radical damage of DNA (Kohanski et al. 2007; Foti et al. 2012). "
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    ABSTRACT: Approximately 80% of adult Cystic Fibrosis (CF) patients become chronically infected with Pseudomonas aeruginosa and consequently require antibiotic therapy at intervals throughout their lives. Achieving lethal concentrations of antibiotics in the lung remains a challenge. Recent evidence from Escherichia coli and Staphylococcus aureus suggests that the generation of hydroxyl radicals by sub-lethal concentrations of antibiotics may induce mutagenesis and confer bacteria with resistance to a wide range of antimicrobials. As P. aeruginosa can persist for many years following colonization of the airways and during this time it is repeatedly exposed to bactericidal antibiotics, we tested whether its exposure to sub-lethal levels increases mutation frequency. We demonstrate that sub-lethal levels of three classes of bactericidal antibiotics commonly used against P. aeruginosa infections, β-lactams, aminoglycosides and quinolones lead to an increase in mutation frequency, varying between ~3-fold increase with aminoglycosides to a ~14-fold increase in mutation frequency with β-lactam antibiotics. These findings could be clinically significant since exposure to sub-lethal concentrations of antibiotics during chronic infection leading to increased mutation frequency may facilitate adaptive radiation of pathogenic bacteria in the heterogeneous environment of the CF lung. © 2012 The Authors Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.
    Letters in Applied Microbiology 12/2012; 56(2). DOI:10.1111/lam.12032 · 1.75 Impact Factor


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