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: 33.61). 04/2012; 336(6079):315-9. DOI: 10.1126/science.1219192
Source: PubMed


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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    • "Recent reports have shown that bacteria treated with lethal doses of aminoglycoside antibiotics undergo a common oxidative damage, which contributes in part to the cell death (Dwyer et al. 2007). These antibiotics promote the overproduction of hydroxyl radicals through changes in cellular respiration involving NADH consumption and increased tricarboxylic acid cycle, ultimately leading to oxidative-stress mediated cell death (Dwyer et al. 2007; Kohanski et al. 2007, 2008, 2010; Foti et al. 2012; Grant et al. 2012; Liu et al. 2012; Wang and Zhao 2009). In mammalian cells, bactericidal antibiotics induce mitochondrial dysfunction by disrupting the electron transport chain, which triggers dose-and time-dependent overproduction of reactive oxygen species (ROS), thereby leading to oxidative stress damages (Kalghatgi et al. 2013). "
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    ABSTRACT: The aminoglycoside antibiotic hygromycin B (Hyg) inhibits prokaryotic, chloroplast and mitochondrial protein synthesis. Because of the toxic effect of Hyg on plant cells, the HPT gene, encoding hygromycin phosphotransferase, has become one of the most widely used selectable markers in plant transformation. Yet the mechanism behind Hyg-induced cell lethality in plants is not clearly understood. In this study, we aimed to decipher this mechanism. With Hyg treatment, rice calli exhibited cell death, and rice seedlings showed severe growth defects, leaf chlorosis and leaf shrinkage. Rice seedlings also exhibited severe lipid peroxidation and protein carbonylation, for oxidative stress damage at the cellular level. The production of reactive oxygen species such as O 2 (·-) , H2O2 and OH(·) was greatly induced in rice seedlings under Hyg stress, and pre-treatment with ascorbate increased resistance to Hyg-induced toxicity indicating the existence of oxidative stress. Overexpression of mitochondrial Alternative oxidase1a gene without HPT selection marker in rice enhanced tolerance to Hyg and attenuated the degradation of protein content, whereas the rice plastidial glutathione reductase 3 mutant showed increased sensitivity to Hyg. These results demonstrate that Hyg-induced cell lethality in rice is not only due to the inhibition of protein synthesis but also mediated by oxidative stress.
    Plant Molecular Biology 09/2015; DOI:10.1007/s11103-015-0380-4 · 4.26 Impact Factor
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    • "Bactericidal drugs kill bacteria partly by induction of ROS formation (Kohanski et al., 2007; Foti et al., 2012). The detection of ROS in treated B. cereus is a common signal in cellular death pathways (Kohanski et al., 2007). "
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    ABSTRACT: Plant polyphenols are known to have varying antimicrobial potencies, including direct antibacterial activity, synergism with antibiotics and suppression of bacterial virulence. We performed the in vitro oligomerization of resveratrol catalyzed by soybean peroxidase, and the two isomers (resveratrol-trans-dihydrodimer and pallidol) produced were tested for antimicrobial activity. The resveratrol-trans-dihydrodimer displayed antimicrobial activity against the Gram-positive bacteria Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus (minimum inhibitory concentration (MIC) = 15.0, 125, and 62.0 µM, respectively) and against Gram-negative Escherichia coli (MIC = 123 µM, upon addition of the efflux pump inhibitor Phe-Arg-β-naphthylamide). In contrast, pallidol had no observable antimicrobial activity against all tested strains. Transcriptomic analysis implied downregulation of ABC transporters, genes involved in cell division and DNA binding proteins. Flow cytometric analysis of treated cells revealed a rapid collapse in membrane potential and a substantial decrease in total DNA content. The active dimer showed >90% inhibition of DNA gyrase activity, in vitro, by blocking the ATP binding site of the enzyme. We thus propose that the resveratrol-trans-dihydrodimer acts to: (1) disrupt membrane potential; and (2) inhibit DNA synthesis. In summary, we introduce the mechanisms of action and the initial evaluation of an active bactericide and a platform for the development of polyphenolic antimicrobials. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Biotechnology and Bioengineering 06/2015; DOI:10.1002/bit.25686 · 4.13 Impact Factor
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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.
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