The Aminoglycoside Antibiotic Kanamycin Damages DNA Bases in Escherichia coli: Caffeine Potentiates the DNA-Damaging Effects of Kanamycin while Suppressing Cell Killing by Ciprofloxacin in Escherichia coli and Bacillus anthracis

Kang, Tina Manzhu; Yuan, Jessica; Nguyen, Angelyn; Becket, Elinne; Yang, Hanjing; Miller, Jeffrey H

doi:10.1128/aac.00066-12

Cited by 42 publications

(53 citation statements)

References 71 publications

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“…a significant role in the lethality of bactericidal antibiotics. These results, along with recent observations that kanamycin can damage DNA bases (26) and induce SOS response in strains with mutated 8-oxo-dG-processing genes (mutM, mutY, and mutT) (96), highlight a DNA-damaging component to aminoglycoside lethality, as is consistent with previous MutT results (25). More broadly, these findings suggest that suppressive alterations to proton motor force cannot wholly explain anaerobic protection against aminoglycoside lethality (49)(50)(51).…”

Section: Fig 5 Ros Contribute To the Lethality Elicited By Bactericsupporting

confidence: 78%

Antibiotics induce redox-related physiological alterations as part of their lethality

Dwyer

Belenky

Yang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

737

683

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Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species. This notion has been supported by many groups but has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H 2 O 2 sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress. Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H 2 O 2 . We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality.reactive oxygen species | DNA repair | mutagenesis T he increasing incidence of antibiotic-resistant infections coupled with a declining antibiotic pipeline has created a global public health threat (1-6). Therefore there is a pressing need to expand our conceptual understanding of how antibiotics act and to use insights gained from such efforts to enhance our antibiotic arsenal. It has been proposed that different classes of bactericidal antibiotics, regardless of their drug-target interactions, generate varying levels of deleterious reactive oxygen species (ROS) that contribute to cell killing (7,8). This unanticipated notion, built upon important prior work (9-11), has been extended and supported by multiple laboratories investigating wide-ranging drug classes (e.g., β-lactams, aminoglycosides, and fluoroquinolones) and bacterial species (e.g., Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Mycobacterium tuberculosis, Bacillus subtilis, Staphylococcus aureus, Acinetobacter baumannii, Burkholderia cepecia, Streptococcus pneumonia, Enterococcus faecalis) using independent lines of evidence (12-39). Importantly,...

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Section: Fig 5 Ros Contribute To the Lethality Elicited By Bactericsupporting

confidence: 78%

Antibiotics induce redox-related physiological alterations as part of their lethality

Dwyer

Belenky

Yang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

737

683

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“…Caffeine has previously been shown to exhibit antibacterial activity against E. coli through interfering with DNA synthesis (108,109). It prevented the removal of thymidine dimers induced by UV radiation, contributing to increased mutational frequency (110).…”

Section: Figmentioning

confidence: 99%

Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays

et al. 2017

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The function and extracellular location of cell envelope proteins make them attractive candidates for developing vaccines against bacterial diseases, including challenging drug-resistant pathogens, such as Neisseria gonorrhoeae. A proteomicsdriven reverse vaccinology approach has delivered multiple gonorrhea vaccine candidates; however, the biological functions of many of them remain to be elucidated. Herein, the functions of six gonorrhea vaccine candidates-NGO2121, NGO1985, NGO2054, NGO2111, NGO1205, and NGO1344 -in cell envelope homeostasis were probed using phenotype microarrays under 1,056 conditions and a ΔbamE mutant (Δngo1780) as a reference of perturbed outer membrane integrity. Optimal growth conditions for an N. gonorrhoeae phenotype microarray assay in defined liquid medium were developed, which can be useful in other applications, including rapid and thorough antimicrobial susceptibility assessment. Our studies revealed 91 conditions having uniquely positive or negative effects on one of the examined mutants. A cluster analysis of 37 and 57 commonly beneficial and detrimental compounds, respectively, revealed three separate phenotype groups: NGO2121 and NGO1985; NGO1344 and BamE; and the trio of NGO1205, NGO2111, and NGO2054, with the last protein forming an independent branch of this cluster. Similar phenotypes were associated with loss of these vaccine candidates in the highly antibiotic-resistant WHO X strain. Based on their extensive sensitivity phenomes, NGO1985 and NGO2121 appear to be the most promising vaccine candidates. This study establishes the principle that phenotype microarrays can be successfully applied to a fastidious bacterial organism, such as N. gonorrhoeae. IMPORTANCE Innovative approaches are required to develop vaccines against prevalent and neglected sexually transmitted infections, such as gonorrhea. Herein, we have utilized phenotype microarrays in the first such investigation into Neisseria gonorrhoeae to probe the function of proteome-derived vaccine candidates in cell envelope homeostasis. Information gained from this screening can feed the vaccine candidate decision tree by providing insights into the roles these proteins play in membrane permeability, integrity, and overall N. gonorrhoeae physiology. The optimized screening protocol can be applied in investigations into the function of other hypothetical proteins of N. gonorrhoeae discovered in the expanding number of whole-genome sequences, in addition to revealing phenotypic differences between clinical and laboratory strains.KEYWORDS Neisseria gonorrhoeae, vaccine antigens, cell envelope, phenotypic microarrays, antibiotics, Biolog I n Gram-negative bacteria, the cell envelope is composed of the outer membrane, the cell wall, and the cytoplasmic or inner membrane, each of which can be further broken down into its constituents (1). The outer membrane is a bilayer composed of a lipopolysaccharide (LPS) or lipooligosaccharide (LOS) outer leaflet facing the extracel-

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“…reported that E. coli cells deficient for alkB , a DNA repair gene, are much more sensitive to the aminoglycoside kanamycin compared to wild type cells [42], suggesting a potential role for DNA damage in cell killing by aminoglycosides. Since alkB is a part of the adaptive response to alkylation [43, 44], prior exposure to very low-levels of alkylating agents induces the expression of alkB as well as other member genes of the adaptive response regulon [45].…”

Section: Resultsmentioning

confidence: 99%

“…Exocyclic DNA adducts such as ethenoadenine are known to be induced by products of lipid peroxidation [46], leading Kang et al . to speculate that aminoglycosides promote membrane lipid peroxidation resulting in the formation of exocyclic adducts targeted by AlkB [42]. However, it should be noted that the range of target lesions repaired by AlkB includes the potent replication-blocking methylated bases 1-methyladenine and 3-methylcytosine, which are formed when endogenous (or exogenous) alkylating agents attack single-stranded DNA [47–49].…”

Section: Resultsmentioning

confidence: 99%

Potential roles for DNA replication and repair functions in cell killing by streptomycin

Humayun

Ayyappan

2013

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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The aminoglycoside streptomycin binds to ribosomes to promote mistranslation and eventual inhibition of translation. Streptomycin kills bacteria, whereas many other non-aminoglycoside inhibitors of translation do not. Because mistranslation is now known to affect DNA replication, we asked if hydroxyurea, a specific inhibitor of DNA synthesis, affects killing, and find that hydroxyurea significantly attenuates killing by streptomycin. We find that the hydroxyl radical scavengers D-mannitol and thiourea have either no effect or only a modest protective effect. The iron chelator 2,2′-dipyridyl eliminated killing by streptomycin, but further investigation revealed that it blocks streptomycin uptake. Prior treatment of cells with low-levels of methyl methanesulfonate to induce the adaptive response to alkylation leads to a significant attenuation of killing, which, together with the hydroxyurea effect, suggests roles for DNA replication and repair functions in cell killing by streptomycin.

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The Aminoglycoside Antibiotic Kanamycin Damages DNA Bases in Escherichia coli: Caffeine Potentiates the DNA-Damaging Effects of Kanamycin while Suppressing Cell Killing by Ciprofloxacin in Escherichia coli and Bacillus anthracis

Cited by 42 publications

References 71 publications

Antibiotics induce redox-related physiological alterations as part of their lethality

Antibiotics induce redox-related physiological alterations as part of their lethality

Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays

Potential roles for DNA replication and repair functions in cell killing by streptomycin

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