Limits...
The communication factor EDF and the toxin-antitoxin module mazEF determine the mode of action of antibiotics.

Kolodkin-Gal I, Sat B, Keshet A, Engelberg-Kulka H - PLoS Biol. (2008)

Bottom Line: We found that antibiotics of both groups caused: (i) mazEF-mediated cell death, and (ii) the production of ROS through MazF action.However, only antibiotics of the first group caused mazEF-mediated cell death that is ROS-dependent, whereas those of the second group caused mazEF-mediated cell death by an ROS-independent pathway.Furthermore, our results showed that the mode of action of antibiotics was determined by the ability of E. coli cells to communicate through the signaling molecule Extracellular Death Factor (EDF) participating in mazEF induction.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.

ABSTRACT
It was recently reported that the production of Reactive Oxygen Species (ROS) is a common mechanism of cell death induced by bactericidal antibiotics. Here we show that triggering the Escherichia coli chromosomal toxin-antitoxin system mazEF is an additional determinant in the mode of action of some antibiotics. We treated E. coli cultures by antibiotics belonging to one of two groups: (i) Inhibitors of transcription and/or translation, and (ii) DNA damaging. We found that antibiotics of both groups caused: (i) mazEF-mediated cell death, and (ii) the production of ROS through MazF action. However, only antibiotics of the first group caused mazEF-mediated cell death that is ROS-dependent, whereas those of the second group caused mazEF-mediated cell death by an ROS-independent pathway. Furthermore, our results showed that the mode of action of antibiotics was determined by the ability of E. coli cells to communicate through the signaling molecule Extracellular Death Factor (EDF) participating in mazEF induction.

Show MeSH

Related in: MedlinePlus

mazEF-Mediated Carbonylation of Cellular Protein Following Treatment by Various Antibiotics(A, C, and E) Protein carbonylation. E. coli strains MC4100relA+ (WT) and its derivative MC4100rel A+ΔmazEF (ΔmazEF) were grown in aerobic conditions. Logarithmic cells were pre-incubated as in Figure 1, and treated with antibiotics as follows: (A) rifampicin (20 μg/ml) for 10 min; (C) nalidixic acid (1 mg/ml) for 10 min; (E) trimethoprim (2 μg/ml) for 1h. Protein carbonylation was determined.(B, D, and F) Relative carbonyl levels of a representative experiment is described in (A, C, and E). The intensity of bands obtained in same time points as in panels (A, C, and E) from three independent experiments was quantified. The numbers express the relative carbonyl levels of each treated strain compared to untreated WT strain.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2602726&req=5

pbio-0060319-g002: mazEF-Mediated Carbonylation of Cellular Protein Following Treatment by Various Antibiotics(A, C, and E) Protein carbonylation. E. coli strains MC4100relA+ (WT) and its derivative MC4100rel A+ΔmazEF (ΔmazEF) were grown in aerobic conditions. Logarithmic cells were pre-incubated as in Figure 1, and treated with antibiotics as follows: (A) rifampicin (20 μg/ml) for 10 min; (C) nalidixic acid (1 mg/ml) for 10 min; (E) trimethoprim (2 μg/ml) for 1h. Protein carbonylation was determined.(B, D, and F) Relative carbonyl levels of a representative experiment is described in (A, C, and E). The intensity of bands obtained in same time points as in panels (A, C, and E) from three independent experiments was quantified. The numbers express the relative carbonyl levels of each treated strain compared to untreated WT strain.

Mentions: Having found that ROS-detoxifying enzymes prevented mazEF-mediated cell death (Figures 1A–1C), we expected that triggering the mazEF module would induce ROS formation that would be reflected in the cellular level of protein carbonylation. To detect oxidized proteins carrying carbonyl groups, we used the immunochemical assay from the Chemicon Oxyblot Kit. The triggering of mazEF was carried out in E. coli MC4100relA+ (wild type (WT)) with rifampicin (Figure 2A and 2B), or nalidixic acid (Figure 2C and 2D) for 10 min, or trimethoprim (Figure 2E and 2F) for 1 h. After removing the antibiotics, we prepared crude protein extracts from the treated cultures. Activating mazEF in WT cells by each of these three antibiotics led to an increase in the level of protein carbonylation within 60 min (Figure 2). Under the same conditions, we observed no such change in the very low basal level of carbonylation in the ΔmazEF derivative strain (Figure 2 and Figure S3). Furthermore, when we quantified the relative levels of protein carbonylation, we found that the DNA-damaging agents nalidixic acid and trimethoprim each induced significantly higher levels of mazEF-dependent carbonylation than did the transcription inhibitor rifampicin. For example, after the addition of trimethoprim, the relative carbonyl level increased by 27 times (Figure 2F), but after the addition rifampicin, it increased only by about 3.5 times (Figure 2B). Note also that, while in the case of rifampicin, mazEF-dependent carbonylation remained constant after 1 h (Figure 2A and 2B), in the case of DNA damage, a gradual increase in the level of carbonylation from 1 to 3 h was observed (Figure 2C–2F).


The communication factor EDF and the toxin-antitoxin module mazEF determine the mode of action of antibiotics.

Kolodkin-Gal I, Sat B, Keshet A, Engelberg-Kulka H - PLoS Biol. (2008)

mazEF-Mediated Carbonylation of Cellular Protein Following Treatment by Various Antibiotics(A, C, and E) Protein carbonylation. E. coli strains MC4100relA+ (WT) and its derivative MC4100rel A+ΔmazEF (ΔmazEF) were grown in aerobic conditions. Logarithmic cells were pre-incubated as in Figure 1, and treated with antibiotics as follows: (A) rifampicin (20 μg/ml) for 10 min; (C) nalidixic acid (1 mg/ml) for 10 min; (E) trimethoprim (2 μg/ml) for 1h. Protein carbonylation was determined.(B, D, and F) Relative carbonyl levels of a representative experiment is described in (A, C, and E). The intensity of bands obtained in same time points as in panels (A, C, and E) from three independent experiments was quantified. The numbers express the relative carbonyl levels of each treated strain compared to untreated WT strain.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2602726&req=5

pbio-0060319-g002: mazEF-Mediated Carbonylation of Cellular Protein Following Treatment by Various Antibiotics(A, C, and E) Protein carbonylation. E. coli strains MC4100relA+ (WT) and its derivative MC4100rel A+ΔmazEF (ΔmazEF) were grown in aerobic conditions. Logarithmic cells were pre-incubated as in Figure 1, and treated with antibiotics as follows: (A) rifampicin (20 μg/ml) for 10 min; (C) nalidixic acid (1 mg/ml) for 10 min; (E) trimethoprim (2 μg/ml) for 1h. Protein carbonylation was determined.(B, D, and F) Relative carbonyl levels of a representative experiment is described in (A, C, and E). The intensity of bands obtained in same time points as in panels (A, C, and E) from three independent experiments was quantified. The numbers express the relative carbonyl levels of each treated strain compared to untreated WT strain.
Mentions: Having found that ROS-detoxifying enzymes prevented mazEF-mediated cell death (Figures 1A–1C), we expected that triggering the mazEF module would induce ROS formation that would be reflected in the cellular level of protein carbonylation. To detect oxidized proteins carrying carbonyl groups, we used the immunochemical assay from the Chemicon Oxyblot Kit. The triggering of mazEF was carried out in E. coli MC4100relA+ (wild type (WT)) with rifampicin (Figure 2A and 2B), or nalidixic acid (Figure 2C and 2D) for 10 min, or trimethoprim (Figure 2E and 2F) for 1 h. After removing the antibiotics, we prepared crude protein extracts from the treated cultures. Activating mazEF in WT cells by each of these three antibiotics led to an increase in the level of protein carbonylation within 60 min (Figure 2). Under the same conditions, we observed no such change in the very low basal level of carbonylation in the ΔmazEF derivative strain (Figure 2 and Figure S3). Furthermore, when we quantified the relative levels of protein carbonylation, we found that the DNA-damaging agents nalidixic acid and trimethoprim each induced significantly higher levels of mazEF-dependent carbonylation than did the transcription inhibitor rifampicin. For example, after the addition of trimethoprim, the relative carbonyl level increased by 27 times (Figure 2F), but after the addition rifampicin, it increased only by about 3.5 times (Figure 2B). Note also that, while in the case of rifampicin, mazEF-dependent carbonylation remained constant after 1 h (Figure 2A and 2B), in the case of DNA damage, a gradual increase in the level of carbonylation from 1 to 3 h was observed (Figure 2C–2F).

Bottom Line: We found that antibiotics of both groups caused: (i) mazEF-mediated cell death, and (ii) the production of ROS through MazF action.However, only antibiotics of the first group caused mazEF-mediated cell death that is ROS-dependent, whereas those of the second group caused mazEF-mediated cell death by an ROS-independent pathway.Furthermore, our results showed that the mode of action of antibiotics was determined by the ability of E. coli cells to communicate through the signaling molecule Extracellular Death Factor (EDF) participating in mazEF induction.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.

ABSTRACT
It was recently reported that the production of Reactive Oxygen Species (ROS) is a common mechanism of cell death induced by bactericidal antibiotics. Here we show that triggering the Escherichia coli chromosomal toxin-antitoxin system mazEF is an additional determinant in the mode of action of some antibiotics. We treated E. coli cultures by antibiotics belonging to one of two groups: (i) Inhibitors of transcription and/or translation, and (ii) DNA damaging. We found that antibiotics of both groups caused: (i) mazEF-mediated cell death, and (ii) the production of ROS through MazF action. However, only antibiotics of the first group caused mazEF-mediated cell death that is ROS-dependent, whereas those of the second group caused mazEF-mediated cell death by an ROS-independent pathway. Furthermore, our results showed that the mode of action of antibiotics was determined by the ability of E. coli cells to communicate through the signaling molecule Extracellular Death Factor (EDF) participating in mazEF induction.

Show MeSH
Related in: MedlinePlus