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

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.

Figure 1. Catalase or Superoxide Dismutase Prevented E. coli mazEF-Mediated Cell Death Induced by Inhibitors of Transcription and/or Translation

E. coli MC4100relA ⁺ (WT), MC4100relA ⁺/pQEkatE (WT+KatE), MC4100relA ⁺/pQEsodA (WT+SodA) or MC4100relA ⁺ΔmazEF (ΔmazEF) were grown aerobically to mid-log phase. Cells were incubated without shaking at 37 °C for 10 min, followed by the addition of: (A) rifampicin (20 μg/ml) for 10 min; (B) spectinomycin (1 mg/ml) for 1 h; (C) chloramphenicol (50 μg/ml) for 20 min; (D) trimethoprim (2 μg/ml) for 1h; and (E) nalidixic acid (1 mg/ml) for 10 min. The wild type strain (WT) is represented by black bars and the derivative ΔmazEF strain is represented by white bars. Error bars indicate standard deviation.

Figure 2. 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.

Figure 3. MazF Overproduction Induces Cellular Protein Carbonylation That is Prevented by Catalase

E. coli strains MC4100relA ⁺/pBAD-mazF (MazF), MC4100relA ⁺/pQEkatE/pBAD-mazF (MazF+KatE), and MC4100relA ⁺/pQEsodA/ pBAD-mazF (MazF+SodA) were grown in M9 minimal medium and MazF was induced. (A) Protein carbonylation; (B) relative carbonyl levels described in (A). The intensity of each band presented in (A) quantified as described in the legend for Figure 2. (C) Percent (%) of survivors was determined 8 h following the induction of mazF expression.

Figure 4. Completely Anaerobic Growth Conditions Prevented mazEF-Mediated Cell Death When mazEF Was Activated by Inhibiting Transcription and/or Translation but Not When Activated by DNA Damage

E. coli MC4100relA ⁺ (WT) and MC4100relA ⁺ΔmazEF (ΔmazEF) were grown under either aerobic or anaerobic conditions. Cells were incubated either aerobically, without shaking, or in an anaerobic jar with: (A) rifampicin (10, 20, or 30 μg/ml) for 10 min or (B) nalidixic acid (2–3 mg/ml) for 10 min.

Figure 5. EDF and mazEF Are Important Determinants in the Mode of Action of Rifampicin in E. coli

Logarithmic changes in the number of colony forming units per milliliter (CFU/ml) following exposure to rifampicin (20 μg/ml) in E. coli strains: (A) MC4100relA ⁺; (B) MC4100relA ⁺ΔmazEF; (C) MG1655 + EDF (0.05 μg/ml); (D) MG1655; and (E) MG1655 + EDF (0.05 μg/ml) + iEDF (0.05 μg/ml).

Figure 6. EDF and mazEF Are Important Determinants in the Mode of Action of Nalidixic Acid in E. coli

Logarithmic changes in the number of colony forming units per milliliter (CFU/ml) following exposure to nalidixic acid (1 mg/ml) in E. coli strains: (A) MC4100relA ⁺; (B) MC4100relA ⁺ΔmazEF; (C) MG1655 + EDF (0.05 μg/ml); and (D) MG1655 .

Figure 7. Alternative Death Pathways Induced by Some Antibiotics in

E. coli (A) Antibiotic induction of EDF-mazEF activates MazF (in red). (a) Antibiotics that inhibit transcription and/or translation cause cell death that is ROS-dependent, probably by inhibition of bulk-protein synthesis, including that of ROS detoxifying enzymes. (b) In contrast, antibiotics causing DNA damage trigger an ROS-independent death pathway(s), probably by the selective synthesis of death proteins. (B) Antibiotic induction of EDF-mazEF-independent death pathways (in blue). (c) Antibiotics that inhibit transcription and/or translation do not cause ROS production. Therefore, they do not kill the cells but cause growth arrest, and thus are bacteriostatic. (d) In contrast, antibiotics that cause DNA damage do induce ROS production, and thereby lead to cell death.