Bacterial strains, compound preparation, and cell culture conditions
Escherichia coli (ATCC 25922) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and ΔLexA, ΔRecA, and ΔdinF mutants were obtained from the E. coli ASKA collection (library of E. coli ORF clones). H2O2 and norfloxacin was purchased from Sigma (St. Louis, MO, USA) and dissolved in H2O and acetic acid, respectively. Sodium pyruvate (10 mM, Sigma), a scavenger of H2O2, was dissolved in H2O and thiourea (150 mM, Sigma), a scavenger of OH∙, was also dissolved in H2O according to previous studies (Choi and Lee 2012, Franco, et al. 2007). For all experiments, bacterial cells were grown in Luria-Bertani (LB, BD Pharmingen, San Diego, CA, USA) agar plates at 37 ℃ and inoculated in LB broth under aerobic conditions at 37 ℃ and 120 rpm. Bacterial strains in the exponential phase were harvested and then resuspended in phosphate-buffered saline (PBS, pH 7.4).
Cell viability measurement
E. coli cells (1 × 106 cells/mL) were incubated with H₂O₂ at four concentrations or with 1.25 μg/mL norfloxacin, including the minimum inhibitory concentration (MIC). H₂O₂ and norfloxacin concentrations were selected based on previously published reports (Kim and Lee 2020, Orrù, et al. 2010). The treatment concentrations of H₂O₂ applied were 25 μg/mL (1/4 MIC), 50 μg/mL (1/2 MIC), 100 μg/mL (MIC), and 200 μg/mL (2 MIC). After 2 h incubation, cultures were spread onto LB agar plates. For colony forming unit (CFU)/mL calculations, 100 μL of culture was centrifuged at 12,000 rpm for 5 min, washed twice with PBS, and serially diluted in PBS. Each dilution was plated onto LB agar and the plates were incubated at 37 ℃ for 24 h. Only those dilutions that yielded between 20 - 200 colonies were counted. CFU/mL is expressed as a percentage of survival using the following formula: [(CFU of sample treated with test compound)/(CFU of untreated control) × 100].
Detection of intracellular hydroxyl radical
3′-(p-hydroxyphenyl) fluorescein (HPF, Molecular Probes, Invitrogen, Carlsbad, CA, USA) was applied to detect hydroxyl radicals (OH∙), which are decomposition product of H2O2. HPF was dissolved in N,N-dimethylformamide (DMF, JUNSEI Chemical Co., Tokyo, Japan). It is nonfluorescent until it reacts with OH∙, becoming fluorescent upon exposure to OH∙. Bacterial cells (1 × 106 cells/mL) were treated with H2O2, H2O2 pretreated with thiourea, or norfloxacin, and incubated for 2 h at 37 °C. Following incubation, cells were centrifuged at 12,000 rpm for 5 min and the supernatant was removed. Cell pallets were then suspended with 1 mL PBS and dyed with 5 μM HPF. After HPF staining, the fluorescence intensity of each sample was assessed by utilizing a FACSVerse flow cytometer (Becton Dickinson, NJ, USA).
Assessment of oxidative DNA damage
Under oxidative stress, the guanine base of DNA is readily damaged, forming 8-hydroxydeoxyguanosine (8-OHdG). Therefore, the level of 8-OHdG is used as a biomarker to determine the degree of DNA oxidative damage. Bacterial cells were incubated for 2 h at 37 ℃ with H2O2, H2O2 pretreated with 10 mM sodium pyruvate, H2O2 pretreated with thiourea, or norfloxacin. Following incubation, DNA was extracted in an extraction solution (containing 100 mM NaCl, 10 mM Tris-Cl , 1 mM EDTA, 1 % SDS, 2 % Triton X-100, and 10 mg/mL proteinase K) as described in our previous study (Lee and Lee 2017). The 8-OHdG levels were estimated by competitive enzyme immunoassay method, using an Oxiselect Oxidative DNA Damage ELISA Kit (Cell Biolabs Inc., San Diego, CA, USA), according to the manufacturer’s instructions. The absorbance of 8-OHdG was measured using an ELISA microplate reader (BioTek Instruments, Winooski, VT, USA) at 450 nm.
Evaluation of lipid peroxidation
The thiobarbituric acid-reactive substances (TBARS) assay was employed to detect malondialdehyde (MDA), which is the final product of lipid peroxidation. MDA levels were evaluated from standard curves (created based on the suggestions of the manufacturer, Sigma). Bacterial cells were treated with H2O2, H2O2 pretreated with sodium pyruvate, H2O2 pretreated with thiourea, or norfloxacin. Following 2 h incubation at 37 °C, cells were centrifuged at 12,000 rpm for 5 min, and cell pellets were then mixed with lysis buffer (pH 8.0, 10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, 2 % Triton-X 100, and 1 % SDS). Next, cells were sonicated on ice using an ultrasonic sonicator (10 pulses of 1 min each at an amplitude of 38) (Sonics, Newtown, CT, USA) followed by centrifugation. The supernatant was mixed with thiobarbituric acid in 5 % trichloroacetic acid. The mixture was heated at 95 ℃ for 30 min, and then cooled on ice. Absorbance of the reaction mixture was measured at 532 nm.
Determination of cell division arrest and cell filamentation
4′,6-diamidino-2-phenylindole (DAPI, Sigma) was used to ascertain bacterial cell division arrest. E. coli wild-type and ΔLexA cells were first treated with H2O2, H2O2 pretreated with sodium pyruvate, H2O2 pretreated with thiourea, or norfloxacin and incubated for 2 h at 37 °C. For chromosomal staining, cells were washed with PBS and then incubated with 1 μg/mL DAPI in a darkroom for 20 min. The intensity of DAPI fluorescence was analyzed with a spectrofluorophotometer (Shimadzu RF-5301PC; Shimadzu, Japan), using excitation and emission wavelengths of 340 nm and 488 nm. To identify whether cell filamentation occurred, bacterial cells (wild-type and ΔLexA) were incubated for 2 h at 37 °C with H2O2, H2O2 pretreated with sodium pyruvate, H2O2 pretreated with thiourea, or norfloxacin. Next, cells were harvested by centrifugation and suspended in PBS. Cell filamentation was observed using an Eclipse Ti-s microscope (Nikon, Tokyo, Japan).
Estimation of caspase-like protein activation
To investigate prokaryotic caspase homologs, the CaspACETM FITC-VAD-FMK In Situ Marker (Promega, Fitchburg, WI, USA) was applied. The marker is a fluorescent analog of the pan-caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) and intends for monitoring caspase activation. E. coli wild-type and ΔRecA cells were treated for 2 h at 37 °C with H2O2, H2O2 pretreated with sodium pyruvate, H2O2 pretreated with thiourea, or norfloxacin. Following incubation, cells were washed twice with PBS and stained with 5 μM FITC-VAD-FMK for 30 min. The total volume was adjusted to 1 mL with PBS, and fluorescence intensity of caspase-like protein activity was analyzed utilizing a FACSVerse flow cytometer.
Measurement of intracellular reactive oxygen species
Intracellular ROS levels were evaluated using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, Molecular Probes). This probe passively diffuses through membranes, remaining in the cell after cleavage by esterases. When oxidation occurs by ROS, nonfluorescent H2DCFDA changes to fluorescent 2′,7′-dichlorofluorescein (DCF). E. coli wild-type and ΔdinF cells were incubated for 2 h at 37 °C with H2O2, H2O2 pretreated with sodium pyruvate, H2O2 pretreated with thiourea, or norfloxacin. After incubation, the cells were centrifuged at 12,000 rpm for 5 min and the supernatant was eliminated. Then, cells were resuspended in PBS and 10 μM H2DCFDA (dissolved in DMSO) was added to the suspension. Following incubation for 1 h at 37 ℃, cells were washed with PBS, and fluorescence intensity was assessed by utilizing a FACSVerse flow cytometer.
Analysis of DNA fragmentation
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was employed to assess DNA cleavage. This assay was performed using an In Situ Cell Death Detection Kit, Fluorescein (Roche Applied Science, Basel, Switzerland). To identify fragmented DNA, the 3′-OH termini of nucleotides were enzymatically labeled and attached to DNA strand breaksby terminal deoxynucleotidyl transferase. E. coli cells were incubated for 2 h at 37 °C with H2O2, H2O2 pretreated with sodium pyruvate, H2O2 pretreated with thiourea, or norfloxacin. Following incubation, cells were washed with PBS and then fixed with 2 % paraformaldehyde for 1 h on ice. Thereafter, the fixed cells were incubated with permeabilization solution (0.1 % Triton X-100 and 0.1 % sodium citrate) on ice for 2 min followed by incubation with TUNEL reaction mixture for 1 h at 37 °C. Fluorescence was assessed by utilizing a FACSVerse flow cytometer.
Detection of membrane depolarization and PS exposure
To assess membrane depolarization, bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC₄(3), Molecular Probes) was employed. E. coli cells were treated for 2 h at 37 °C with H2O2, H2O2 pretreated with sodium pyruvate, H2O2 pretreated with thiourea, or norfloxacin. Following incubation, cells were suspended in PBS and stained with 5 μg/mL DiBAC₄(3). The fluorescence intensity was analyzed using a FACSVerse flow cytometer. To detect PS exposure, an Annexin V–FITC Apoptosis Detection kit (BD Pharmingen) was used. Cells were incubated for 2 h at 37 °C with H2O2, H2O2 pretreated with sodium pyruvate, H2O2 pretreated with thiourea, or norfloxacin. After incubation, cells were harvested and resuspended in 100 μL of 1× Annexin V binding buffer, followed by the addition of 50 μL/ml Annexin V–FITC. The mixtures were incubated for 15 min in the dark and fluorescence was measured by utilizing a FACSVerse flow cytometer.
Statistical analysis
All experiments were performed in triplicate and values were expressed as mean ± standard deviation. After confirming normality of distributions using the Shapiro-Wilk test, statistical comparisons between various groups were carried out by analysis of variance followed by Tukey's post-hoc test for three-group comparisons using SPSS software (version 25, SPSS/IBM. Chicago, IL, USA). Intergroup differences were considered statistically significant at p < 0.05.