“On demand” redox buffering by H2S contributes to antibiotic resistance revealed by a bacteria-specific H2S donor

Enhancement of hydrogen sulfide in bacteria reveals a key role for this gas in mediating antibiotic resistance.


Measurement of H 2 S using BODIPY-based dye 4a:
A stock solution of 1a-1g (2.5 mM) was prepared in DMSO and 10 mM NADH was prepared in HEPES buffer pH 7.4. A stock solution of commercially available E. coli nitroreductase (NTR) was prepared using 1 mg of a lyophilized powder dissolved in HEPES buffer (2 mL).
A solution of 4a (1 mM) was prepared in DMSO and stored under dark conditions. The reaction mixture was prepared by adding 1a-1g (4 μL, 2.5 mM), NADH (10 μL, 10 mM) and NTR (4 μL stock as previously prepared) in HEPES buffer of pH 7.4 (162 μL). The resulting mixture was incubated at 37 °C for 5 min. 20 μL of 4a (1 mM) was added to the above mixture and incubated for 10 min at 37 °C under dark conditions and fluorescence (excitation 444 nm; emission 520 nm) from reaction mixture was measured using microtiter plate reader. In the control experiment 1c was co-treated with competitive substrate of nitroreductase 5 (5 μL, 20 mM). Data presented are an average of three independent experiments. The yields reported are determined by a dose-response curve generated with Na 2 S (see above).
Detection of H 2 S from 1c using mBBr assay: A 10 mM stock solution of mBBr was prepared in degassed CH 3 CN and stored at -20 °C under dark conditions. A 100 mM sodium sulfide solution was prepared in degassed water and diluted it further to 1 mM in degassed water. Reaction mixture was prepared by adding sodium sulfide (30 μL, 1 mM), reaction buffer (70 μL, 100 mM Tris-HCl buffer pH 9.5 with 0.1 mM DTPA) and S5 mBBr (50 μL, 0.4 mM) under ambient conditions. The resulting mixture was incubated at room temperature for 30 min under dark conditions and quenched with 50 μL (1M HCl) solution. In the experiments of 1c with NTR, the reaction mixture was prepared by adding 1c (4 μL, 2.5 mM), NADH (10 μL, 10 mM) NTR (4 μL as prepared previously) in reaction buffer (82 μL) pH 9.5. The resulting mixture was incubated for 10 min at 37 °C. To this incubated mixture, mBBr (50 μL, 0.4 mM) was added and incubated at rt for 30 min under dark conditions. The mixture was quenched with 50 μL (1 M HCl) solutions. The resulting mixture was filtered (0.22 μm) and injected (10 μL Measurement of H 2 S using NBD-fluorescein: Stock solutions of 1c and 5 (2.5 mM) were prepared in DMSO and 10 mM NADH was prepared in HEPES buffer pH 7.4. A stock solution of commercially available E. coli nitroreductase (NTR) was prepared using 1 mg of a lyophilized powder dissolved in HEPES buffer (2 mL). A solution of NBD-fluorescein (1 mM) was prepared in DMSO and stored under dark conditions. The reaction mixture was prepared by adding 1c or 5 (4 μL, 2.5 mM), NBD-fluorescein (2 μL, 1 mM), NADH (10 μL, 10 mM) and NTR (8 μL stock as previously prepared) in HEPES buffer of pH 7.4 (176 μL). The resulting mixture was incubated at 37 °C for 2 h. Fluorescence (excitation 490 nm; emission 514 nm) from reaction mixture was measured using microtiter plate reader. In the control experiment NTR + NADH were co-incubated with NBDfluorescein dye and the fluorescence value was subtracted from each data point (except for Na 2 S). Data presented are an average of three independent experiments. Figure S1. A) Structure of NBD-fluorescein. B) Hydrogen sulfide generated during incubation of 1c with NTR in HEPES buffer pH 7.4 was estimated using NBD-Fluorescein.

S6
Time course of H 2 S release from 1c: A stock solution of 1c, 4a, NADH, and E. coli nitroreductase (NTR) were prepared as mentioned above. A reaction mixture was prepared by adding 1c (4 μL, 2.5 mM), NADH (10 μL, 1 mM) and NTR (4 μL) in HEPES buffer (162 μL) pH 7.4. The resulting mixture was incubated at 37 °C. At different time interval (5, 15, 30 and 45 min) 20 μL (1 mM) 4a was added to the above mixture and incubated under dark conditions for 10 min at 37 °C. The fluorescence (excitation 444 nm; emission 520 nm) from reaction mixture was measured using microtiter plate reader. A calibration curve was used to quantify hydrogen sulfide produced during incubation of 1c with NTR. In the control experiment 1c (4 μL, 2.5 mM) was incubated with NADH (10 μL, 10 mM) in HEPES buffer (166 μL) pH 7.4 and at different time interval 4a (20μL, 1 mM) was added. The fluorescence from reaction mixture was measured. Data presented are an average of three independent experiments.
Chemoreduction of 1e with Zn and ammonium formate: Compound 1e (10 L, 10 mM) was dissolved in 980 L of a 1:1 solution of methanol and phosphate buffer (10 mM, pH 7.4). To this mixture, ammonium formate (10 L, 100 mM) and zinc dust (1 mg) were added and the reaction mixture was incubated at 37 ºC for 30 min. The reaction mixture was filtered (0.22 μm) and injected (50 μL) in a high-performance liquid chromatograph (HPLC) attached with a diode-array detector (the detection wavelength was 250 nm) and a Zorbax SB C-18 reversed-phase column (250 mm × 4.6 mm, 5 μm). A mobile phase of water/acetonitrile (25: 75) was used with a flow rate of 1 mL/min for 20 min. The authentic compound 2e was injected to identify the peak position of the reaction product. Two independent experiments were conducted, each carried out in duplicate and representative data is presented below. Figure S2. Representative HPLC traces of chemoreduction of 1e using Zn and ammonium formate in MeOH: pH 7.4 phosphate buffer (1 mL, 1:1 v/v) after 30 min: a) Authentic 1e (100 M) in acetonitrile; (Note: The peak at RT 12 min is likely the partially reduced compound, but we were unable to characterize it) b) 100 M reaction mixture after 30 min; c) Authentic acetophenone, 2e (100 M) in acetonitrile. The suspension was centrifuged to aspirate out any excess of the compound and/or azo-BODIPY in the medium. The bacterial pellet was washed with HEPES buffer pH 7.4 (1 mL × 3) and centrifuged. The collected bacterial pellet was re-suspended with acetonitrile and the cells were lysed by vortexing for 1 min. The cell lysate was then removed by centrifugation and the supernatant acetonitrile (50 L) was injected in an Agilent high performance liquid chromatography (HPLC) attached with a fluorescence detector (excitation at 444 nm; emission at 520 nm). The HPLC method used was as described previously.   Oxidative stress measurement using roGFP2 redox sensor To examine the effect of H 2 O 2 or antibioticsinduced oxidative stress exposure and protective role of H 2 S, we used variable concentrations of Ampicillin, Amikacin and Ciprofloxacin for specific time points. roGFP2 expressing E.coli cells were firstly grown aerobically till mid-exponential phase (OD 600 of 0.6) and diluted to OD 600 of 0.2 followed by pre-treatment with 1c for 20 min. Specific concentration of antibiotics were used and biosensor response (405/488 ratio at a fixed emission of 520 nm) was measured. Increase in 405/488 ratio indicated that antibiotic exposure leads towards the oxidative stress induction. Figure S6. Reduction-oxidation sensitive GFP (roGFP2) was used to measure dynamic changes in cytoplasmic redox potential of E. coli upon exposure to: H 2 O 2 , 1 mM; 1c, 100 M; Na 2 S, 100 M and 5, 100 M. Figure S7. Dynamic changes in cytoplasmic redox potential of E. coli upon exposure to Cip 5 μg/mL; 1c, 100 μM.
Time-kill assay. Cells were grown aerobically till mid-exponential phase (OD 600 of 0.6) and diluted to OD 600 of 0.2 in LB-broth and pre-exposed to 1c (100 µM) was given for 20 min. Antibiotics and H 2 O 2 treatments were given for certain time points with their selective concentrations. For survival analysis, serial dilutions of cultures were plated at each time points on LB-Agar plates and plates were maintained at 37°C. CFUs were counted carefully and the results were calculated as bacterial survival percentage against their respective untreated control.
qRT-PCR analysis: E. coli cells were grown till mid-exponential phase (OD 600 of 0.6) and diluted to OD 600 of 0.2, followed by pre-treatment with 1c for 20 min. Antibiotic treatment was given for 1h and total RNA was isolated using Trizol method. First-strand cDNA synthesis was performed using 200 ng of the total RNA with iScript Select cDNA Synthesis Kit (Bio-Rad) using random oligonucleotide primers. PCR was performed using gene specific primers (Table S2). Gene expression was analyzed with real-time PCR using iQTM SYBR GreenSupermix (Bio-Rad) and a CFX96 RT-PCR system (Bio-Rad). Data analysis was performed with the CFX ManagerTM software (Bio-Rad).