Bacterial strain and culture conditions

Deinococcus radiodurans R1 (ATCC 13939) was obtained from the American Type Culture Collection (ATCC) and were cultured at 30 °C in tryptone glucose yeast extract (TGY) broth comprising 0.5% tryptone (Difco Laboratories, Detroit, MI, USA), 0.3% yeast extract (Difco Laboratories), and 0.1% glucose (Sigma–Aldrich, St. Louis, MO, USA) or on TGY plates with 1.5% Bacto-agar (Difco Laboratories). Antibiotics (8 μg/mL kanamycin; Sigma–Aldrich) were added to the medium, if necessary.

Isolation and purification of R1-MVs

Deinococcus radiodurans strains were grown at 30 °C for 72 h under static conditions for isolation and purification of the R1-MVs. Briefly, after culturing in TGY broth for 72 h, the bacteria-free culture supernatants were harvested by centrifugation (10,000 × g, 30 min, 4 °C). The supernatant was filtered through a 0.45 μm bottle-top vacuum filter system (Corning, Merck KGaA, Darmstadt, Germany) using a Minimate™ tangential flow filtration (TFF) system with an Omega™ 300 K membrane capsule (Pall Scientific, NY, USA). The R1-EV pellets were harvested by ultracentrifugation (100,000 × g, 2 h, 4 °C), washed in sterile phosphate buffer saline (PBS; pH 7.4), and then purified by centrifugation using Optiprep density gradient medium (Sigma, #D1556, Steinheim, Germany). The R1-MVs were purified by ultracentrifugation (18 h, 170,000 × g, 4 °C; under no brake condition) in a discontinuous 60% Optiprep density gradient medium [step gradient ranging from 10 to 60% (w/v)]. The final MV pellet was resuspended in PBS and stored at − 80 °C. The protein content of R1-MVs was assessed using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific Pierce, Rockford, IL, USA) according to the manufacturer’s instructions.

Characterization of R1-MVs

The hydrodynamic size of the R1-MVs was analyzed by dynamic light scattering (DLS) using a Zetasizer Nano ZS Zen3600 (Malvern, UK). For transmission electron microscopy (TEM), the samples were dispersed in ethanol, mounted onto a carbon support film on a 150-mesh nickel grid, and dried. For field-emission transmission electron microscopy (FE-TEM), the analysis was performed using a field-emission transmission electron microscope (JEM-2100F; JEOL Ltd., Japan) at an acceleration voltage of 200 kV. The samples were fixed with 3.7% glutaraldehyde (Sigma–Aldrich GmbH, Taufkirchen, Germany) in PBS for 15 min and used for scanning electron microscopy using an ESEM Quanta 400 scanning electron microscope (FEI, Hillsboro, Oregon, USA). After washing twice with PBS, the fixed samples were dehydrated using an ascending sequence of ethanol (40%, 60%, 80%, and 96–98%). After evaporation of ethanol, the samples were left to dry at room temperature (RT) for 24 h on a glass substrate and then analyzed by SEM after gold–palladium sputtering.

Cell culture conditions

The immortalized human epidermal keratinocyte (HaCaT) cell line was obtained from Lonza and Korean Cell Line Bank (Seoul, Korea). The HaCaT cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Biowest, Nuaille, France) supplemented with 10% fetal bovine serum (FBS, Biowest), and 1% penicillin and streptomycin (P/S, GIBCO, Carlsbad, CA, USA) at 37 °C in a humidified chamber with 5% CO2.

2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay

Assays using DPPH (Sigma-Aldrich) were carried out to investigate the free radical scavenging activity of R1-MVs. Briefly, 100 μL of the DPPH solution was added to 100 μL of R1-MVs (15.6, 31.3, 62.5, and 125 μg/mL). The mixture was incubated for 30 min at RT in the dark. DPPH solution is decolorized from deep violet to light yellow, upon receiving a hydrogen atom from an antioxidant sample. Absorbance was measured at 520 nm using a microplate reader (Biotek, Winooski, VT, USA). Vitamin C (300 μM) was used as a positive control. All measurements were performed in triplicate. The percent scavenging activity (%SA) was calculated using the following equation:

$$\mathrm= [(}_} - }_})/}_}] \times 100$$

Ferric-reducing/antioxidant power (FRAP) assay

The FRAP assay was conducted as previously described [65]. The method is based on the reduction of a ferric 2,4,6-tripyridyl-s triazine complex (Fe3+-TPTZ) by antioxidants to the ferrous form (Fe2+-TPTZ). Briefly, the FRAP reagent (Sigma-Aldrich) comprising 10 mM TPTZ (ferrous iron) and 40 mM HCl was added to 300 mM sodium acetate buffer (pH 3.6) at 37 °C for 15 min. Reactions were started by adding 750 μL freshly prepared FRAP reagent to 50 μL R1-MVs. Vitamin C (300 μM) was used as a positive control. The absorbance was measured at 593 nm using a microplate reader (Biotek). All measurements were performed in triplicate.

Effects of R1-MVs on cell viability of HaCaT keratinocytes

The viability of R1-MVs-treated HaCaT cells was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) assay [66]. HaCaT cells were cultured at a density of 3 × 104 cells/well in 96-well plates and incubated at 37 °C for 24 h. After culturing, the medium was discarded, and the cells were washed with PBS. The cells were treated with R1-MVs at concentrations of 1, 5, 10, 30, 50, and 100 μg/mL for 12 h. As a positive control, 0.5% DMSO was used. After culturing, the cells were washed, and MTT (0.5 mg/mL) was added to the wells at 37 °C for 4 h. Subsequently, the media was discarded, and 150 μL DMSO was added to each well to solubilize the formazan crystals. Formazan absorbance was analyzed at 540 nm using a microplate reader (Biotek). The viability of HaCaT cells is presented as a percentage of the control cell group.

Effects of H2O2 on cell viability of HaCaT keratinocytes

To determine the optimal concentration of H2O2 that induced oxidative damage in vitro, HaCaT cells were treated with different concentrations of H2O2 (50, 100, 200, 300, 400, and 500 μM) for 12 h, and cell viability was measured using the MTT assay. Next, 300 μM H2O2 correspond to 70% cell viability and were chosen as optimal injury concentration.

Determination of effect of R1-MVs on the viability of HaCaT cells under H2O2 -induced oxidative stress

To explore the protective effects of R1-MVs against oxidative stress, HaCaT cells were pretreated with nominal concentrations of R1-MVs (1, 5, 10, 30 and 50 μg/mL) for 12 h before H2O2-induced oxidative damage. The cells were then exposed to H2O2 (0.3 mM) for 12 h, and HaCaT cell viability was detected using the MTT method.

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay

HaCaT cells were seeded on glass slides for 24 h and then exposed to H2O2 (0.3 mM) in the presence or absence of R1-MVs for 12 h. The cells were fixed in 3.7% paraformaldehyde in 1 × PBS buffer for 30 min and were then permeabilized in 0.2% Triton X-100/PBS (Sigma, Germany) for 5 min. The glass slides were washed twice using PBS and 100 μL of the Equilibration Buffer was added for 10 min at 4 °C. Thereafter, the samples were cultured in 50 μL of TdT reaction mixture for 1 h at 37 °C in the dark. To stop the reaction, the glass slides were immersed in 2 × SSC for 15 min. Finally, DAPI nuclear stain was added along with a mounting medium and the samples were analyzed using a confocal laser scanning microscope (LSM510, Carl Zeiss, Jena, Germany).

Determination of intracellular ROS contents in HaCaT cells

HaCaT cells were sequentially treated with R1-MVs (10 and 30 μg/mL) and H2O2 (0.3 mM) for 12 and 12 h, respectively. Subsequently, the supernatant was aspirated and cultured in an FBS-free medium containing dichlorodihydrofluorescein diacetate (DCFH-DA) (100 μM) for 30 min at 37 °C in the dark. Fluorescence intensity was analyzed using a confocal laser scanning microscope (LSM510), and quantitative analysis was performed using the ImageJ software. For flow cytometry analysis, the cells were detached by trypsinization (Trypsin–EDTA, Gibco, Paisley, UK) and resuspended in PBS. The fluorescence intensity of oxidized DCF was detected using a FACSVerse™ flow cytometer and FlowJo software.

Measurement of the intracellular antioxidant molecules and malondialdehyde (MDA) levels

HaCaT cells were seeded at 1.0 × 107 cells/well in a 100 mm dish for 24 h. After washing with serum-free media, the cells were treated with different concentrations of R1-MVs (5, 10, and 30 μg/mL) for 12 h. Then, H2O2 (0.3 mM) was added, and the cells were incubated at 37 °C for 12 h. A lysis buffer was used to resuspend the cells at 4 °C for 5 min after culturing, followed by centrifugation at 13,000 × g at 4 °C for 5 min to determine antioxidant molecule activities and MDA levels in the cell lysate. The activities of SOD, CAT, glutathione (GSH), and the level of MDA were measured using respective assay kits (BioVision, Milpitas, CA, USA).

Detection of changes in the mitochondrial membrane potential (MMP)

MMP changes were measured using the JC-1 probe. JC-1 emits red fluorescence in non-apoptotic cells and green fluorescence in apoptotic or necrotic cells. Briefly, HaCaT cells were cultured on glass coverslips coated with poly-L-lysine (0.5 mg/mL)-coated glass coverslips for 12 h. After incubation, the cells were pretreated with R1-MVs (10 and 30 μg/mL) and exposed to H2O2 (0.3 mM) for 12 and 12 h, respectively. The medium was aspirated and incubated with 10 μg/mL of JC-1 (Thermo Fisher Scientific, Waltham, MA, USA) solution for 20 min at 37 °C in the dark, aspirated with the staining solution, and resuspended in the PBS. The fluorescence intensity was assessed using a confocal laser scanning microscope (LSM510). Red fluorescent JC-1 aggregates were detected by a 561 nm PE channel, while the monomeric green fluorescent form of JC-1 was detected by a 488 nm FITC channel. Quantification of JC-1 fluorescence was performed using the ImageJ software. The results are represented as a percentage of the control cells.

Western blotting analysis

HaCaT cells were seeded in a 6-well plate and treated with H2O2 (0.3 mM) in the presence or absence of R1-MVs (10 and 30 μg/mL). Cytosolic and nuclear proteins were extracted using cell lysis buffer (RIPA buffer, Pierce, Rockford, IL, USA) and the CelLytic NuCLEAR Extraction Kit (Sigma-Aldrich), respectively. Protein concentration was measured using the BCA protein assay. Proteins were isolated using 10% SDS–PAGE and electrically transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk and incubated with the respective primary antibodies (1:1000 dilution; anti-Bcl-2, anti-Bax, anti-cytochrome C, anti-cleaved-caspase 3, anti-cleaved-caspase 8, anti-cleaved-caspase 9, anti-PARP, anti-p38, anti-ERK, anti-JNK, anti-p38, phosphorylated (p)-ERK, p-JNK, p-p38, Nrf2, β-actin, and anti-α-tubulin antibodies) overnight at 4 °C. Thereafter, the membranes were incubated with an HRP-conjugated secondary antibody (anti-rabbit Ab, 1:5000 dilution) for 1 h at RT. Proteins were visualized using an electrochemiluminescence advance kit (Millipore, Merck KGaA, Darmstadt, Germany). Primary and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and Calbiochem (San Diego, CA, USA), respectively.

ΔDR2577 deletion mutant construction

ΔDR2577 deletion mutants were constructed using the deletion mutagenesis method as previously described [67]. Briefly, polymerase chain reaction (PCR)-amplified fragments from the upstream and downstream regions of DR2577 were digested with the appropriate restriction enzymes and ligated into the corresponding sites of pKatAPH3. The recombinant plasmid was then transformed into D. radiodurans cells. The mutant strains were selected on TGY agar plates (0.5% tryptone, 0.3% yeast extract, and 0.1% glucose) supplemented with 8 μg/mL kanamycin (Sigma-Aldrich). All constructs were confirmed using diagnostic PCR and nucleotide sequencing. The primers used in this study are listed in Table S1.

Annexin V and propidium iodide (PI) staining

To investigate apoptosis, HaCaT cells were treated with 30 μg/mL of R1-MVs or ΔDR2577-R1-MVs for 12 h prior to treatment with H2O2 (0.3 mM) for 12 h at 37 °C and were analyzed using Annexin V/PI staining (BD Bioscience, San Jose, CA, USA). Cells were harvested and stained with annexin V (1:50 dilution with annexin V binding buffer, BD Bioscience, San Jose, CA, USA) for 15 min at RT. After washing with annexin V binding buffer, cells were stained with PI (1:25 dilution with annexin V binding buffer) for 10 min at RT. Necrotic, late apoptotic, and apoptotic cell death were assessed by analyzing cells positive for Annexin V, PI, or both, respectively, using a FACSverse cytometer and FlowJo software (V10, BD Bioscience).

Determination of mitochondrial ROS contents in HaCaT cells

The generation of ROS by mitochondria was analyzed using the MitoSOX mitochondrial superoxide indicator (Thermo Fisher Scientific). HaCaT cells were incubated with R1-MVs (30 μg/mL) or ΔDR2577-R1-MVs (30 μg/mL) for 12 h prior to treatment with H2O2 (0.3 mM) for 12 h at 37 °C The HaCaT cells were cultured with 5 μM MitoSOX reagent for 10 min at 37 °C in the dark, washed, and resuspended in PBS. The samples were analyzed using a FACSverse cytometer and FlowJo software (V10, BD Biosciences).

Statistical analysis

Statistical analyses were performed using Tukey’s multiple comparison test or an unpaired t-test using GraphPad Prism 7 (2018, GraphPad, San Diego, CA, USA). Data are expressed as the mean ± SD. P-values of < 0.05 were considered statistically significant.