Peptides and protein chemistry techniques

If not otherwise stated, all chemicals and materials used in this work were from Merck (Darmstadt, Germany) while all solutions were prepared using ultrapure, endotoxin-free water from Purelab Maxima system (ELGA LabWater, High Wycombe, United Kingdom).

BacSp222 bacteriocin, its succinylated form (suc-K20-BacSp222), as well as bacteriocin deprived of N-terminal formyl-methionine (-fM-BacSp222) were obtained and analyzed strictly as described in our previous work [15]. As is presented in the above-cited study, the purity (over 99%), homogeneity, and identity of the peptides were checked by an analytical reversed-phase high-pressure liquid chromatography (RP-HPLC) and mass spectrometry. At the same time, the possible contamination by a Gram-negative endotoxin, LPS, was excluded during an E-TOXATE assay.

Additionally and independently, the possible contamination of peptides by LPS and LTA (a Gram-positive endotoxin) was carefully verified by total phosphorus determination performed by a modified method originally developed by Huang and Zhang [18]. This method involves converting the total dissolved organic phosphorus (present in all forms of the mentioned bacterial endotoxins) into an inorganic phosphate, determined spectrophotometrically after subsequent reaction with a mixture of molybdate-antimony reagent. In the present study, this technique has been adapted to the microplate format as follows: 238 µL portions of appropriate peptide solutions or an LTA standard (LTA from Staph. aureus, cat. No L2515, Merck, Darmstadt, Germany) were pipetted into the wells of a 96-well flat-bottom polystyrene cell culture microplate (Nest, Wuxi, China) and mixed with 48 µL portions of K2S2O8 solution (2 g in 40 mL H2O). The wells were sealed tightly using a MicroAmp film (Applied Biosystems/Thermo, Waltham, MA, USA) and incubated overnight at 90 °C in a thermal cycler (model C1000 Touch, Bio-Rad, Hercules, CA, USA). After cooling down, the content of the wells was mixed with 71 µL of a 1:1 mixture of ascorbic acid solution (0.5 g in 50 mL H2O) and molybdate-antimony solution (0.12 g N6H24Mo7O24 · 4H2O, 46 mL H2O, 1.25 mL 98% w/v H2SO4, 2.5 mL 0.3% w/v K2C8H4O12Sb2 · 3H2O). After 8 min incubation at room temperature, the absorbance was read at 890 nm using a microplate reader (model Sunrise, Tecan, Männedorf, Switzerland).

The fluorescent labeling of BacSp222 on carboxyl groups has been carried out using a CF488A dye (cat. No SCJ4600015, Merck, Darmstadt, Germany). 1 mg of CF488A was dissolved in 1 mL of 0.2 M 2-(N-morpholino) ethanesulfonic acid (MES) pH 5.0, and to this solution successively was added 20 µL portion of BacSp222 solution (500 µg in water), and then 50 µL portion of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride solution (EDC-HCl, cat. No PG82079, Pierce/Thermo, Waltham, MA, USA, 5.14 mg in 0.2 M MES pH 5.0). The mixture was mixed at room temperature for 2 h. After this the labeled peptide has been purified by RP-HPLC chromatography using a Discovery Bio Wide Pore C8 4.6 × 250 mm column and two buffers: A, containing 0.1% (v/v) trifluoroacetic acid (TFA) as well as B, containing 0.07% TFA and 80% (both v/v) acetonitrile. The linear gradient 45% to 100% of buffer B was developed for 10 min, the flow rate was 1 mL/min, and the labeled peptide has been manually collected as a broad peak eluting at 9 to 13 min. The collected fraction has been evaporated using a vacuum centrifuge and dissolved in water. As verified by bactericidal radial diffusion assay as well as by NO release experiment on macrophage-like cells (data not shown) the bacteriocin labeled by CF488A at carboxyl groups (BacSp222-CF488) maintained its full biological activity.

The concentration of all peptides used in this study was determined by an amino acid analysis as described earlier [19].

Eukaryotic cells used in experiments

Murine monocyte/macrophage RAW 264.7 cell line (ATCC TIB-71) and murine monocyte/macrophage P388.D1 cell line (ATCC CCL-46) were obtained from the American Type Culture Collection (Manassas, VA, USA). Human TLR2/NF-κB/SEAP (HEK-Blue hTLR2), TLR4/NF-κB/SEAP (HEK-Blue hTLR4), TLR5/NF-κB/SEAP (HEK-Blue hTLR5), TLR2+TLR1/NF-κB/SEAP (HEK-Blue hTLR2 and hTLR1), and TLR2+TLR1/NF-κB/SEAP (HEK-Blue hTLR2 and hTLR6) reporter HEK293 cells were purchased from InvivoGen (San Diego, CA, USA). The passages of all TLR overexpressing cells were from 3 to 8.

The cells were grown in an incubator, at 5% CO2, 37 °C, and > 95% humidity, in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose (GIBCO, Paisley, UK) and 5% (v/v) fetal bovine serum (FBS, GIBCO, Paisley, UK), (for RAW 264.7 and P388.D1 cells) or in DMEM containing 4.5 g/L glucose, 10% (v/v) FBS, 50 units/mL penicillin, and 50 µg/mL streptomycin (GIBCO, Paisley, UK) (in case of TLR family overexpressing HEK293 cells).

The interaction of BacSp222-CF488 with P388.D1 cells: confocal microscopy imaging of live cells

AxioObserver Z.1 inverted optical microscope with a laser scanning confocal module LSM 880 (Carl Zeiss, Germany) was used to analyze the interaction of BacSp222-CF488 with P388.D1 cells. First, the cells (2 × 106 in the volume of 2 mL DMEM supplemented with 5% FBS) were seeded on a 12 mm Nunc glass base dish (Thermo Scientific, Rochester, NY, USA). After overnight culture and prior imaging, the medium was replaced with fresh DMEM. Then the cells were transferred to an incubation chamber of the microscope (under temperature and CO2 control) and stimulated with 1 µM BacSp222-CF488 or 1 µM BacSp222 (for 5 or 30 min). Next, the cells were gently rinsed with PBS, and 2 mL of PBS containing 1.73 µM sulforhodamine B was added. Imaging was performed using oil immersion and Plan-Neofular 40 × NA1.3 objective. The argon laser line of 488 was used for BacSp222-CF488 excitation, and emission in the range 493–556 nm was recorded as the green channel. For sulforhodamine, 561 nm was used for excitation, and 566–703 nm emission was recorded as the red channel. ImageJ 1.53c software (National Institutes of Health, Bethesda, MD, USA) was used for image processing.

The interaction of BacSp222-CF488 with P388.D1 cells: confocal microscopy imaging of fixed cells

P388.D1 cells (2 × 106 cells per well) were seeded in 1 mL of DMEM/5% (v/v) FBS on poly-l-lysine-coated glass coverslips placed in the wells of a 12-well plate and grown overnight. Next, the medium was replaced with a fresh DMEM. The cells were stimulated for 5 or 30 min with 1 µM BacSp222 or 1 µM BacSp222-CF488 and fixed in a 4% (v/v) solution of methanol-free formaldehyde (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 15 min at room temperature (RT). In the next step, the cells were washed 3 times with PBS, and the nuclei were stained with 4′,6-diamidyno-2-fenyloindol (DAPI, Thermo Fisher Scientific, Waltham, MA, USA) in the dark at RT for 15 min. Then the cells were washed with PBS, and the samples were mounted onto slides in ProLong Glass Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA) and stored for 24 h in the darkness. The cells were observed using AxioObserver Z.1 inverted optical microscope with a laser scanning confocal module LSM 880 (Carl Zeiss, Germany). Imaging was performed using oil immersion and Plan-Neofular 40 × NA1.3 objective. The argon laser line of 488 was used for BacSp222-CF488 excitation, and emission in the range 495–630 nm was recorded as the green channel. For DAPI, 405 nm was used for excitation, and emission in the range 410–495 nm was recorded as the blue channel. ImageJ 1.53c software (National Institutes of Health, Bethesda, MD, USA) was used for image processing.

Inhibition of FPR1 and FPR2 by selected antagonists

RAW 264.7 or P388.D1 cells were grown overnight on 96-well plates in 100 µL of DMEM enriched with 5% (v/v) FBS (the density of cells was 3 × 104 cells/well). Next, the medium was replaced with fresh DMEM supplemented with 2% (v/v) FBS (control) or fresh DMEM supplemented with 2% (v/v) FBS containing: 50 µM WRW4 (Millipore Corporation, affiliated by MERCK, Germany) or 5 µM Boc-2 (MP-Bio, France), and the cells were incubated for 30 min. After incubation RAW 264.7 or P388.D1 cells were treated for 6 h with: (1) 50 µM WRW4, (2) 5 µM Boc-2, (3) 1 µM fMLP, (4) 1 µM WKYMVM, (5) 1 µM BacSp222, (6) 1 µM suc-K20-BacSp222, (7) 1 µM -fM-BacSp222, (8) 5 µM Boc-2 and 1 µM fMLP, (9) 50 µM WRW4 and 1 µM fMLP, (10) 5 µM Boc-2 and 1 µM WKYMVM, (11) 50 µM WRW4 and 1 µM WKYMVM, (12) 5 µM Boc-2 + 1 µM BacSp222, (13) 50 µM WRW4 + 1 µM BacSp222, (14) 5 µM Boc-2 + 1 µM suc-K20-BacSp222, (15) 50 µM WRW4 + 1 µM suc-K20-BacSp222, (16) 5 µM Boc-2 + 1 µM -fM-BacSp222, (17) 50 µM WRW4 + 1 µM -fM-BacSp222. Next, the media were collected for TNF concentration measurement using the ELISA test (ELISA MAX™ Standard Set Mouse TNF; Biolegend, San Diego, CA, USA) according to the protocol provided by the manufacturer. The viability of the cells was determined and analyzed using a MTT assay as described previously [15]. The absorbance was measured at 450 nm for ELISA and 545 nm for MTT assays using the Synergy H1 Hybrid plate reader controlled by Gene5 version 2.00.18 software (BIOTEK Instruments, Winooski, VT, USA).

Stimulation of overexpressing TLR receptors cells

HEK-Blue hTLR2, HEK-Blue hTLR4, HEK-Blue hTLR5, HEK-Blue hTLR2 and hTLR1, as well as HEK-Blue hTLR2 and hTLR6, were seeded on a 96-well plate at density 2.5 × 104 cells per well in 100 µL DMEM containing 10% (v/v) FBS, 50 units/mL penicillin, 50 µg/mL streptomycin (control) or in 100 µL DMEM containing 10% (v/v) FBS, 50 units/mL penicillin, 50 µg/mL streptomycin and (1) 5 × 107 Heat Killed Listeria monocytogenes cells/mL (HKLM, InvivoGen, San Diego, CA, USA) or 10 ng/mL ultrapure LPS from Escherichia coli 0111:B4 (InvivoGen, San Diego, CA, USA), or 10 ng/mL ultrapure flagellin from Bacillus subtilis (InvivoGen, San Diego, CA, USA), or 10 ng/mL FSL-1 (InvivoGen, San Diego, CA, USA), or 20 µM CU-T12-9 (InvivoGen, San Diego, CA, USA) (the positive controls for HEK-Blue hTLR2, HEK-Blue hTLR4, HEK-Blue hTLR5, HEK-Blue hTLR2 and hTLR1, HEK-Blue hTLR2 and hTLR6, respectively), (2) 1 µM BacSp222, (3) 1 µM suc-K20-BacSp222, (4) 1 µM -fM-BacSp222. After 17 h, the media were collected for secreted embryonic alkaline phosphatase (SEAP) detection according to the protocol described in the section below. The viability of the cells was determined and analyzed using an MTT assay as described previously [15].

Inhibition of human TLR2 and TLR6 heterodimer by the selected antagonists

HEK-Blue hTLR2 and HEK-Blue hTLR6 cells were seeded on a 96-well plate at density 2.5 × 104 cells per well in 100 µL DMEM containing 10% (v/v) FBS, 50 units/mL penicillin, 50 µg/mL streptomycin (control) or in 100 µL DMEM containing 10% (v/v) FBS, 50 units/mL penicillin, 50 µg/mL streptomycin and (1) 20 µM sparstolonin B, (2) 200 µM TL2-C29 (InvivoGen, San Diego, CA, USA). After 1 h to each well was added 10 µL of (1) water (control), (2) FSL-1 (to a final concentration of 10 ng/mL), (3) BacSp222 (to a final concentration of 1 µM), (4) suc-K20-BacSp222 (to a final concentration 1 µM), or (5) -fM-BacSp222 (to a final concentration 1 µM). After 17 h, the media were collected for SEAP detection according to the protocol described below. The viability of the cells was determined using an MTT assay as described previously [15].

Assay for detection of secreted embryonic alkaline phosphatase (SEAP)

Activation of NF-κB upon TLRs stimulation was analyzed by measurement of SEAP activity secreted by the cells to the media in response to bacteriocins. 10 µL of culture media were collected from the cells and transferred to a 96-well plate containing 90 µL Cell Culture Medium for SEAP Detection (InvivoGen, San Diego, CA, USA). The plate was incubated at 37 °C for 1 h, and the absorbance was measured at 620 nm using a microplate reader Synergy H1 Hybrid plate reader controlled by Gene5 version 2.00.18 software (BIOTEK Instruments, Winooski, VT, USA).

Inhibition of murine TLR2 by the selected antagonists

RAW 264.7 cells were grown overnight on 96-well plates in 100 µL of DMEM enriched with 5% (v/v) FBS (the density of the cells was 3 × 104 cells/well), while P388.D1 cells were grown overnight on 48-well plates in 200 µL of DMEM enriched with 5% FBS (v/v) (the density of the cells was 6 × 104 cells/well). Next, the medium was replaced with 100 µL fresh DMEM with 2% (v/v) FBS or fresh DMEM with 2% (v/v) FBS containing: (1) 6 µM sparstolonin B or (2) 200 µM TL2-C29 (InvivoGen, San Diego, CA, USA). After 30 min, the medium was replaced again with fresh DMEM supplemented with 2% (v/v) FBS or DMEM supplemented with 2% (v/v) FBS and containing (1) 2 µg/mL LTA, (2) 1 µM BacSp222, (3) 1 µM suc-K20-BacSp222, (4) 1 µM -fM-BacSp222, (5) 6 µM sparstolonin B, (6) 6 µM sparstolonin B and 2 µg/mL LTA, (7) 6 µM sparstolonin B and 1 µM BacSp222, (8) 6 µM sparstolonin B and 1 µM suc-K20-BacSp222, (9) 6 µM sparstolonin B and 1 µM -fM-BacSp222, (10) 200 µM TL2-C29, (11) 200 µM TL2-C29 and 2 µg/mL LTA, (12) 200 µM TL2-C29 and 1 µM BacSp222, (13) 200 µM TL2-C29 and 1 µM suc-K20-BacSp222, (14) 200 µM TL2-C29 and 1 µM -fM-BacSp222. After 6 h, the media were collected for TNF concentration analysis using the ELISA test described above.

Analysis of NO production by the cells

RAW 264.7 cells were seeded on 96-well plates in 100 μL DMEM supplemented with 5% (v/v) FBS at 3 × 104 cells/well density. After 16 h, the medium was replaced with fresh DMEM with 2% (v/v) FBS or DMEM containing 2% (v/v) FBS and LTA in the concentration range between 0,005 µg/mL to 2 µg/mL. Each stimulation was in the presence or absence of 10 ng/mL mouse interferon γ (IFN-γ, Biolegend, San Diego, CA, USA). The nitrate levels were measured as described previously [15].

Statistical analysis and data presentation

Experiments were performed with three independent replications. The data were presented as mean ± standard deviation (SD). The Statistica software (Tibco Software, version 13.3) was used to perform statistical analysis. The statistical significance of differences between the particular results was calculated by a one-way ANOVA with Tukey’s HSD (honestly significant difference) post hoc test and shown in figures as * for p < 0.05 and # for p < 0.001.