All chemicals were purchased from Merck KGaA (Darmstadt, Germany), unless specified otherwise.
GVF27, FAM-GVF27 and LL-37 were synthesized by the solid-phase method (CASLO ApS, Lyngby, Denmark). FAM-GVF27 presents 5,6 - carboxyfluoresceine fluorochrome conjugated to the N-terminus of peptide. The purity of all the three peptides was determined by analytical HPLC to be higher than 95%. Trifluoroacetic acid (TFA) was removed and the molecular weight was confirmed by MALDI-TOF mass spectrometry. Peptide sequences are reported in Table 1.
Bacterial strains and growth conditions
Clinical isolates Burkholderia cenocepacia LMG 18863 and Burkholderia multivorans LMG 17582 were kindly provided by Dr. Alessandra Bragonzi (Infection and CF Unit, San Raffaele Scientific Institute, Milan, Italy). Both bacterial strains were grown in Muller Hinton Broth (MHB, Becton Dickinson Difco, Franklin Lakes, NJ) and on Tryptic Soy Agar (TSA; Oxoid Ltd., Hampshire, UK). In all the experiments, bacteria were inoculated and grown overnight in MHB at 37 °C. The next day, bacteria were transferred to a fresh MHB tube and grown to mid-logarithmic phase.
The antimicrobial activity of GVF27, FAM-GVF27 and LL-37 on B. cenocepacia LMG 18863 and B. multivorans LMG 17582 was determined by using the broth microdilution method, as previously described . Bacteria were grown to mid-logarithmic phase in MHB at 37 °C, then diluted to 2×106 CFU/mL in Difco 0.5X Nutrient Broth (Becton-Dickenson, Franklin Lakes, NJ) and mixed 1:1 v/v with two-fold serial dilutions of peptides. Following over-night incubation, MIC100 values were determined as the lowest peptide concentration responsible for no visible bacterial growth. All the experiments were carried out in three independent replicates.
Cell Culture and Differentiation
Human monocytic cells THP-1 (ATCC, TIB-202) were cultured in Roswell Park Memorial Institute 1640 medium (RPMI) completed with 10% fetal bovine serum, 100 μg/mL penicillin and 100 μg/mL streptomycin and stored in a humidified 37 ºC incubator with 5% CO2/95% air. Cells were periodically checked at the microscope to ensure normal morphology.THP-1 cells were differentiated using 2 nM phorbol 12-myristate 13-acetate (PMA) for 96 h at 37 °C with 5% CO2/95% air.
Cytotoxic effects of GVF27 on undifferentiated or PMA-differentiated THP-1 cells were determined by performing the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide reduction inhibition assay (MTT assay), designed to be used for the spectrophotometric quantification of cell proliferation. Briefly, 2×104 cells were seeded into a 96-well plate and incubated at 37 °C in the presence of 5% CO2. Medium was then replaced with 100 μL of fresh medium containing peptide solution at a final concentration ranging from 0 to 40 μM/well. After 24 h of incubation at 37 °C, the peptide-containing medium was removed, and 100 μL of tetrazolium MTT diluted at 0.5 mg/mL in DMEM without red phenol was added. After 4 h of incubation at 37 °C, the resulting insoluble formazan salts were solubilized in 0.04 M HCl in anhydrous isopropanol and quantified by measuring the absorbance at λ = 570 nm, using an automatic plate reader spectrophotometer (Synergy HTX Multi-Mode Reader-BIOTEK, Winooski, Vermont, United States). Cell survival was expressed as means of the percentage values compared to control. Analyses were performed at last 3 times.
Anti-biofilm activity assays
Bacteria were grown over-night at 37 °C and then diluted to 4×108 CFU/mL in 0.5x MHB. Incubations with increasing concentrations of GVF27 (from 0.078 to 20 μM) were carried out either for 4 h, in order to test peptide effects on cells attachment, or for 24 h, in order to test peptide effects on biofilm formation, as previously described . When the effects of peptides on preformed biofilm were evaluated, bacterial biofilm was formed for 24 h at 37 °C, and then treated with GVF27. To carry out the crystal violet assays, bacterial biofilms were washed with PBS 1X and then incubated with the dye (0.04%) for 20 min at room temperature. At the end of the incubation and after a further wash with PBS 1X, the dye bound to cells was dissolved in 33% acetic acid. Spectrophotometric analyses were then carried out at a wavelength of 600 nm.
Confocal laser scanning microscopy analyses in static conditions were performed by using Thermo Scientific™ Nunc™ Lab-Tek™ Chambered Coverglass systems (Thermo Fisher Scientific, Waltham, MA, USA). Viability of the cells embedded into biofilm structure was evaluated by sample staining with either LIVE/DEAD® Bacterial Viability kit (Molecular Probes Thermo Fisher Scientific, Waltham, MA, USA) or with FilmTracer™ SYPRO™ Ruby Biofilm Matrix Stain (Invitrogen™) accordingly to manufacturers’ instructions. Biofilm images were captured by using a confocal laser scanning microscope (Zeiss LSM 710, Zeiss, Germany) and a 63X objective oil-immersion system. Biofilm architecture was analysed by using the Zen Lite 2.3 software package. Each experiment was performed in triplicate. All the images were taken under identical conditions.
Minimal agglutination concentration (MAC)
B. cenocepacia LMG 18863 cells were grown at 37 °C to an OD600 nm of 0.2, centrifuged at 5,000 g for 2 min, and resuspended in NaP buffer (pH 7.4) to give an absorbance at 600 nm of 1. An aliquot of 300 µL of the bacterial suspension was treated with increasing peptide concentrations (from 0 to 40 µM) in the Lab-Tek™ Chambered Cover glass systems and incubated at room temperature for 1 h. The aggregation behavior was observed by staining with wheat germ agglutinin-Oregon Green 488 conjugate (50 µg/mL) by confocal laser scanning microscopy. The agglutinating activity is expressed as the minimum agglutinating concentration (MAC) of the samples tested. Each experiment was performed in triplicate.
Scanning Electron Microscopy
To perform scanning electron microscopy (SEM) analyses, B. cenocepacia LMG 18863 biofilm was incubated with increasing concentrations of GVF27 (2.5 to 40 μM) for 24 h at 37 °C. Following incubation, bacterial biofilm was fixed in 2.5% glutaraldehyde. Following over-night incubation, bacterial biofilm was washed three times in distilled water and then dehydrated with a graded ethanol series: 25% ethanol (1 × 10 min); 50% ethanol (1 × 10 min); 75% ethanol (1 × 10 min); 95% ethanol (1 × 10 min); 100% anhydrous ethanol (3 × 30 min). Bacterial biofilm deposited onto glass substrate was sputter coated with a thin layer of Au-Pd (Sputter Coater Denton Vacuum DeskV) to allow subsequent morphological characterization using a FEI Nova NanoSEM 450 at an accelerating voltage of 5 kV with Everhart Thornley Detector (ETD) and Through Lens Detector (TLD) at high magnification.
B. cenocepacia J2315 cells were grown over-night in NB at 37 °C, harvested by centrifugation and freeze-dried. Afterwards, these cells were then submitted to the petroleum ether-chloroform-phenol (PCP) extraction method; the remaining pellet was further extracted according to the hot water-phenol method. Each phase was dialyzed against distilled water to remove phenol, freeze-dried, and then screened by SDS-PAGE to detect the presence of LPS by using silver staining procedure [46, 47]. LPS was identified in the water phase of the hot water-phenol extraction (yield 77.2 mg/g of cells). This material was further purified through an enzymatic digestion using DNAse, RNAse and proteinase K  followed by centrifugation at 6,000 rpm for 30 min at 4 °C. The purified LPS was isolated in the pellet (yield 15.0 mg/g of cells).
Isothermal Titration Calorimetry
Isothermal titration calorimetry (ITC) was performed on the Low Volume NanoITC (TA Instruments-Waters LLC, New Castle, DE, USA) to determine interaction between LPSs from B. cenocepacia or E. coli and GVF27. Briefly, both GVF27 (200 µM) and LPS (0.188 mg/ml ~ 12,5 µM) were diluted in 10 mM HEPES, pH 7. The chamber was filled with 164 µL of LPS, and 2 µl of the peptide was titrated into the chamber every 300 seconds. Experiments were performed at 37 °C and analysed using the Nano Analyze software (TA instruments-Waters LLC). All the experiments were performed in duplicate after which calculated binding characteristics of both experiments were averaged.
Fluorescence Displacement Assay
Association of GVF27 with LPS has been determined as described in . Briefly, the synthetic FAM-GVF27 (1.25 mM) was added to increasing concentrations of LPSs extracted from B. cenocepacia (from 0 to 150 mg/mL) and the fluorescence was monitored at an excitation wavelength of 495 nm and an emission wavelength of 520 nm in 5 mM ammonium acetate (pH 5.0).
LPS neutralization assay by ELISA
Anti-inflammatory effects of GVF27 were analysed in PMA differentiated and undifferentiated THP-1 cells treated with LPS. Cells were plated into 96-well plates at a density of 2×104 cells in 100 µL of medium per well. Following incubation with 5 or 20 µM GVF27 and 1µg/mL LPS, medium was collected to quantify cytokines levels. TNF-α and MCP-1 levels in collected supernatants were determined by using human immunoassay kits (DuoSet ELISA kits, R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Samples optical density was measured using an ELISA reader set at 450 nm with a wavelength correction set at 540 nm. All experiments were performed in triplicate.
Data were analysed with GraphPad Prism, version 5.0 software (GraphPad Inc., San Diego, CA) by using Student’s t-test. A p-value of 0.05 or less was considered statistically significant.