The authors have declared that no competing interests exist.
Conceived and designed the experiments: MB H. Frøkiær H. Franzyk CO JMW HI. Performed the experiments: MB BK SBC NT MM. Analyzed the data: MB H. Frøkiær NT JMW HI. Contributed reagents/materials/analysis tools: BK H. Frøkiær H. Franzyk CO. Wrote the paper: MB H. Frøkiær H. Franzyk CO JMW HI.
Strains used in this study, and their sources are listed in
Strain | Description | Source / Reference |
---|---|---|
PC322 | S.J.Foster, [ |
|
PC203 | S.J.Foster, [ |
|
SH101F7 | S.J.Foster, [ |
|
NCTC 8325–4 | S.J.Foster, [ |
|
RN6607 | R. Novick [ |
|
RN4850 | R. Novick, [ |
|
M0Z53 | R. Novick, [ |
|
RN6911 | R. Novick, [ |
|
FPR3757 | CA-MRSA USA 300 | ATCC Boras,Sweden |
RN10829 | R. Novick, [ |
|
NCFM | Danisco, CPH, DK | |
O6:K5:H1 | SSI, CPH, DK | |
DU1090 | O’Reilly [ |
|
WCFS1 | NIZO Food Research, Ede, NL [ |
For the synthesis of analogues SolB-NaI (
For the synthesis of the lactam analogues Am15-D (
Synthesis of solonamides A and B (SolA (
The reporter assay was conducted as described by Nielsen et al. [
The method used is described by Nielsen et al. 2014 [
Untreated 96-well plates (Nunc 265301) were incubated with 100 μL per well of 10 μg/mL fibronectin from human plasma for 24 h while shaking at 4°C. The plates were then washed three times with 1% bovine serum albumin in phosphate-buffered saline (PBS).
A starter culture of each strain was grown in TSB to an OD600 0.5. From this culture 50 μL was withdrawn and diluted 10-fold (from 10−1 to 10−5) in 0.9% NaCl solution. 5 μL of each dilution was inoculated into 200 μL TSB. Compound in DMSO was added to each respective well to a final concentration range of 5, 10, 20, 40 and 80 μg/mL. DMSO and no cells were used as controls. The microtiter plates were incubated for approximately 20 h at 37°C without shaking. The biofilm was then washed twice with 0.9% NaCl (200 μL), dried in a LAF bench, stained with 125 μL crystal violet (0.1%) for 30 min, followed by a 3× final wash with 200 μL 0.9% NaCl. To quantify the biofilm formation the stained biofilm was solubilized in 200 μL 95% ethanol, of which 100 μL was transferred to a new microtiter plate and the absorbance measured at 590 nm.
All animals used as a source of bone marrow cells were housed under conditions approved by the Danish Animal Experiments Inspectorate (Forsøgdyrstilsynet) according to The Danish Animal Experimentation Act; LBK no. 474 from 15/05/2014, and experiments were carried out in accordance with the guidelines of ‘The Council of Europe Convention European Treaty Series (ETS) 123 on the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes’. The source of bone marrow cells was female 4–6 month old C57/Black6 –Jtac mice (Taconic, Ejby, Denmark). All mice were sacrificed by cervical dislocation prior to bone marrow extraction for dendritic cell isolation. Dendritic cells were isolated and prepared as previously described by Christensen et al. [
The human PBMC assays were approved by Wageningen University Ethical Committee and performed according to the principles of the Declaration of Helsinki. PBMCs were isolated and prepared as previously described [
Isolated PBMCs were counted and adjusted to a concentration of 1×106 cells/mL. They were then spun down at 300×g for 5 min and the pellet was resuspended in 1 mL sterile PBS + 0.1% BSA containing 50 μg/mL CFDA/SE (Carboxyfluorescein diacetate succinimidyl ester, Cayman Chemicals) and allowed to incubate for 10 min at 37°C. 5 mL IMDM + 10% FBS was added to the cells which were then placed on ice for a further 5 min prior to spinning down and washing 3x with IMDM + 10% FBS. Washed PBMCs were resuspended in complete culture medium to a final concentration of 1×106 cells/mL and 500 μl/well were seeded in 48-well tissue culture plates. PBMCs were then stimulated with the lymphocyte proliferation inducers aCD3 and aCD28 (BD Pharmingen) at concentrations inducing 100% or 25% T-cell proliferation (10 ng/mL and 0.4 ng/mL respectively for aCD3 and 0.6 ng/mL and 0.024 ng/mL respectively for aCD28). PBMCs were then further co-stimulated with either compound or vehicle alone to a final concentration of 10 μg/mL, or thawed aliquots of the
DC, PBMC and T-cell viability was assessed by using the commercially available Annexin V: PI Apoptosis Detection Kit APC (eBiosciences) according to the manufacturer’s instructions. Viability was assessed by flow cytometry and analyzed using FACS Diva software. Significance was tested using one-way ANOVA.
For DCs levels of IL-12, TNF-α, IL-6 and IL-10 (all purchased from R&D Systems, Minneapolis, MN, USA) were detected in culture supernatants by commercially available enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions. For PBMC and T-cell cytokine analysis, cytokines (IL-12, TNF-α, IL-6, IL-10, IL-8 and IL-1β) were measured by BD Cytometric Bead Array Flexset (BD Biosciences) using a FACS CantoII flow cytometer, according to the manufacturer’s instructions and analyzed using the BD FCAP software. Significance was tested using one-way ANOVA.
All cells used for the generation DCs were generated from bone marrow cells isolated from mice sacrificed by cervical dislocation. The use of mice was approved by Danish Animal Experiments Inspectorate (Forsøgdyrstilsynet). This is the ethical committee, who approves all animal experiments to be performed in Denmark as well as all the experimental animal facilities in Denmark. All animals used as a source of bone marrow cells were housed under conditions approved by the Danish Animal Experiments Inspectorate (Forsøgdyrstilsynet) according to The Danish Animal Experimentation Act; LBK no. 474 from 15/05/2014, and experiments were carried out in accordance with the guidelines of ‘The Council of Europe Convention European Treaty Series (ETS)123 on the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes. The human PBMC assays were approved by Wageningen University Ethical Committee and performed according to the principles of the Declaration of Helsinki. PBMCs were isolated and prepared as previously described [
To explore the structure–activity relationships of solonamides, total syntheses of solonamides A (
Structures of autoinducing peptides (AIP-I and III) used in the study as well as tested depsipeptides and their modified lactam analogues.
The ability of synthetic solonamides and lactam analogues to repress
Agar plates containing the
Interference with the
Cultures of RN10829 (P2-agrA:P3-blaZ) containing the pagrC-I-WT, were grown to an OD600 of 0.4–0.5 where a 1/10 volume of AIP-I containing supernatant was added and solonamides and analogues in DMSO to a final concentration of 10 μg/mL. Samples obtained at 15 min time intervals after addition of test solutions were analysed for β-lactamase activity. Each bar represents the average of 3 replicates and the error bars represent the standard deviation. Comparisons were made for each individual time point between AIP and AIP + Compound samples. ns (no significance); *, p<0.05; **, p<0.01; ***, p<0.001.
As
Upon exposure of
A dilution series of an 8325–4 culture were inoculated (5 μL) into wells of a 96-well microtiter plate containing 200 μL TSB. SolB (A), ESB (C) or Am16-L (C) was added to final concentrations of 5, 10, 20, 40, and 80μg/mL. Inoculum alone, DMSO and no inoculum were used as controls. Biofilm formation was assessed by crystal violet staining and OD590 nm measurement. Each bar represents the average of 3 experiments, and the error bars represent the standard deviation. For statistical significance, comparisons were made between untreated versus vehicle and treated (black bracket), and between vehicle control versus compound treated (red square). ns (no significance);*, p<0.05; **, p<0.01; ***, p<0.001.
Solonamide B was previously shown to display undetectable toxicity to both human and bovine neutrophils [
As the selected anti-virulence compounds did not affect viability of human PBMCs, proliferated T-cells or murine DCs, we investigated whether they would modulate cytokine and chemokine responses in unstimulated and microbially stimulated antigen-presenting cells as well as proliferating T cells. Cytokines IL-6, TNF-α, IL-12, and IL-10 were measured for DCs, and for the PBMCs and aCD3 and aCD28 stimulated T-cells, IL-1β and the chemokine IL-8 were also measured. The tested compounds had no effect on the cytokine and chemokine secretion in non-stimulated DCs, PBMCs or T-cells activated with aCD3 and aCD28 compared to untreated controls, indicating that the compounds alone do not stimulate an immune response. Furthermore, the selected solonamides and analogues exerted no immune-modulating effect on cytokine secretion by
As
Bone marrow derived murine dendritic cells (A) and human peripheral blood mononuclear cells (B) were stimulated at an MOI of 10 with
One reported mechanism of
CFDA/SE (25μg/mL) stained human PBMCs (1x106/ml) were co-stimulated with aCD3/aCD28 (0.4ng and 0.024ng/mL respectively) and S. aureus either treated or not with 10μg/mL SolB, ESB or Am16-L at an MOI of 10 for 4 days. Harvested cells were stained with aCD4-PE and analyzed by Flow Cytometry using FACS Diva software. Lymphocytes were gated based on the expression of CD4, and the number of cell divisions was gated according to the CFDA/SE FITC excitation. The data represent the average of 3 donors, and the error bars represent the standard deviation.
In summary our investigations show that solonamide B and analogues harbor no toxicity towards immune cells, and they suggest that no adverse effects are anticipated when using these compounds to target
Human PBMCS were stimulated with 10 μg/mL solB (2), ESB (4) or the analogue Am16-L (9) for 24 h (A) and 4 days (B) or stimulated for T-Cell proliferation with aCD3/aCD28 and co-stimulated with the selected compounds for 4 days (C) Culture media and DMSO were used as controls. PBMCs and T-cells were harvested; stained with Annexin V/PI, and cell viability measured by flow cytometry. The results are representative of 3 different donors.
(TIF)
Human PBMCS were stimulated with a concentration gradient of 5, 10, 20 and 40 μg/mL of solB (2), ESB (4) or the analogue Am16-L (9) for 4 days. Culture media and DMSO were used as controls. PBMCs were harvested; stained with Annexin V/PI, and cell viability measured by flow cytometry. The results are representative of 3 different donors.
(TIF)
Bone-marrow-derived DCs were stimulated with
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(TIF)
CFDA/SE (25
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The authors thank Anni Mehlsen for skilled technical assistance as well as Lisbeth D. Lund and Anita Nielsen for expert guidance.