In Vitro Antibacterial Studies Group A Streptococcus (GAS) was grown to mid-log phase in Todd-Hewitt Broth (THB, Sigma). A final concentration of 105 CFU/ml were incubated with synthetic d-toxin at 0, 1, 2, 4, 8, and 16 mM in the presence and absence of LL-37 (0, 4, and 8 mM), CRAMP (0, 2, and 4 mM), hBD2 (0, 2, and 4 mM), and hBD3 (0, 2, and 4 mM). GAS was incubated with peptides in 25% THB and 75% 1x dulbecco phosphate buffered saline (dPBS) for 4 hours at 37uC and then plated on Todd-Hewitt agar for 18 hours before colony enumeration. Graphs are representative of two independent experiments performed in duplicate. Whole Blood Assay Group A Streptococcus (GAS) was grown to mid-log phase in Todd-Hewitt Broth (THB, Sigma). A final concentration of 105 CFU/ml was incubated with heparanized human whole blood containing synthetic d-toxin at 0, 1, 2, or 4 mM. GAS was plated and colonies enumerated at 0.5, 1, 1.5, and 2 hours post inoculation. Graph is representative of two independent experi- ments performed in triplicate. Spectroscopic Measurements Synthetic d-toxin (QCB) at 5 mM, was incubated with 5 mM hBD2 (Peptide International), hBD3 (Peptide International), CRAMP, LL-37, or 16 mg/ml genomic neutrophil DNA in 20 mM potassium phosphate buffer, pH 7.3. Fluorescence mea- surements were taken on a Jobin Yvon SPEX FL3-11 spectroflu- orometer equipped with an R928 photomultiplier tube. Conditions for tryptophan fluorescence were as follows: excitation wavelength of 290 nm, emission excitation and emission bandpass of 8 nm and 4 nm, respectively, 1 nm steps, and an integration time of 1s. Spectroscopic curves of host peptide hBD2, hBD3, CRAMP, or LL- 37 were subtracted from respective d-toxin + host peptide curves. Similarly, the spectroscopic curve of DNA was subtracted from the d-toxin + DNA curve. Immunoblot and Precipitation of Antimicrobial Peptides Staphylococcus epidermidis ATCC 12228 was grown for 18 hours to stationary phase in Todd-Hewitt Broth (THB, Sigma). Cells were centrifuged for 5 mins at 50006g. Supernatants were harvested and filtered through 0.22 mm PES Express Millex GP filter. 1 mM final concentration of synthetic CRAMP or LL-37 was added to 1ml of the filtered supernatant and incubated on nutator at room temperature for 7 hours. Supernatants containing peptides were precleared with 1 mg control IgG antibody and 20 mL Protein A/ G-Plus agarose (Santa Cruz Biotechnology) for 1.5 hours at 4uC rotating. Beads were pelleted by centrifugation at 2,5006g for 5 minutes and supernatant was removed and placed in new tube. The 1 ml precleared supernatant was divided into 2 tubes with 500 mL each. To one tube, 1 mg mouse anti-d-toxin (Abcam) was added. To the other tube 1 mg IgG control was added. Tubes were incubated overnight at 4uC, rotating. 20 mL Protein A/G-Plus agarose were added for 4 hours at 4uC, rotating. Beads were pelleted by centrifugation at 2,5006g for 5 minutes and washed three time in ice cold 1x PBS, spinning down after each wash as described. After the last wash, the supernatant was removed, and beads were resuspended in sample buffer for direct immunoblot. Beads were boiled for 5 minutes and loaded into 16% SDS-PAGE gel along with synthetic peptide as positive control and biotinylated protein ladder (Amersham) and run at 100V for 1.5 hours. Proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad) for 2 hours at 4uC. The membrane was blocked for 1 hour in blocking buffer (PBS, 5% nonfat milk with PSMs Cooperate with Host AMPs 3% bovine serum albumin) at room temperature. Incubation in primary antibody rabbit anti-LL-37 at 1:1000 or rabbit anti- CRAMP at 1:5000 was performed in blocking buffer overnight at 4uC. Membranes were then incubated in the appropriate horseradish peroxidase-conjugated secondary antibody goat anti- rabbit IgG for 1 hour at 1:5,000 for 1 hour at room temperature, followed by enhanced chemiluminescence (ECL) detection with the ECL detection kit (Amersham Biosciences). Cell Culture and NETs Killing Assay Neutrophils were purified from healthy donors using the PolymorphPrep TM system (Axis-Shield, Fresnius). 46104 cells were added to chamber slides in 500 uL RPMI (Biowhitaker). 26105 cells were added to 96 well plates in RPMI and 5% heat inactivated plasma. NET killing assays were performed as previously described [26], with the exception of adding 6 uM d- toxin to NETs after 4 hours of PMA stimulation. d-toxin was allowed to bind to NETs for 1 additional hour, so that the total PMA stimulation time was 5 hours. Media was replaced from NETs, GAS NZ131 was added, spun down for 10 minutes at 800 g and incubated for 30 minutes. GAS was recovered and plated for CFU enumeration. Fluorescence Immunohistochemistry of NETs Freshly isolated neutrophils in chamber slides were incubated with 25 nM PMA in DMEM for 4 hours at standard tissue cul- ture conditions to induce neutrophil extracellular trap formation. Media was carefully removed and replaced with media con- taining 5 mM d-toxin for 1 hour. NHEKs, grown on chamber slides, were incubated with 1 mM d-toxin for 1 hour. Neutrophils and NHEKs were fixed with 4% PFA for 20 minutes at room temperature, washed three time with 16 phosphate-buffered saline (PBS), blocked for 1 hour at room temperature with 3% bovine serum albumin in 1x PBS, and stained with primary antibodies mouse anti-d-toxin (Abcam), rabbit anti-LL-37, rabbit anti-CRAMP (Quality controlled biochemicals, QCB), rabbit anti-hBD2, and goat anti-hBD3 in 3% bovine serum albumin in 1x PBS overnight at 4uC. After washing the cells 3 times with PBS, the cells were stained with secondary antibodies goat anti- mouse IgG (Sigma), anti-rabbit IgG, and anti-goat IgG in 3% bovine serum albumin in 1x PBS for 1 hour at room temperature. Slides were mounted in ProLong Anti-Fade reagent (Molecular Probes). In Vivo Mouse Experiment The dorsal skin of wild-type C57BL/6 mice, 8–10 weeks old, was removed by shaving and Nair (a depilating agent). 4 mm full thickness punch biopsies were performed on the hair-free dorsal skin. 5 mL PBS or 5 mL 16 mM d-toxin was added to wounds for 30 minutes. 5 mL of 26106 CFU/ml was added to treated wounds for an additional 30 minutes. Wounds were harvested using a 6 mm punch biopsy 18 hours post-infection. Wounds were placed in 1 mL PBS with 1 mm zirconia beads. Samples were bead- beated for 1 minute, placed on ice, and bead-beated for an additional minute. Supernatant was serial diluted and plated for CFU on Todd-Hewitt Agar (THA) as well as assayed for Mip-2 levels using ELISA kit following manufacturer’s instructions (R&D Systems). Author Contributions Conceived and designed the experiments: ALC KY JEK RLG. Performed the experiments: ALC JM. Analyzed the data: ALC KMS RLG. Contributed reagents/materials/analysis tools: KMS LECA JT YL JEK VN RLG. Wrote the paper: ALC VN RLG. PLoS ONE | www.plosone.org 6 January 2010 | Volume 5 | Issue 1 | e8557