Figure 2. d-toxin induces NET formation. Freshly isolated human neutrophils were cultured with media only (unstimulated), 25 nM PMA, or 6 mM d-toxin for 4 hours. Like PMA, d-toxin induced NET formation as seen by the DNA strands (white arrows). doi:10.1371/journal.pone.0008557.g002 formation of NETs in vitro. Here, freshly isolated human neutrophils were stimulated with d-toxin, or PMA as a positive control. Like PMA, d-toxin was able to induce NETs formation (Figure 2). Thus, these data demonstrate that d-toxin derived from S. epidermidis is deposited on the skin and induces formation of and interacts with NETs. d-Toxin Enhances Endogenous Antimicrobial Activity To determine whether d-toxin was able to cooperatively enhance bacterial killing of host AMPs, GAS survival was assessed in the presence of d-toxin and CRAMP, hBD2, and hBD3. The presence of d-toxin and low concentrations of host AMPs increased GAS killing in a cooperative manner (Figure 1b and 1c; Figure 3a). Since d-toxin enhanced the activity of AMPs, we investigated whether d-toxin could enhance antimicrobial activity of whole blood. For this, d-toxin was added to whole blood containing GAS at concentrations below the calculated in vitro MIC against this pathogen. Over time, GAS was able to grow in blood without d-toxin, bacteriostasis was achieved in blood containing 2–4 mM d-toxin (Figure 3d), As d-toxin’s ability to increase bacterial killing by whole blood may in part be due to the contribution of neutrophil extracellar traps (NETs), and d-toxin and closely related peptides have been known to lyse red blood cells and PSMs Cooperate with Host AMPs neutrophils [19], we next examined if the activity of d-toxin in the presence of neutrophils is aided by DNA forming the NET. Treatment of NETs and d-toxin with DNAse eliminated the capacity of both the NETs alone, or NETs with d-toxin, to inhibit GAS survival (Figure 3e). Hence, the addition of d-toxin to whole blood or to NETs increases their killing capacity. d-Toxin Physically Binds to Host-Derived AMPs Since previous studies suggest that AMPs form heterodimeric and homodimeric complexes [21], and because we found that d-toxin colocalizes with LL-37 in NETs, we hypothesized that d-toxin physically binds to the host derived AMPs. In order to determine whether d-toxin could associate with LL-37 and CRAMP, synthetic host AMPs were added to S. epidermidis cell- free, stationary phase supernatant, naturally containing d-toxin. Precipitation of d-toxin followed by an immunoblot for LL-37 and CRAMP demonstrated that d-toxin physically bound to the host derived peptides (Figure 4a). In order to further confirm interaction between d-toxin and the host AMPs, tryptophan spectroscopy was performed on d-toxin in the presence and absence of host AMPs. Unlike d-toxin, that has a tryptophan at position 15, LL-37, CRAMP, hBD2, and hBD3 are spectroscopically silent because they lack a tryptophan. Due to the spectroscopic silence of the host AMPs, a maximal tryptophan emission shift in d-toxin during incubation with a host AMP would signify an interaction with and structural change of d-toxin. To determine the ability of the host AMPs to interact with d-toxin, the peptide was incubated with host AMPs and tryptophan emission was monitored upon excitation at 290 nm (Figure 4b and 4c). We found that d-toxin maximal tryptophan emission shifted from 341 nm to 344 nm in the presence of LL-37 and CRAMP, indicating a greater exposure of the tryptophan to the aqueous environment. Conversely, in the presence of hBD2 and hBD3, d-toxin’s maximal tryptophan emission shifted from 341 nm to 339 nm, indicating that the tryptophan resides in a more hydrophobic and embedded environment (Figure 4b and 4c). In addition, we observed that the emission spectra intensity increased Figure 3. d-toxin cooperates with host antimicrobial peptides and enhances whole blood and NET killing of GAS. d-toxin cooperates with host antimicrobial peptides CRAMP (a), hBD2 (b), and hBD3 (c) to kill GAS. d, in whole blood, increasing concentrations of synthetic d-toxin rendered GAS bacteriostatic. e, d-toxin added to NETs showed greater GAS killing than NET killing alone. The effect was abrogated by DNase. Data are representative of 2 individual experiments performed in triplicate. *p,0.05. Student’s t-test. doi:10.1371/journal.pone.0008557.g003 PLoS ONE | www.plosone.org 3 January 2010 | Volume 5 | Issue 1 | e8557