PSMs Cooperate with Host AMPs Figure 4. d-toxin physically binds to host derived antimicrobial peptides. Synthetic 1 mM LL-37 or 1 mM CRAMP was added to S. epidermidis supernatants. a, d-toxin was precipitated and immunoblotted for LL-37 or CRAMP. Immunoblots show co-precipitation of LL-37 or CRAMP with d- toxin indicating binding. The left and right panels were stained for LL-37 or CRAMP, respectively. The LL-37 or CRAMP positive control standards are shown in the left lane of each panel. The middle and right lanes show relative quantities of LL-37 or CRAMP that precipitated with d-toxin using anti d-toxin (right lane) or IgG control to anti d-toxin (middle lane) b, emission spectra of d-toxin’s tryptophan in buffer or in the presence of LL-37, CRAMP, hBD2, or hBD3. c, table of maximal wavelength emission and shift in wavelength upon addition of host antimicrobial peptide. Data are representative of 2 individual experiments. doi:10.1371/journal.pone.0008557.g004 upon incubation with LL-37 and CRAMP and decreased upon incubation with hBD2 and hBD3 (Figure 4b). Similarly, we observed that d-toxin undergoes a conformation change in the presence of genomic keratinocyte DNA. Overall, these data illustrate that d-toxin directly interacts with, and binds to, host AMPs. d-Toxin Reduces GAS Survival and Inflammation in Mouse Wounds As we have shown that PSMs may contribute to whole blood and neutrophil killing of GAS, we sought to determine if PSMs present in a wound could have a similar protective effect against bacteria, we utilized a mouse wound model. d-toxin or PBS control was added to 4 mm full-thickness fresh mouse wounds. After only 30 minutes, GAS was added to the wounds to mimic an infected wound. After 18 hours, the infected wounds and surrounding fascia were harvested. GAS survival was significantly decreased in mouse wounds pretreated with d-toxin but not PBS (Figure 5a). Paralleling the GAS infection, Mip-2 (CXCL2) was significantly decreased in mouse wounds treated with d-toxin (Figure 5b). These data suggest that d-toxin reduces GAS survival in vivo and may contribute to the innate immune system. Discussion We have previously demonstrated that the PSMd and PSMc (d-toxin) from S. epidermidis exhibit antimicrobial activity over pathogens. This activity appeared to result from membrane disruption, a trait common among many antimicrobials [21]. These data support studies suggesting a role of S. aureus PSMs in virulence, as the peptides induce pore formation in cells such as neutrophils [19]. Although the PSMs produced by Staphylococcal sp. PLoS ONE | www.plosone.org 4 January 2010 | Volume 5 | Issue 1 | e8557