Figure 5. d-toxin reduces GAS survival and inflammation in mouse wounds. d-toxin or PBS was added to mouse wounds. After 30 minutes, the treated wounds were challenged with GAS. Wounds were excised, homogenized and plated for GAS CFU/ml (a) or Mip-2 protein levels by ELISA (b). Data are representative of 3 individual experiments performed with n = 3–4. *p,0.05, Student’s t-test. doi:10.1371/journal.pone.0008557.g005 are clearly pore-forming, the impact on the host is likely in part contingent upon the nature of the bacterium and area of residence in the host. The antimicrobial effect of PSMs on skin pathogens and enhancement of host derived AMPs suggests a role for S. epidermidis in the innate immune system of the skin [15]. Other staphylococ- cal species produce additional or different PSMs that may have other functions or differ in their activity [19]. AMPs have been previously shown to be crucial components of the innate immune system, as illustrated by the susceptibility of Camp2/2 mice to GAS [2]. In addition, bacteria have been shown to benefit the human host, as suggested by the hygiene hypothesis and other studies on the gut microflora. Here, we suggest that S. epidermidis benefits the host and provides an additional layer of protection against skin pathogens. S. epidermidis rather than acting alone, is able to kill pathogens by complementing the host’s innate immune system. This close interaction of d-toxin with the host AMPs insinuates the presence of a mutual relationship between host and bacteria. We have shown that d-toxin is deposited in the epidermis and sparsely in the dermis in normal healthy human skin. During scenarios such as injury, d-toxin may then be able to interact with a variety of cell types including neutrophils. Neutrophils expressing AMPs and forming NETs facilitate eradication of potentially dangerous bacteria [6]. In fact, it has also been shown that injury PSMs Cooperate with Host AMPs itself increases AMP production and it is not yet known if injury also induces NETs in circulating neutrophils. As S. epidermidis lives on the skin, injury would bring the bacterium and products in close proximity to the innate immune cells. In this occasion, d-toxin may increase antimicrobial NET activity and likelihood of pathogen eradication. The NETs illustrate that the host AMPs and d-toxin intertwine with host DNA. The DNA binding that occurs by AMPs to the NETs may be more than an artifact of charge attraction, but rather suggests a novel role for AMPs. Many studies demonstrate that AMPs cause transcriptional and translational changes in cells. Of course, AMPs may bind receptors, or affect signaling pathways, but these peptides, including PSMs, may directly affect host transcription at the level of DNA binding. Such DNA binding capacity of d-toxin also suggests a potential role for S. epidermidis PSMs in bacterial gene regulation, although S. aureus PSMs are not implicated in such [22]. Phenomena of heterologous complex formation and synergy of AMPs from different species have been explored only minimally. Yet AMP synergy has been well documented between AMPs from the same species [16,17]. Though, one study demonstrated that bacteriocin synergistically enhanced pleurocidin activity against E. coli [18]. Although these synergy studies suggest an important relationship between different peptides, a physical interaction between the peptides has not yet been proven. Here, we have shown for the first time, that AMPs from the resident S. epidermidis interacts physically with the host AMPs. Co-immunoprecipitation of d-toxin and CRAMP (or LL-37), along with the tryptophan spectroscopy demonstrated a direct AMP binding. Interestingly, the precipitated CRAMP peptide, ran slightly higher than the synthetic peptide positive control. This may have occurred due to the binding of d-toxin to CRAMP causing a slower migration. To show that S. epidermidis d-toxin may contribute to the antimicrobial response in vivo, in a scenario in which the peptide enters a wound, we added synthetic d-toxin to mimic a wound in human skin. Significantly less GAS survived in the mouse wounds that were pretreated with d-toxin as compared to the vehicle (PBS) control. We also evaluated the levels of a proinflammatory cytokine Mip-2, as a monitor of infection severity. Mip-2, like the number of GAS recovered, indicated an increase in bacterial clearance and general reduction of an inflammatory infection. d-toxin has been shown to actually induce rather than suppress proinflammatory signaling [23,24], thus indicating that the reduction in Mip-2 purely parallels the GAS burden. These data suggest that d-toxin decreases GAS survival in a wound environment. The mechanism responsible for decreased survival of GAS is not clear, but may include neutrophil killing, cooperation with the host AMPs, a surfactant- based effect or activation of the host’s innate immune response. Overall, we show here that d-toxin is able to bind to host AMPs. Moreover, d-toxin enhances blood and NET killing of GAS. Finally, the functional reduction of GAS survival in a mouse wound pretreated with d-toxin suggests a beneficial innate immune role and mutual role for S. epidermidis, a common constituent of the skin microbiome. Materials and Methods Peptides Formylated d-toxin was commercially synthesized and purified by HPLC (Quality Controlled Biochemicals, Hopkinton, MA). Mouse cathelicidin-related antimicrobial peptide (CRAMP) and LL-37 were commercially synthesized and purified by HPLC as previously described [25]. Human b-defensins hBD2 and hBD3 were commercially synthesized and HPLC purified (Peptide International). PLoS ONE | www.plosone.org 5 January 2010 | Volume 5 | Issue 1 | e8557