Staphylococcus epidermidis Antimicrobial d-Toxin (Phenol-Soluble Modulin-c) Cooperates with Host Antimicrobial Peptides to Kill Group A Streptococcus Anna L. Cogen1,2, Kenshi Yamasaki1, Jun Muto1, Katheryn M. Sanchez3, Laura Crotty Alexander4, Jackelyn Tanios1, Yuping Lai1, Judy E. Kim3, Victor Nizet4,5, Richard L. Gallo1,4* 1 Division of Dermatology, University of California San Diego, San Diego, California, United States of America, 2 Department of Bioengineering, University of California San Diego, San Diego, California, United States of America, 3 Department of Chemistry and Biochemistry, University of California San Diego, San Diego, California, United States of America, 4 Department of Pediatrics, School of Medicine, University of California San Diego, San Diego, California, United States of America, 5 Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, San Diego, California, United States of America Abstract Antimicrobial peptides play an important role in host defense against pathogens. Recently, phenol-soluble modulins (PSMs) from Staphylococcus epidermidis (S. epidermidis) were shown to interact with lipid membranes, form complexes, and exert antimicrobial activity. Based on the abundance and innocuity of the cutaneous resident S. epidermidis, we hypothesized that their PSMs contribute to host defense. Here we show that S. epidermidis d-toxin (PSMc) is normally present in the epidermis and sparsely in the dermis of human skin using immunohistochemistry. Synthetic d-toxin interacted with neutrophil extracellular traps (NETs) and colocalized with cathelicidin while also inducing NET formation in human neutrophils. In antimicrobial assays against Group A Streptococcus (GAS), d-toxin cooperated with CRAMP, hBD2, and hBD3. In whole blood, addition of d-toxin exerted a bacteriostatic effect on GAS, and in NETs, d-toxin increased their killing capacity against this pathogen. Coimmunoprecipitation and tryptophan spectroscopy demonstrated direct binding of d-toxin to host antimicrobial peptides LL-37, CRAMP, hBD2, and hBD3. Finally, in a mouse wound model, GAS survival was reduced (along with Mip-2 cytokine levels) when the wounds were pretreated with d-toxin. Thus, these data suggest that S. epidermidis–derived d-toxin cooperates with the host-derived antimicrobial peptides in the innate immune system to reduce survival of an important human bacterial pathogen. Citation: Cogen AL, Yamasaki K, Muto J, Sanchez KM, Crotty Alexander L, et al. (2010) Staphylococcus epidermidis Antimicrobial d-Toxin (Phenol-Soluble Modulin-c) Cooperates with Host Antimicrobial Peptides to Kill Group A Streptococcus. PLoS ONE 5(1): e8557. doi:10.1371/journal.pone.0008557 Editor: Frank R. DeLeo, National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States of America Received September 2, 2009; Accepted December 10, 2009; Published January 5, 2010 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This work was supported by National Institutes of Health grants P01HL057345, R56AI083358, and R01AR45676, and a VA Merit award (RLG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rgallo@ucsd.edu Introduction Antimicrobial peptides (AMPs) are critically important in the host’s defense against infections. In particular, cathelicidins are an important class of mammalian AMPs as cathelicidin-deficient mice have enhanced susceptibility to skin and other infections, providing evidence that AMPs are critical to the innate immune defense[1,2,3,4]. AMPs have been shown to be important in multiple cell types, including, but not limited to, macrophages, neutrophils, mast cells, and epithelial cells such as skin keratino- cytes. Pathogens come into contact with AMPs via host secretory mechanisms and phagocytosis. In addition, it has recently been suggested that microbes can also be killed by binding to insoluble released nucleic acids, histones and associated with AMPs. This structure can be produced by mast cells or neutrophils, where it is known as neutrophil extracellular traps (NETS) [5,6]. It has been suggested that NETs entwine and kill circulating pathogens in a web of DNA, histones, and AMPs. Cathelicidins have multiple functionalities. The peptides provide protection through immuno-modulation, inducing chemotaxis, and through direct pore-formation of a broad range of infectious agents including fungi, viruses, and bacteria. LL-37, a active peptide form of human cathelicidin, is suggested to form an amphipathic a-helix that subsequently inserts into negatively charged membranes forming veritable holes [7]. Previous reports suggest that cellular perforation requires multiple LL-37 peptides which coalesce to form oligomeric complexes [8]. Although complexes have been shown to form, it is not known as to whether LL-37 physically forms heterologous complexes with other antimicrobial a-helical peptides. Staphylococcus epidermidis (S. epidermidis), the most prevalent of many cutaneous resident microflora, is generally innocuous. Several studies have identified S. epidermidis as a common colonizer of healthy human skin [9,10] and mouse skin [11]. Much like the gut, the microbiota of the skin is hypothesized to play a mutually beneficial role in the cutaneous niche. We have previously demonstrated that peptides produced by S. epidermidis exhibit antimicrobial properties, potentially acting as an additional antimicrobial shield. The physico-chemical properties of the S. epidermidis PSM, d-toxin, is comparable to those properties of the Staphylococcus aureus d-toxin. The peptides are similarly a-helical and form complexes, yet the S. aureus d-toxin, unlike the S. epidermidis d-toxin, lacks antimicrobial activity [12,13,14]. The PLoS ONE | www.plosone.org 1 January 2010 | Volume 5 | Issue 1 | e8557