Antimicrobial peptides serve as a first line of innate immune defense against invading organisms such as bacteria and viruses. In this study, we hypothesized that peptides produced by a normal microbial resident of human skin, Staphylococcus epidermidis, might also act as an antimicrobial shield and contribute to normal defense at the epidermal interface. We show by circular dichroism and tryptophan spectroscopy that phenol-soluble modulins (PSMs) γ and δ produced by S. epidermidis have an α-helical character and a strong lipid membrane interaction similar to mammalian AMPs such as LL-37. Both PSMs directly induced lipid vesicle leakage and exerted selective antimicrobial action against skin pathogens such as Staphylococcus aureus. PSMs functionally cooperated with each other and LL-37 to enhance antimicrobial action. Moreover, PSMs reduced Group A Streptococcus (GAS) but not the survival of S. epidermidis on mouse skin. Thus, these data suggest that the production of PSMγ and PSMδ by S. epidermidis can benefit cutaneous immune defense by selectively inhibiting the survival of skin pathogens while maintaining the normal skin microbiome.

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 δ-toxin (PSMγ) is normally present in the epidermis and sparsely in the dermis of human skin using immunohistochemistry. Synthetic δ-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), δ-toxin cooperated with CRAMP, hBD2, and hBD3. In whole blood, addition of δ-toxin exerted a bacteriostatic effect on GAS, and in NETs, δ-toxin increased their killing capacity against this pathogen. Coimmunoprecipitation and tryptophan spectroscopy demonstrated direct binding of δ-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 δ-toxin. Thus, these data suggest that S. epidermidis–derived δ-toxin cooperates with the host-derived antimicrobial peptides in the innate immune system to reduce survival of an important human bacterial pathogen.

Refolding curves of the integral membrane protein Outer Membrane Protein A (OmpA) were measured to determine the conformational stabilities of this model system for membrane protein folding. Wild-type OmpA exhibits a free energy of unfolding ( ) of 10.5 kcal/mol. Mutants, containing a single tryptophan residue at the native positions 7, 15, 57, 102, or 143, are less stable than wild-type OmpA, with values of 6.7, 4.8, 2.4, 4.7, and 2.8 kcal/mol, respectively. The trend observed here is discussed in terms of non-covalent interactions, including aromatic interactions and hydrogen bonding. The effect of the soluble tail on the conformational stability of the transmembrane domain of OmpA was also investigated via truncated single-trp mutants; values for four of the five truncated mutants are greater by >2.7 kcal/mol relative to the full-length versions, suggesting that the absence of the soluble domain may destabilize the unfolded transmembrane domain. Finally, dynamic light scattering experiments were performed to measure the effects of urea and protein on vesicle size and stability. Urea concentrations greater than 1 M cause an increase in vesicle size, and these diameters are unaltered in the presence of protein. These dynamic light scattering results complement the fluorescence studies and illustrate the important effects of vesicle size on protein conformational stability. KeywordsOuter Membrane Protein A; membrane protein folding; tryptophan fluorescence; conformational stability; dynamic light scattering; refolding curves Membrane proteins adopt unique three-dimensional structures in cell membranes to be biologically active. Like soluble proteins, membrane proteins that form aberrant structures lead to a number of prevalent diseases, such as cystic fibrosis and type II diabetes (1,2). A great deal of experimental and theoretical research has recently been devoted towards elucidating the molecular mechanisms, thermodynamics, and kinetics of protein folding in lipid bilayers (3-7). Despite these efforts, our knowledge of membrane protein folding remains inferior to our fundamental understanding of soluble protein folding. A number of factors contribute to the inherent difficulties in the study of membrane protein folding. First, in contrast to soluble proteins, a limited number of membrane protein systems are available to serve as models for membrane protein folding (5)(6)(7)(8). An additional challenge is the membrane itself; lipid bilayers *Corresponding author: Judy E. Kim, Assistant Professor, Department of Chemistry & Biochemistry, University of California, San Diego, 9500 Gilman Drive MC 0314, La Jolla, CA 92093-0314, judyk@ucsd.edu, Phone: (858) Fax: (858) . Supporting Information Available: A supporting figure of gel-shift refolding/unfolding curves. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 August 10. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript are complex systems, with a high degree of chemical heterogeneity (6...

Aromatic amino acids of membrane proteins are enriched at the lipid-water interface. The role of tryptophan on the folding and stability of an integral membrane protein is investigated with ultraviolet resonance Raman and fluorescence spectroscopy. We investigate a model system, the β-barrel outer membrane protein A (OmpA), and focus on interfacial tryptophan residues oriented toward the lipid bilayer (trp-7, trp-170, or trp-15) or the interior of the β-barrel pore (trp-102). OmpA mutants with a single tryptophan residue at a nonnative position 170 (Trp-170) or a native position 7 (Trp-7) exhibit the greatest stability, with Gibbs free energies of unfolding in the absence of denaturant of 9.4 and 6.7 kcal/mol, respectively. These mutants are more stable than the tryptophan-free OmpA mutant, which exhibits a free energy of unfolding of 2.6 kcal/mol. Ultraviolet resonance Raman spectra of Trp-170 and Trp-7 reveal evolution of a hydrogen bond in a nonpolar environment during the folding reaction, evidenced by systematic shifts in hydrophobicity and hydrogen bond markers. These observations suggest that the hydrogen bond acceptor is the lipid acyl carbonyl group, and this interaction contributes significantly to membrane protein stabilization. Other spectral changes are observed for a tryptophan residue at position 15, and these modifications are attributed to development of a tryptophan-lipid cation-π interaction that is more stabilizing than an intraprotein hydrogen bond by ∼2 kcal/mol. As expected, there is no evidence for lipid-protein interactions for the tryptophan residue oriented toward the interior of the β-barrel pore. These results highlight the significance of lipid-protein interactions, and indicate that the bilayer provides more than a hydrophobic environment for membrane protein folding. Instead, a paradigm of lipid-assisted membrane protein folding and stabilization must be adopted.

The vibrational structure of native anchoring tryptophan (Trp) and tyrosine residues in an integral membrane protein, bacterial outer membrane protein A (OmpA), have been investigated using UV resonance Raman (UVRR) spectroscopy for the first time. Spectra of native OmpA, a single-Trp mutant, and a Trp-less mutant were recorded in folded and unfolded states, and reveal significant changes in tryptophan structure and local environment. Salient alterations upon folding include loss of hydrogen-bonding character of indole N1H, evidenced by a shift in W17 frequency from 874 and 878 cm(-1), and growth in hydrophobicity of the local tryptophan environment, supported by increase in the ratio I1361/I1340. In addition to these site-specific changes in a single tryptophan residue, modification of the vibrational structure of the remaining native tryptophan and tyrosine amino acids is also evident. Finally, the UVRR data presented here indicate that the structures of OmpA folded in vesicle and folded in detergent may differ, and provide important foundations for ongoing studies of membrane protein folding.