Selective antimicrobial action is provided by phenol-soluble modulins derived from Staphylococcus epidermidis, a normal resident of the skin

Abstract

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) gamma and delta produced by S. epidermidis have an alpha-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 PSMgamma and PSMdelta by S. epidermidis can benefit cutaneous immune defense by selectively inhibiting the survival of skin pathogens while maintaining the normal skin microbiome.

Figure 1. Phenol-soluble modulins have structural similarities and strongly interact with synthetic lipid membranes

(a) Sequences of PSMγ and PSMδ, highlighting tryptophan in PSMγ. (b) Helical wheel plots show sequestration of hydrophobic residues for PSMγ, PSMδ, and LL-37. Circular dichroism spectra of 20 μm PSMδ (c) or PSMγ (d) in the presence and absence of 1 mm of 2:1 POPC/POPG lipid vesicles in 20 mm potassium phosphate buffer, pH 7.3, show an α-helical structure and structural changes of PSMδ and PSMγ in the presence of lipid vesicles. Tryptophan fluorescence spectra of PSMγ in the presence and absence of 1 mm of 2:1 POPC/POPG vesicles in 20 mm potassium phosphate buffer, pH 7.3 (e), or in the presence of 5.5 M urea (f). (g) Table displaying the maximum emission wavelength of PSMγ’s tryptophan. POPC/POPG vesicles in 20 mm of potassium phosphate buffer, pH 7.3 (Kpi), cause a blue shift in the tryptophan’s maximal emission indicating an embedment of PSMγ in the lipid membrane.

Figure 2. S. epidermidis PSMs form multimeric complexes in solution

(a), Tryptophan spectra of 5, 10, and 25 μm PSMγ in 20 mm potassium phosphate buffer, pH 7.3. Tryptophan shift in phenol-soluble modulin-γ in the presence and absence of vesicles and urea. (b) Table of slope, midpoint, and $\Delta G_{\left({\mathrm{H}}_2\mathrm{O}\right)}^{\mathrm{o}}$ from unfolding curves of PSMγ at 5 and 25 μm. Increased concentrations of PSMγ results in a shift of the $\Delta G_{\left({\mathrm{H}}_2\mathrm{O}\right)}^{\mathrm{o}}$ from 1.43 to 1.57 kcal mol⁻¹, which indicates greater stability and complex formation. (c) Circular dichroism spectra of 20 μm PSMγ in the presence and absence of 5 or 10 μm PSMδ. PSMδ curves were subtracted from PSMγ curves. Curves show structural changes of PSMγ due to interaction with PSMδ.

Figure 3. Phenol-soluble modulins disrupt artificial membrane vesicles and selectively kill skin pathogens

Synthetic vesicles encapsulating ANTS (fluorophore) and DPX (quencher) show dose-dependent fluorescence (leakage) in the presence of increasing concentrations of (a) PSMγ or (b) PSMδ. Both PSMs exhibit selective dose-dependent inhibition. GAS, but not S. epidermidis (SE), is susceptible to (c) PSMγ and (d) PSMδ. Similarly, (e) PSMγ and (f) PSMδ exert selective antimicrobial killing with S. aureus (SA) but not SE. (g) TEM analysis of SE, GAS, and SA membranes after incubation with PBS, PSMγ, or CRAMP (with GAS only). GAS and SA, but not SE, showed membrane blebbing after incubation with PSMγ. The effect of PSMγ on GAS membranes was similar to the blebbing that occurred after incubation with CRAMP. Images at original magnification × 30 000 and bars represent 50 nm.

Figure 4. LL-37 and phenol-soluble modulin-γ induce similar dose-dependent changes in keratinocyte membrane permeability

Normal human epidermal keratinocytes, incubated with increasing concentrations of LL-37 or PSMγ for 18 h, were stained with propidium iodide (PI) to evaluate disruption in membrane permeability. PI uptake was measured by manually counting PI-positive fluorescent cells per microscopic field. Data represent mean ±SEM of three random fields and are representative of two independent experiments.

Figure 5. Phenol-soluble modulins cooperate with each other and host AMPs to kill GAS

(a) Co-incubation of GAS with PSMγ and PSMδ shows cooperative antimicrobial effect. Co-incubation of GAS with LL-37 and PSMγ (b) or PSMδ (c) shows cooperative antimicrobial effect. Data representative of two individual experiments performed in triplicate. Data are mean ±SEM of a single experiment performed in duplicate and are representative of two independent experiments.

Figure 6. Phenol-soluble modulins selectively kill GAS but not S. epidermidis on the skin’s surface

(a) Survival of S. epidermidis on mouse skin treated with PSMs at 0.16 or 0.32 nmol. Addition of PSMs on skin did not affect colonization of S. epidermidis. (b) GAS survival on skin was greatly reduced by the addition of PSMs at 0.16 or 0.32 nmol. Data are means of two independent experiments performed in triplicate. P-values were calculated using ANOVA with Bonferroni post hoc test when applicable.