A novel lipopeptide from skin commensal activates TLR2/CD36-p38 MAPK signaling to increase antibacterial defense against bacterial infection

Abstract

Staphylococcus epidermidis (S.epidermidis) plays important protective roles by directly producing or by stimulating hosts to produce antimicrobial peptides (AMPs) against pathogenic infections. Although several AMPs from S.epidermidis have been identified, molecules that stimulate hosts to produce AMPs remain largly unknown. Here we demonstrate that a new lipopeptide (named LP01) purified from S.epidermidis culture media has a unique structure with heneicosanoic acid (21 carbons) binding to lysine(11) of a peptide chain. In vitro LP01 increased the expression of β-defensin 2(hBD2) and hBD3 in neonatal human epidermal keratinocytes(NHEK), leading to increased capacity of cell lysates to inhibit the growth of S.aureus. In vivo LP01 induced the expression of mouse β-defensin 4(mBD4) to decrease the survival of local S.aureus in skin and systemic S.aureus survival in liver. The induction of beta-defensins by LP01 was dependent on TLR2 as Tlr2-deficient mice had decreased mBD4. Furthermore, knockdown of CD36 decreased the expression of hBD2 and hBD3, and p38 MAPK inhibitor significantly inhibited the expression of hBDs induced by LP01.Taken together, these findings demonstrate that lipopeptide LP01 from normal commensal S.epidermidis increases antimicrobial peptide hBD2 and hBD3 expression via the activation of TLR2/CD36-p38 MAPK, thus enhancing antimicrobial defense against pathogenic infections.

Figure 1. The identification of the lipopeptide from Staphylococcus epidermidis.

A. The analysis of the lipopeptide from S.epidermidis by thin-layer chromatography. Duplicate samples were loaded on TLC plate and molecules with different hydrophobicity were separated. Water was used to show hydrophobicity of lipopeptides and ninhydrin was used to show that the lipopeptide contains amino acids. The arrows indicate LP01. B. Q-TOF MS/MS analysis of lipopeptide in positive-ion model. Q-TOF MS/MS analysis showed that the amino acid sequence of the lipopeptide was DIISTIGDLVKWIIDTVIIDATE. C&D. Two possible structures of the lipopeptide. Aspartic acid (D¹) at N-terminus and lysine (K¹¹) are two amino acids with free NH₃ ⁺, and free -COOH of heneicosanoic acid might react with NH₃ ⁺ to form CO–NH. Thereby heneicosanoic acid may bind to ¹D or ¹¹K of the peptide chain.

Figure 2. LP01 induces β-defensin expression in primary keratinocytes. A&B.

Quantification of hBD2 and hBD3 mRNA expression in NHEKs treated with LP01 or SECM. C&D. hBD2 and hBD3 protein expression in cell lysates of NHEKs treated with LP01 by ELISA. E&F. Quantification of mBD4 and mBD14 mRNA expression stimulated with 15 µg/mL LP01 in primary murine keratinocytes. Primary murine keratinocytes were isolated from C57BL/6 mice. G&H. Quantification of hBD2 and hBD3 mRNA expression in NHEKs stimulated by LP01 with shortened peptide chain. I&J. Quantification of hBD2 and hBD3 mRNA expression in NHEKs stimulated by LP01 with shortened fatty acid chain. *P<0.05, **P<0.01 and ***P<0.001, n.s., no significance. P values were determined by one-way ANOVA or two-tailed t test. Data are the means ± SEM of n = 3 and representative of two independent experiments.

Figure 3. LP01 increases antibacterial activity of keratinocytes.

The growth of S.aureus (A), E.coli DH5a (B), P.acnes (C) and S.epidermidis (D) after exposure to lysates of LP01-treated neonatal human epidermal keratinocytes or the growth of S.aureus after exposure to cell culture medium of LP01-treated NHEKs (E). The bacterial were serially diluted 10-fold with PBS and then counted by colony formation. *P<0.05; n.s., no significance. P values were determined by t-test. All data are representative of three independent experiments with n = 3 and are means ± SEM.

Figure 4. LP01 protects mice from S.aureus infection.

A. Photograph of skin lesions caused by S.aureus at 3 days after S.aureus injection. B. ImageJ analysis of the lesional size of A. C. ImageJ analysis of the lesional size of PBS- or scrambled lipopeptide-treated mice. Local S.aureus survival in skin (D and E) and systemic S.aureus survival in liver (F) and spleen (G) of PBS- or scrambled lipopeptide- or LP01- pretreated mice. **P<0.01; ***P<0.001. P values were determined by two-tailed t test or two-way ANOVA. All data are the means ± SEM of n = 6 and representative of two independent experiments.

Figure 5. The induction of beta-defensins by LP01 is dependent on TLR2.

A, B. The expression of hBD2 and hBD3 in NHEKs treated with 15 µg/mL of LP01 in the presence or absence of TLR2 inhibitor OxPAPC. C. mBD4 expression in mouse ears 24 h after injection by 2 mg/kg of LP01. D. Photograph of skin lesions caused by S.aureus at 3 days after S.aureus injection in Tlr2^+/+ and Tlr2^–/– mice. E. S.aureus survival in skin of PBS- and LP01- pretreated Tlr2^+/+ and Tlr2^–/– mice. F–G. Quantification of hBD2 and hBD3 expression in NHEK cells stimulated with LP01 after CD14 or CD36 was silenced. *P<0.05, ***P<0.001. P values were determined by one-way or two-way ANOVA. All data are representative of two independent experiments with n = 3–6 and are the means ± SEM.

Figure 6. The activation of p38 MAPK is required for the induction of beta-defensins by LP01.

A&B. TLR2 inhibitor OxPAPC inhibited p38 phosphorylation induced by 15 µg/mL of LP01 in NHEK cells. C&D. p38 MAPK inhibitor SB202190 inhibited p38 phosphorylation induced by 15 µg/mL of LP01 in NHEK cells. E&F. p38 MAPK inhibitor SB202190 completely inhibited hBD2 and hBD3 expression induced by 15 µg/mL of LP01 in NHEK cells. G. p38 MAPK inhibitor SB202190 completely inhibited mBD4 expression induced by 15 µg/mL of LP01 in murine primary keratinocytes. ***P<0.001. P values were determined by one-way ANOVA. Data are the means ± SEM of n = 3 and representative of two to three independent experiments.