S-Layer Protein of Lactobacillus helveticus SBT2171 Promotes Human β-Defensin 2 Expression via TLR2-JNK Signaling

Abstract

Antimicrobial peptides that contribute to innate immunity are among the most important protective measures against infection in many organisms. Several substances are known to regulate the expression of antimicrobial peptides. In this study, we investigated the factors in lactic acid bacteria (LAB) that induce antimicrobial peptide expression in the host. We found that Lactobacillus helveticus SBT2171 (LH2171) induced the expression of human β-defensin (hBD)2 in Caco-2 human colonic epithelial cells. Specifically, surface layer protein (SLP) of LH2171 stimulated hBD2 expression by activating c-Jun N-terminal kinase (JNK) signaling via Toll-like receptor (TLR)2 in Caco-2 cells. SLPs extracted from other lactobacilli similarly increased hBD2 expression, suggesting that this stimulatory effect is common feature of Lactobacillus SLPs. Interestingly, Lactobacillus strains that strongly induced hBD2 expression also potently activated JNK signaling. Thus, upregulation of hBD2 induced by TLR2-JNK signaling contributes to protection of the host against infection.

Keywords: Lactobacillus helveticus SBT2171; antimicrobial peptide; c-Jun N-terminal kinase; human β-defensin 2; lactic acid bacteria; surface-layer protein; toll-like receptor 2.

FIGURE 1

hBD2 upregulation in epithelial cells by strain LH2171 and its components. Cells were cultured with LH2171 or its cellular components [DNA, cell wall (CW), peptidoglycan (PGN), Surface layer protein (SLP)] (A,C,D), with LH2171 (non-treated) or SLP-deficient LH2171 (LiCl-treated) (B), and with SLP, heated SLP, or proteinase K-treated SLP (proK-SLP) (F). hBD2 mRNA levels were evaluated by quantitative real time-PCR. Each experiment was performed in triplicate; data are shown as mean ± SD (A–D,F). Samples were added at a concentration of 10 μg/ml (A,B,F). ^∗∗∗P < 0.001 vs. LH2171 (one-way ANOVA and Dunnett’s post hoc test) (A). ^∗∗∗P < 0.001 according to the Student’s t-test (B). Values not sharing a common letter are significantly different (P < 0.05 by one-way ANOVA and Tukey–Kramer post hoc test) (C,D,F). SDS–PAGE profile of digested SLP (E).

FIGURE 2

Characterization of LH2171 SLP. Deduced amino acid sequence of LH2171 SLP (full length). Shaded area (residues 1–30) represent the signal peptide (A). SDS–PAGE profile of purified and recombinant SLP (B). Caco-2 cells were cultured with LH2171 or SLP. hBD2 mRNA levels were evaluated by quantitative real time-PCR. Each experiment was performed in triplicate; data are shown as mean ± SD. Values not sharing a common letter are significantly different (P < 0.05 by one-way ANOVA and Tukey–Kramer post hoc test) (C).

FIGURE 3

Mechanisms of hBD2 upregulation by LH2171 SLP. Caco-2 cells were pre-incubated with anti-TLR2 antibody (a-TLR2), isotype control antibody (IgA2) (A,C,D), or the indicated inhibitor (SB202190 for p38, SP600125 for JNK, PD98059 for ERK, and JSH-23 for NF-κB) (B) and then treated with LH2171 SLP. hBD2 mRNA levels were evaluated by quantitative real time-PCR (A,B), and protein levels of total JNK and phospho-JNK (P-JNK) (C) or total c-Jun and phospho-c-Jun (D) in total cell lysates were analyzed by western blotting. Each experiment was performed in triplicate; data are shown as mean ± SD (A–D). ^∗∗P < 0.01 and ^∗∗∗P < 0.001 vs. IgA2 + SLP (A) or SLP (B) (one-way ANOVA and Dunnett’s post hoc test). Values not sharing a common letter are significantly different (P < 0.05 by one-way ANOVA and Tukey–Kramer post hoc test) (C,D).

FIGURE 4

Comparison of SLP activity between LH2171 and other Lactobacillus strains. Caco-2 cells were cultured with each SLP, and hBD2 mRNA levels were evaluated by quantitative real time-PCR. Each experiment was performed in triplicate; data are shown as mean ± SD. Values not sharing a common letter are significantly different (P < 0.05 by one-way ANOVA and Tukey–Kramer post hoc test) (A). Protein levels of total JNK and phospho-JNK (P-JNK) (B) or total c-Jun and phospho-c-Jun (C) in total cell lysates were analyzed by western blotting.

FIGURE 5

Phylogenetic analysis of Lactobacillus SLPs based on primary amino acid sequences. Amino acid sequences of the predicted mature forms of LH2171 SLP, L. helveticus JCM1120^T S-layer protein (accession no. CAB46985.1), L. acidophilus JCM1132^T slpA (accession no. CAA50535.1), L. helveticus CNRZ892 slpH1 (accession no. CAA62606.1), and L. helveticus CP790 prtY (accession no. BAA86287.1) were analyzed with Clustal Omega and a phylogenetic tree was constructed with TreeView X.

FIGURE 6

Schematic illustration of the mechanism underlying hBD2 upregulation by LH2171SLP. LH2171 SLP activates JNK signaling via TLR2 stimulation, leading to increased expression of hBD2.