The Triacylated ATP Binding Cluster Transporter Substrate-binding Lipoprotein of Staphylococcus aureus Functions as a Native Ligand for Toll-like Receptor 2

Abstract

Some synthetic lipopeptides, in addition to native lipoproteins derived from both Gram-negative bacteria and mycoplasmas, are known to activate TLR2 (Toll-like receptor 2). However, the native lipoproteins inherent to Gram-positive bacteria, which function as TLR2 ligands, have not been characterized. Here, we have purified a native lipoprotein to homogeneity from Staphylococcus aureus to study as a native TLR2 ligand. The purified 33-kDa lipoprotein was capable of stimulating TLR2 and was identified as a triacylated SitC lipoprotein, which belongs to a family of ATP binding cluster (ABC) transporter substrate-binding proteins. Analyses of the SitC-mediated production of cytokine using mouse peritoneal macrophages revealed that the SitC protein (3 nm) induced the production of tumor necrosis factor-alpha and interleukin-6. Moreover, analysis of knock-out mice showed that SitC required TLR2 and MyD88, but not TLR1 or TLR6, for the induction of cytokines. In addition to the S. aureus SitC lipoprotein, we purified two other native ABC transporter substrate-binding lipoproteins from Bacillus subtilis and Micrococcus luteus, which were both shown to stimulate TLR2. These results demonstrate that S. aureus SitC lipoprotein is triacylated and that the ABC transporter substrate-binding lipoproteins of Gram-positive bacteria function as native ligands for TLR2.

FIGURE 1.

Identification of S. aureus 33-kDa TLR2-stimulating protein. A–H, the CHO/hCD14/hTLR2 reporter cell line harboring the NF-κB-driven hCD25 gene was stimulated for 16 h, and the resulting surface expression of CD25 was analyzed by flow cytometry. Bacteria were precultured at 30 °C in A and B or at 37 °C in C–E. The stimulation was induced by S. aureus whole cells of the parent RN4220 (A and C), by its LTA-deficient ΔltaS mutant (B), by its lipoprotein maturation-deficient Δlgt mutant harboring an empty plasmid (D), and by the Δlgt mutant harboring the pSlgt plasmid (E). In A–E, the ratio of CHO cells to bacterial cells was 1:10 (blue) and 1:100 (magenta) in (A) and (B) and 1:5 (blue) and 1:50 (magenta) in C–E. Also, the CHO/hCD14/hTLR2 cells were stimulated with the soluble PGN fraction (F), with the Triton X-114 detergent fraction (G), and with its aqueous fraction after Triton X-114 treatment (H). The amount of soluble PGN used for cell stimulation was 0.7 μg/ml (blue) or 7 μg/ml (magenta) in F. The Triton X-114 fractions containing 24 ng (blue) or 240 ng (magenta) proteins were treated to cells (G). One-fold (blue) or 10-fold (magenta) aqueous fractions of soluble PGN amounts were used to treat cells (H). The black line with gray area indicates mock treatment (A–H). I, SDS-PAGE analyses of the soluble PGN fraction (60 μg dry weight; lane 1), aqueous phase (lane 2), and Triton X-114 detergent phase concentrated by 10-fold (lane 3), which were obtained from S. aureus RN4220 harboring the empty plasmid pKE515. The arrowheads and arrow indicate Protein A and 33-kDa protein, respectively.

FIGURE 2.

The 33-kDa protein is identified as triacylated SitC lipoprotein. A, indicates the whole amino acid sequence of the S. aureus SitC protein (box, signal peptide; asterisk, lipid binding cysteine; underlined letters, amino acids determined by Edman degradation sequencing; red letters, amino acid sequences determined by liquid chromatography-MS/MS; double underlined blue letters, amino acids determined by MALDI-TOF MS). B, the SitC protein fragments obtained by lysylendopeptidase digestion in an SDS-polyacrylamide gel slice were measured by MALDI-TOF MS. A series of mass peaks harboring 14 mass differences were numbered 1–8 in the left panel and described in the right panel. The obtained m/z of peak 1 corresponded to theoretical m/z of tripalmitic acid (Pam₃)-modified N-acyl-S-(diacyl-propyl)-cysteinyl-peptide, and those of peaks 2–8 corresponded with Pam₃-N-terminal peptides harboring increasing numbers of methylene (CH₂) groups in their fatty acids. C and D, CHO/hCD14/hTLR2 cells (C) or CHO/hCD14/hTLR4 (D) were stimulated in the absence (black line with gray area) or in the presence of 2 ng/ml (blue) or 20 ng/ml (magenta) of the purified S. aureus SitC protein. Surface expression of NF-κB-driven hCD25 was analyzed by flow cytometry.

FIGURE 3.

TLR2 stimulation by the purified SitC and SitC mutant bacteria. A and B, peritoneal macrophages (1 × 10⁵ cells) prepared from the parent C57BL/6 (bar 1), TLR1^–/– (bar 2), TLR2^–/– (bar 3), TLR6^–/– (bar 4), and MyD88^–/– (bar 5) mice were stimulated for 24 h with 1 μg/ml lipopolysaccharide, 10 ng/ml synthetic Pam₃CSK₄, 100 ng/ml MALP-2, or 100 ng/ml or 1 μg/ml of purified SitC protein. The concentrations of tumor necrosis factor-α (A) and interleukin-6 (B) in the culture supernatants were measured using enzyme-linked immunosorbent assay. The symbols are the same between A and B. The data were shown as the mean ± S.D. using at least two independent mice. N.D., not detected. C, SDS-PAGE analysis of the aqueous phase (lanes 1 and 3) and Triton X-114 detergent phase (lanes 2 and 4) of solubilized PGN. Each fraction was obtained from S. aureus ΔsitC mutant harboring the empty plasmid pKE515 (lanes 1 and 2) and the ΔsitC mutant harboring a plasmid containing the sitC gene, pSsitC (lanes 3 and 4). D and E, CHO/hCD14/hTLR2 cells were stimulated with S. aureus whole cells of the parent RN4220 harboring an empty plasmid (D) or the ΔsitC mutant harboring an empty plasmid (E). Surface expression of NF-κB-driven hCD25 was analyzed by flow cytometry. The black line with gray area indicates mock treatment; a ratio of CHO cells to bacterial cells of 1:5 (blue) and 1:50 (magenta), respectively.

FIGURE 4.

ABC transporter substrate-binding lipoproteins of two another Gram-positive bacteria activate TLR2. A, SDS-PAGE analysis of purified lipoproteins of S. aureus SitC (lane SitC), B. subtilis YfmC (lane 1), and M. luteus GluB (lane 2). B–E, CHO/hCD14/hTLR2 (B and D) or CHO/hCD14/hTLR4 (C and E) cells were stimulated in the absence (black line with gray area) or in the presence of 2 ng/ml (blue) or 20 ng/ml (magenta) of the B. subtilis YfmC lipoprotein (B and C) or of the M. luteus GluB lipoprotein (D and E). The experiments were performed as described in the legend to Fig. 1 and were representative of at least two independent experiments.