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. 2011 May 20;286(20):17736-45.
doi: 10.1074/jbc.M110.200113. Epub 2011 Mar 28.

A Novel Exopolysaccharide From the Biofilm of Thermus Aquaticus YT-1 Induces the Immune Response Through Toll-like Receptor 2

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A Novel Exopolysaccharide From the Biofilm of Thermus Aquaticus YT-1 Induces the Immune Response Through Toll-like Receptor 2

Miao-Hsia Lin et al. J Biol Chem. .
Free PMC article

Abstract

Bacterial polysaccharides are known to induce the immune response in macrophages. Here we isolated a novel extracellular polysaccharide from the biofilm of Thermus aquaticus YT-1 and evaluated its structure and immunomodulatory effects. The size of this polysaccharide, TA-1, was deduced by size-exclusion chromatography as 500 kDa. GC-MS, high performance anion-exchange chromatography with pulsed amperometric detection, electrospray ionization-MS/MS, and NMR revealed the novel structure of TA-1. The polysaccharide is composed of tetrasaccharide-repeating units of galactofuranose, galactopyranose, and N-acetylgalactosamine (1:1:2) and lacked acidic sugars. TA-1 stimulated macrophage cells to produce the cytokines TNF-α and IL-6. Screening of Toll-like receptors and antibody-blocking experiments indicated that the natural receptor of TA-1 in its immunoactivity is TLR2. Recognition of TA-1 by TLR2 was confirmed by TA-1 induction of IL-6 production in peritoneal macrophages from wild-type mice but not from TLR2(-/-) mice. TA-1, as a TLR2 agonist, could possibly be used as an adjuvant and could enhance cytokine release, which increases the immune response. Furthermore, TA-1 induced cytokine release is dependent on MyD88/TIRAP.

Figures

FIGURE 1.
FIGURE 1.
Serious spectra of NMR analysis of the EPS TA-1 from T. aquaticus YT-1 (A–F). A, shown is an 1H NMR (600 MHz, D2O, 350 K) spectrum. B, two-dimensional total correlation spectroscopy (600 MHz, D2O, 350 K, 150 ms) spectrum is shown. C, HMQC (600 MHz, D2O, 350 K) is shown. D, HMBC (600 MHz, D2O, 350 K) is shown. E, two-dimensional NOE spectroscopy (600 MHz, D2O, 500 ms) is shown. F, shown is the structure of TA-1. In all figure parts, the residues are as follows: A = 3,4-substituted N-acetyl-galactosamine, B = 3-substituted galactopyranose, C = 3-substituted N-acetyl-galactosamine, and D = terminal galactofuranose.
FIGURE 2.
FIGURE 2.
Electrospray ionization-MS/MS fragmentation spectra of depolymerized TA-1 (A) and primary structure of TA-1 (B). A, the MS/MS fragments revealed the TA-1 primary sequence unambiguously. B, the labeled molecular weights corresponded to the MS/MS fragments in A.
FIGURE 3.
FIGURE 3.
Stimulation of macrophages by TA-1 (A–C). RAW264.7 cells were treated with TA-1 at the dosages indicated. TNF-α (A), IL-6 (B), and NO (C) in culture medium were measured using ELISA (A and B) or Griess reagent (C). Data represent the mean ± S.D. of triplicates from one of at least three independent experiments.
FIGURE 4.
FIGURE 4.
Pretreatment of murine macrophages with polymyxin B to exclude LPS contamination of assays. Effect of LPS and TA-1 on TNF-α secretion by Raw 264.7 macrophages after pretreatment with polymyxin B is shown. The control was pretreated with polymyxin B and not treated with LPS or TA-1.
FIGURE 5.
FIGURE 5.
Activation of NF-κB reporter plasmid by TA-1 TLRs (A–F). HEK293T cells were transfected with p5xNF-κB-luc, pcDNA3.1-β-galactosidase, and either the vector pcDNA3.1 (empty vector) or this vector expressing one of six TLR genes as indicated TLR2 (A), TLR3 (B), TLR5 (C), TLR7 (D), TLR8 (E), and TLR9 (F). Twenty-four hours after transfection, the cells were treated with TA-1 (50 μg ml−1) or left untreated for 6 h; specific TLR ligands were also added as positive controls. The cells were lysed, and the lysates were used for luciferase activity measurement. Data represent the mean ± S.D. of triplicates from one of at least two independent experiments.
FIGURE 6.
FIGURE 6.
Activation of NF-κB by TA-1 via TLR2. HEK293T cells were transfected with p5xNF-κB-luc, pcDNA3.1-β-gal, and either pcDNA3.1 (empty vector) or pcDNA3.1 carrying the TLR2 gene. Twenty-four hours after transfection, the cells were treated with TA-1 at the concentrations indicated or left untreated for 6 h. The cells were lysed, and the lysates were used for luciferase activity measurement. Data represent the mean ± S.D. of triplicates from one of at least two independent experiments.
FIGURE 7.
FIGURE 7.
Blocking of TA-1 induced IL-6 and NO productions by TLR2-specific antibody (A and B). RAW264.7 cells were treated with TLR2 antibody, the isotype control IgG, or left untreated for 1 h followed by TA-1 and pamcsk4 treatments for 24 h. The pam3csk4 treatment was served as positive control. IL-6 (A) or NO (B) production by the macrophages in culture medium was measured by using ELISA or Griess reagent, respectively. Data represent the mean ± S.D. of triplicates from one of at least two independent experiments.
FIGURE 8.
FIGURE 8.
Effect of TLR4 on the TA-1-induced immunoregulation (A and B). HeNC2 (A) and GG2EE (B) cells (1 × 106/ml) were incubated with TA-1 for 24 h. The TNF-α concentration was determined by ELISA. TNF-α secretion stimulated by Pam3csk4 (1 μg ml−1) served as positive control, and the response was considered as 100% to normalize differences in the two cell lines.
FIGURE 9.
FIGURE 9.
Production of IL-6 in peritoneal macrophages of wild-type and TLR2−/− mice induced by TA-1. Peritoneal macrophages were isolated from wild-type or TLR2−/− mice and treated with TA-1 or the TLR2 ligand pam3csk4 at the indicated concentrations for 24 h. IL-6 production in culture medium was measured using ELISA. Data represent the mean ± S.D. of triplicates from one of at least two independent experiments.
FIGURE 10.
FIGURE 10.
Effect of MyD88 or TIRAP knockdown on IL-6 production in Raw264.7 macrophages. A, the protein expression level of scrambled siRNA, MyD88 siRNA, and TIRAP siRNA-transfected Raw264.7 cells was analyzed using Western blot with α-tubulin as the loading control. B, scrambled siRNA, MyD88 siRNA, and TIRAP siRNA-transfected Raw264.7 cells were treated with the TLR2 ligand, pam3csk4, and TA-1 polysaccharide for 24 h. The IL-6 level in the culture supernatants was measured by ELISA. Data represent the mean ± S.D. of triplicates from one of at least two independent experiments.
FIGURE 11.
FIGURE 11.
MyD88 played a key role in TA-1 function. THP-1 and MyD88-deficient THP-1 cells were treated with TA-1 or pam3csk4 (1 μg ml−1) for 16 h, and the amount of TNF-α in the medium was measured by ELISA. Data represent the mean ± S.D. of triplicates from one of at least two independent experiments.

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