Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 May 17;288(20):14178-14188.
doi: 10.1074/jbc.M112.447060. Epub 2013 Apr 2.

CD14 protein acts as an adaptor molecule for the immune recognition of Salmonella curli fibers

Glenn J Rapsinski  1 , Tiffanny N Newman  1 , Gertrude O Oppong  1 , Jos P M van Putten  2 , Çagla Tükel  3
Affiliations

CD14 protein acts as an adaptor molecule for the immune recognition of Salmonella curli fibers

Glenn J Rapsinski et al. J Biol Chem. .

Abstract

Amyloids, protein aggregates with a cross β-sheet structure, contribute to inflammation in debilitating disorders, including Alzheimer's disease. Enteric bacteria also produce amyloids, termed curli, contributing to inflammation during infection. It has been demonstrated that curli and β-amyloid are recognized by the immune system via the Toll-like receptor (TLR) 2/TLR1 complex. Here we investigated the role of CD14 in the immune recognition of bacterial amyloids. We used HeLa 57A cells, a human cervical cancer cell line containing a luciferase reporter gene under the control of an NF-κB promoter. When HeLa 57A cells were transiently transfected with combinations of human expression vectors containing genes for TLR2, TLR1, and CD14, membrane-bound CD14 enhanced NF-κB activation through the TLR2/TLR1 complex stimulated with curli fibers or recombinant CsgA, the curli major subunit. Similarly, soluble CD14 augmented the TLR2/TLR1 response to curli fibers in the absence of membrane-bound CD14. We further revealed that IL-6 and nitric oxide production were significantly higher by wild-type (C57BL/6) bone marrow-derived macrophages compared with TLR2-deficient or CD14-deficient bone marrow-derived macrophages when stimulated with curli fibers, recombinant CsgA, or synthetic CsgA peptide, CsgA-R4-5. Binding assays demonstrated that recombinant TLR2, TLR1, and CD14 bound purified curli fibers. Interestingly, CD14-curli interaction was specific to the fibrillar form of the amyloid, as demonstrated by using synthetic CsgA peptides proficient and deficient in fiber formation, respectively. Activation of the TLR2/TLR1/CD14 trimolecular complex by amyloids provides novel insights for innate immunity with implications for amyloid-associated diseases.

Keywords: Amyloid; Bacteria; Bacterial Pathogenesis; Biofilm; CD14; Curli; Salmonella; TLR2; Toll-like Receptors (TLR).

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
CD14 enhances NF-κB activation by the TLR2-TLR1 complex upon curli stimulation. HeLa 57A cells were transfected with an empty human expression vector or human expression vector carrying the indicated TLRs and CD14. Cells were stimulated with 50 ng of Pam3CSK4 (A), 10 μg of purified curli fibers (B), or GST or GST-CsgA fusion peptide (C) and lysed 8 h after the start of stimulation. NF-κB activation was monitored by measuring the luciferase activity as relative luminescence units (RLU). Bars represent averages mean ± S.E. from three independent experiments.
FIGURE 2.
FIGURE 2.
Soluble CD14 enhances recognition of curli fibers by the TLR2-TLR1 complex. HeLa 57A cells were transfected with an empty human expression vector or a human expression vector carrying the indicated TLRs and CD14. Cells were stimulated with 10 μg of curli fibers or 10 μg of curli fibers that had been incubated overnight with 10 μg/ml of soluble CD14. Cells were lysed 8 h after the start of stimulation, and NF-κB activation was monitored by measuring relative luminescence units (RLU). Bars represent mean ± S.E. from three independent experiments.
FIGURE 3.
FIGURE 3.
Membrane-bound CD14 contributes to IL-6 and nitrite production by BMDMs. BMDMs from C57BL/6, TLR2-deficient (TLR2−/−), and CD14-deficient (CD14−/− mice) were stimulated with 50 ng of Pam3CSK4 (A), 7 μg of purified curli fibers (B), or GST or GST-CsgA fusion peptide (C) for 6 h. Supernatants were collected at 6 h, and ELISA was conducted to measure IL-6 production. D, BMDMs were also stimulated with 10 μg of purified curli fibers for 24 h, and nitrite levels in the supernatant were measured. Bars represent mean ± S.E. from three independent experiments.
FIGURE 4.
FIGURE 4.
CD14, TLR2, and TLR1 bind purified curli fibers. A, ELISA plates coated with 5 μg/ml of recombinant CD14, TLR2, or TLR1 were incubated with 50 μg/ml of purified curli fibers for 2 h. α-CsgA antibody was used as primary antibody, and alkaline phosphatase-conjugated goat anti-rabbit IgG antibody was used for secondary antibody for detection and visualization of binding of curli to each receptor. LPS binding to CD14 was utilized as a positive control. B, an ELISA plate coated with 5 μg/ml of recombinant TLR2 or TLR1 was incubated with an increasing concentration of purified curli fibers (10 μg/ml, 30 μg/ml, 50 μg/ml, 75 μg/ml, and 100 μg/ml). α-CsgA antibody was used as a primary antibody, and alkaline phosphatase-conjugated goat anti-rabbit IgG antibody was used for secondary antibody for detection. C, an ELISA plate coated with 5 μg/ml of recombinant CD14 was incubated with increasing concentrations of purified curli fibers (10 μg/ml, 30 μg/ml, 50 μg/ml, 75 μg/ml, and 100 μg/ml). α-CsgA antibody was used as a primary antibody, and alkaline phosphatase-conjugated goat anti-rabbit IgG antibody was used for secondary antibody for detection. Curve data points and bars represent mean ± S.E. from at least four independent experiments. D, ELISA plates coated with 2.5 μg/ml of recombinant CD14, TLR2, or TLR1 were incubated with 5.0 μg/ml of recombinant GST-CsgA mixed with increasing concentrations of purified curli fibers (0.1 μg/ml, 0.5 μg/ml, 1 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml, and 50 μg/ml) for 2 h. α-CsgA antibody was used as a primary antibody and alkaline phosphatase-conjugated goat anti-rabbit IgG antibody was used for secondary antibody for detection and visualization of binding of curli to each receptor.
FIGURE 5.
FIGURE 5.
CD14 binds synthetic curli fibers and enhances IL-6 production by BMDMs. Polymerization of 50 μm CsgA R4–5 (A) and CsgA R4–5N122A was monitored by measuring fluorescence intensity (excitation, 440; emission, 490) in a BMG Omega Polar Star plate reader. Equal volumes of peptides and 10 μm ThT were mixed and incubated at 37 °C. B, ELISA plates coated with CD14 were incubated with 50 μg/ml of synthetic peptides, CsgA R4–5, and CsgA R4–5N122A, and binding of the synthetic peptides to CD14 was detected using α-CsgA antibody and the secondary antibody, alkaline phosphatase-conjugated goat anti-rabbit IgG antibody. Absorbance was read at 405 nm. C, BMDMss from C57BL/6 were stimulated with 90 μg/ml of polymerized CsgA R4–5 and CsgA R4–5N122A for 6 h, and IL-6 in the supernatant was measured using ELISA. Bars represent mean ± S.E. from at least three independent experiments. D, differentiated BMDMs from C57BL/6 mice and CD14-deficient (CD14−/−) mice were stimulated with polymerized CsgA R4–5 (90 μg/ml) for 6 h, and IL-6 in the supernatant was measured using ELISA. Bars represent mean ± S.E. from at least three independent experiments. E, dot blot of 5 μg of α-CsgA antibody, CsgA R4–5, CsgA R4–5N122A, BSA, and purified curli fibers. The primary antibody used for detection was α-CsgA antibody. Goat anti-rabbit IgG antibody conjugated to IRDye® 800CW was used as secondary antibody.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Toyama B. H., Weissman J. S. (2011) Amyloid structure. Conformational diversity and consequences. Annu. Rev. Biochem. 80, 557–585 - PMC - PubMed
    1. Mok K. H., Pettersson J., Orrenius S., Svanborg C. (2007) HAMLET, protein folding, and tumor cell death. Biochem. Biophys. Res. Commun. 354, 1–7 - PubMed
    1. Theos A. C., Truschel S. T., Raposo G., Marks M. S. (2005) The Silver locus product Pmel17/gp100/Silv/ME20. Controversial in name and in function. Pigment Cell Res. 18, 322–336 - PMC - PubMed
    1. Leonhardt R. M., Vigneron N., Rahner C., Van den Eynde B. J., Cresswell P. (2010) Endoplasmic reticulum export, subcellular distribution, and fibril formation by Pmel17 require an intact N-terminal domain junction. J. Biol. Chem. 285, 16166–16183 - PMC - PubMed
    1. Pfefferkorn C. M., McGlinchey R. P., Lee J. C. (2010) Effects of pH on aggregation kinetics of the repeat domain of a functional amyloid, Pmel17. Proc. Natl. Acad. Sci. U.S.A. 107, 21447–21452 - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources