Formation of amyloid fibrils the CsgA QYGGNN hexapeptide. Formation of amyloid fibrils by synthetic QYGGNN hexapeptide (A), synthetic QYGGNA hexapeptide (B) and synthetic β-amyloid 1-40 peptide from amyloid plaques of Alzheimer’s disease (C) was investigated by negative staining and visualized by electron microscopy.

Pro-inflammatory responses elicited by amyloids of bacterial and host origin in macrophages. (A) Stimulation (4 hours) of human THP-1 cells with purified curli fibrils or β-amyloid 1-40 in the presence (open bars) or absence (gray bars) of inhibitory anti-TLR2 monoclonal antibody. Data are shown as average fold increases over non-stimulated THP-1 cells ± standard error from three independent experiments. (B) Stimulation (4 hours) of murine BMDM from control mice (C57BL/6, gray bars) or TLR2-deficient mice (TLR2^−/−, open bars) with IFN-γ and purified curli fibrils (1μg/ml) or synthetic peptides (β-amyloid 1-40, β-amyloid 1-42 or scrambled β-amyloid 1-42; 20μM). Data are shown as average fold increases in mRNA levels over those detected in non-stimulated BMDM (i.e. in cells not stimulated with IFN-γ or amyloids) ± standard error from three independent experiments.

Characterization of the TLR2 agonist activity of CsgA. Production of IL-8 was determined 24 hours after stimulation of HEK293-TLR2CD14 cells with different affinity purified fusion proteins or synthetic peptides (Pam₃CSK4) using ELISA. Data are shown as averages ± standard error from at least three independent experiments. (A) Stimulation with increasing concentrations of synthetic lipopeptide (Pam₃CSK4), GST or GST-CsgA protein. Stars indicate that IL-8 production was significantly (P < 0.05) increased compared to stimulation with GST control. (B) Stimulation with affinity purified fusion proteins between GST and truncated forms of CsgA (0.15μM). Brackets and P values indicate results from statistical analysis

Effect of an N122A substitution on the ability of affinity purified GST-CsgA-R1-5 to form amyloids and serve as TLR2 agonist. (A) Increasing concentrations of GST-CsgA-R1-5 and GST-CsgA-R1-5_N122A fusion proteins were stained with the amyloid specific dye Thioflavin T (ThT) and fluorescence measured (438nm excitation, 495nm emission). Data are shown as averages ± standard error from three independent experiments. Stars indicate that ThT fluorescence of GST-CsgA-R1-5 was significantly (p<0.05) increased compared to GST-CsgA-R1-5_N122A. (B) Analysis of the far ultra violet spectrum (from 190 to 260 nm) of GST-CsgA-R1-5 and GST-CsgA-R1-5_N122A by CD spectroscopy. (C) Stimulation of HEK293-TLR2CD14 cells with increasing concentrations of GST-CsgA-R1-5 and GST-CsgA-R1-5_N122A. Production of IL-8 was determined 24 hours later using ELISA. Data are shown as averages ± standard error from three independent experiments. Brackets and p values indicate results from statistical analysis.

Amyloidogenicity and TLR2 agonist activity of synthetic peptides. (A) Synthetic peptides were stained with the amyloid specific dye Thioflavin T (ThT) and fluorescence was measured (438nm excitation, 495nm emission) to assess their amyloidogenicity. Data are shown as averages ± standard error from three independent experiments. Statistical significance of differences are indicated by a bracket. (B) Expression of TLR2 on by murine microglia cells detected by flow cytometry using anti-TLR2 antibody (anti-TLR2-FITC) or an isotype control (mIgG-FITC). (C) Nos2 expression in murine microglia cells that were either untreated (gray bars) or treated with anti-TLR2 antibody (open bars). Microglia cells were stimulated with IFN-γ (200units/ml) and synthetic peptides (CsgA-R4-5, CsgA-R4-5_N122A, β-amyloid 1-42, scrambled β-amyloid 1-42 or Pam₃CSK₄, each at a concentration of 10μM). Data are shown as averages of fold increases in Nos2 expression over non-stimulated microglia cells ± standard error from three independent experiments. Statistical significance of differences are indicated by a bracket. NS, not significant.

Identification of CsgA residues required for its TLR2 agonist activity. Production of IL-8 was determined 24 hours after stimulation of HEK293-TLR2CD14 cells with different affinity purified fusion proteins using ELISA. Data are shown as averages ± standard error from at least three independent experiments. (A) Stimulation with GST-CsgA fusion proteins (0.15μM) from S. Typhimurium, E. coli and S. sonnei. ns, not significant. (B) Primary sequence of the amyloidogenic C-terminal domain of S. Typhimurium CsgA (residues 43-151), which contains 5 repeat regions (R1-R5). A gray background indicates residues that are conserved between CsgA proteins from different members of the Enterobacteriaceae. A dark gray background indicates residues that were subjected to alanine scanning mutagenesis. A box indicates the amyloidogenic QYGGNN hexapeptide. (C) Stimulation with affinity purified GST-CsgA-R1-5 fusion proteins (0.15μM) carrying the indicated amino acid substitutions. Brackets and P values indicate results from statistical analysis.

Contribution of curli fibrils to iNOS production during S. Typhimurium sepsis. (A) Nos2 mRNA levels observed in the liver of mice 8 hours after intraperitoneal infection of wild type mice or TLR2 deficient mice with 10⁸ colony forming units (CFU) of S. Typhimurium (wild type), a csgBA mutant or a msbB mutant. Each bar represents the average fold increases in Nos2 expression of S. Typhimurium-infected mice (n=4) compared to mock-infected mice (n=4) ± standard error. Statistical significance of differences is indicated by a bracket. NS, not significantly different. (B) Nitrite production by BMDM from C57BL/6 mice in response to stimulation with increasing concentrations of purified curli fibrils in the presence (closed circles) or absence (open circles) of IFN-γ (200units/ml). Data represent averages ± standard error from at least three independent experiments. (C) Nitrite production by IFN-γ treated BMDM from C57BL/6 mice (closed circles) or TLR2 deficient mice (open circles) after stimulation with purified curli fibrils or synthetic lipopeptide (Pam₃CSK₄). Data represent averages ± standard error from at least three independent experiments.