In vitro polymerization of a functional Escherichia coli amyloid protein

Abstract

Amyloid formation is characterized by the conversion of soluble proteins into biochemically and structurally distinct fibers. Although amyloid formation is traditionally associated with diseases such as Alzheimer disease, a number of biologically functional amyloids have recently been described. Curli are amyloid fibers produced by Escherichia coli that contribute to biofilm formation and other important physiological processes. We characterized the polymerization properties of the major curli subunit protein CsgA. CsgA polymerizes into an amyloid fiber in a sigmoidal kinetic fashion with a distinct lag, growth, and stationary phase. Adding sonicated preformed CsgA fibers to the polymerization reaction can significantly shorten the duration of the lag phase. We also demonstrate that the conversion of soluble CsgA into an insoluble fiber involves the transient formation of an intermediate similar to that characterized for several disease-associated amyloids. The CsgA core amyloid domain can be divided into five repeating units that share sequence and structural hallmarks. We show that peptides representing three of these repeating units are amyloidogenic in vitro. Although the defining characteristics of CsgA polymerization appear conserved with disease-associated amyloids, these proteins evolved in diverse systems and for different purposes. Therefore, amyloidogenesis appears to be an innate protein folding pathway that can be capitalized on to fulfill normal physiological tasks.

FIGURE 1. In vitro polymerization of CsgA measured by ThT fluorescence, CD, and TEM

A, the fluorescence of freshly purified CsgA mixed with 25 μm ThT was measured in 10-min intervals at 495 nm after excitation at 438 nm. B, circular dichroism analysis of 10 μm CsgA immediately after purification, 2 days after purification, 7 days after purification, and 15 days after purification. CsgA was incubated at room temperature without shaking after purification. C–E, TEM micrographs of 30 μm CsgA after incubation at room temperature for the indicated times. Scale bar, 500 nm.

FIGURE 2. Detection of transient conserved intermediate species during CsgA polymerization

A, ThT fluorescence (top) and immunoblotting (bottom) of 80 μm CsgA incubated for the indicated time after purification. At the indicated times, samples were removed, ThT was added to a final concentration of 20 μm, and fluorescence was measured. Samples were blotted onto a nitrocellulose membrane and probed with the A11 antibody, and after stripping, probed with the anti-CsgA antibody. a.u., arbitrary units. B, CsgA denatured with 8 M GdnHCl was blotted onto nitrocellulose and probed with the A11 and anti-CsgA antibodies (left). GdnHCl (GuHCl) was removed using a Sephadex G25 column (final buffer: 50 mm KPi, pH 7.2) and then immediately blotted onto nitrocellulose and probed with the A11 and anti-CsgA antibodies (right). RT, room temperature; Ret., retentate; Filt., filtrate. C, Amicon ultra filters were used to separate CsgA solutions prior to probing with the A11 and anti-CsgA antibodies. The molecular weight cutoff of the filters is indicated. Retentates and filtrates were immediately blotted onto nitrocellulose and probed with the A11 antibody, and after stripping, probed with the anti-CsgA antibody.

FIGURE 3. CsgA fibers can catalyze self-polymerization

A, CsgA (40 μm) fluorescence in the absence (solid line) or presence of 2.5% by weight of sonicated CsgA fibers (dashed line). Samples were incubated at room temperature, collected at the indicated times, and amended with 20 μm ThT prior to excitation at 438 nm and measurement at 495 nm. a.u., arbitrary units. B, samples were removed at the indicated times and immediately blotted to nitrocellulose. Blots were probed with the A11 antibody, and after stripping, probed with an anti-CsgA antibody.

FIGURE 4. Three CsgA intramolecular peptide repeats can assembly into amyloid fibers

A, alignment of internally conserved residues. The CsgA primary sequence shows the repeated consensus sequences. The identical amino acid residues of five repeats are in gray, and the conserved polar amino acid residues are enclosed in four boxes. N, N terminus; C, C terminus. B, oligopeptides of R1, R2, R3, R4, and R5 at 0.2 mg/ml in KPi were incubated at room temperature for 5 days before ThT fluorescence measurements were taken. The error bar indicates the S.E. for at least three separate experiments. CsgA fibers were diluted to 0.2 mg/ml and assayed for ThT fluorescence. C–E, TEM. 0.5 mg/ml R1, R3, and R5 in pH 7.2 KPi was incubated at room temperature for 5 days. Samples of different peptide solutions were directly applied on Formvar-coated grids and visualized with negative staining electron microscopy. The scale bar is equal to 500 nm.