Volume 57, Issue 3, Pages 445-455 (February 2015) The Bacterial Curli System Possesses a Potent and Selective Inhibitor of Amyloid Formation  Margery L. Evans, Erik Chorell, Jonathan D. Taylor, Jörgen Åden, Anna Götheson, Fei Li, Marion Koch, Lea Sefer, Steve J. Matthews, Pernilla Wittung-Stafshede, Fredrik Almqvist, Matthew R. Chapman  Molecular Cell  Volume 57, Issue 3, Pages 445-455 (February 2015) DOI: 10.1016/j.molcel.2014.12.025 Copyright © 2015 Elsevier Inc. Terms and Conditions

Molecular Cell 2015 57, 445-455DOI: (10.1016/j.molcel.2014.12.025) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Secretion-Deficient Mutants Induce an Amyloid Inhibitory Activity in the Periplasm (A and B) Periplasmic extracts (PEs) were harvested from BW25113 ΔcsgG (A) or complete curli deletion (Δcsg; B) and incubated with soluble CsgA for 24 hr. CsgA amyloid formation was monitored by ThT fluorescence. (C) Soluble and insoluble CsgA were separated by centrifugation after 24 hr of incubation alone or in the presence of periplasmic protein extracts from a ΔcsgD or ΔcsgG mutant. Samples were suspended in SDS loading buffer with or without HFIP pretreatment. Samples were separated on a 15% SDS-PAGE gel and stained with Coomassie (top panel) or analyzed by western blot for CsgA (lower panel). See also Figure S1. Molecular Cell 2015 57, 445-455DOI: (10.1016/j.molcel.2014.12.025) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 Amyloid Inhibition Requires Expression of csgC (A) PEs from a BW25113 ΔcsgD mutant with pTrc99A (vector) or pTrc99A-csgD (pcsgD) were assayed for CsgA amyloid inhibition in vitro. (B and C) PEs from BW25113 mutants (ΔcsgA, ΔcsgB, ΔcsgC) or BW25113 combination mutants (ΔcsgBΔcsgA; ΔcsgAΔcsgC; ΔcsgBΔcsgAΔcsgC) were assayed for CsgA amyloid inhibition. CsgA amyloid formation was monitored by ThT fluorescence. See also Figure S2. Molecular Cell 2015 57, 445-455DOI: (10.1016/j.molcel.2014.12.025) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 ΔcsgGΔcsgC Mutants Accumulate Intracellular CsgA Amyloid Aggregates (A) BW25113 cells were grown on Congo red indicator plates at 26°C for 48 hr to assess curli production in vivo. (B) Cells grown under curli-inducing conditions were subject to western blot analysis with CsgG, CsgA, CsgC, and Sigma70 antibodies. (C) Intact or lysed cells were analyzed by dot blot western using CsgA antibodies. See also Figure S3. Molecular Cell 2015 57, 445-455DOI: (10.1016/j.molcel.2014.12.025) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 Purified CsgC Inhibits CsgA Amyloid Formation at Substoichiometric Concentrations (A) Polymerization of CsgA was monitored by ThT fluorescence over time in the presence or absence of purified CsgC. (B–D) (B) The secondary structure of CsgA was monitored by CD every 24 hr for 4 days in the absence (closed symbols) or presence (open symbols) of CsgC (1:10, CsgC:CsgA). Sequential 1H NMR spectra of 10 μM CsgA alone (C) or in 150-fold molar excess over CsgC (D). The methyl proton spectral region is shown for clarity. (E) The amplitude of the strongest methyl peak in the CsgA (C) and CsgA+CsgC (D) spectra (0.83 ppm) plotted over time. (F) Polymerization of CsgA was seeded by the addition of 2% (by weight) CsgA seeds in the absence of CsgC (open squares) or the presence of CsgC (open circles and triangles). (G) Dot blot analysis of CsgA alone, CsgC alone, or CsgA+CsgC (1:3, CsgA:CsgC) immediately after purification (t = 0 hr) or after 24 hr of incubation (t = 24 hr) with anti-CsgA, anti-CsgC, and A11 antibodies. (H) Native gel electrophoresis and western blot of 20 μM soluble CsgA without and with 5 min of boiling, 20 μM CsgA that has been incubated at 25°C for 24 hr, 6.7 μM CsgC alone, or 20 μM CsgA that has been incubated with 2 μM CsgC for 24 hr. See also Figure S4. Molecular Cell 2015 57, 445-455DOI: (10.1016/j.molcel.2014.12.025) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 Amyloid Inhibition by CsgC Is Specific (A–E) Using the ThT assay, E. coli CsgC was tested for amyloid inhibition against CsgA from S. enterica (A), E. coli CsgB (B), Aβ42 (C), α-synuclein (D and E). (F) Sequence alignment of E. coli CsgA, CsgB, human α-synuclein, and the α-synuclein alanine variant used in (E). See also Figure S5. Molecular Cell 2015 57, 445-455DOI: (10.1016/j.molcel.2014.12.025) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 6 Model of CsgC-Mediated Amyloid Inhibition A predicted energy landscape of CsgA amyloidogenesis where CsgA forms dynamic, amorphous aggregates before assembling into amyloid-like, prefibrillar oligomers. Our data support a model where CsgC inhibits CsgA amyloid formation by transiently interacting with a prefibrillar CsgA species that results in keeping CsgA in an amorphous conformation. Molecular Cell 2015 57, 445-455DOI: (10.1016/j.molcel.2014.12.025) Copyright © 2015 Elsevier Inc. Terms and Conditions