Structural Insights into Curli CsgA Cross-β Fibril Architecture Inspire Repurposing of Anti-amyloid Compounds as Anti-biofilm Agents

Abstract

Curli amyloid fibrils secreted by Enterobacteriaceae mediate host cell adhesion and contribute to biofilm formation, thereby promoting bacterial resistance to environmental stressors. Here, we present crystal structures of amyloid-forming segments from the major curli subunit, CsgA, revealing steric zipper fibrils of tightly mated β-sheets, demonstrating a structural link between curli and human pathological amyloids. D-enantiomeric peptides, originally developed to interfere with Alzheimer's disease-associated amyloid-β, inhibited CsgA fibrillation and reduced biofilm formation in Salmonella typhimurium. Moreover, as previously shown, CsgA fibrils cross-seeded fibrillation of amyloid-β, providing support for the proposed structural resemblance and potential for cross-species amyloid interactions. The presented findings provide structural insights into amyloidogenic regions important for curli formation, suggest a novel strategy for disrupting amyloid-structured biofilms, and hypothesize on the formation of self-propagating prion-like species originating from a microbial source that could influence neurodegenerative diseases.

Fig 1. Crystal structures of CsgA spine segments.

High-resolution crystal structures of CsgA segments are shown. In each structure, the left view is down the fibril axis, with residues shown as sticks, and the right view is perpendicular to the fibril axis, with β-strands, shown as ribbons, running horizontally. Eight layers of β-strands are shown, while actual fibrils contain thousands of layers. The carbons of each β-sheet are colored either gray or purple; heteroatoms are colored by atom type (nitrogen in blue, oxygen in red). The ₄₅LNIYQY₅₀, ₄₇IYQYGG₅₂ and ₁₃₇VTQVGF₁₄₂ segments formed the classical class 1 steric zipper architecture of tightly mated parallel β-sheets with individual subunits (peptides) situated perpendicular to the fibril axis. Two possible dry interfaces in the crystal packing are displayed. The ₁₂₉TASNSS₁₃₄ segment formed extended β-strands that stacked in an anti-parallel manner, but with a small contact area between two facing β-sheets and lacking a dry interface. S3–S6 Figs provide a detailed description of the four crystal structures.

Fig 2. D-peptides inhibit fibrillation of CsgA but not of PSMα3.

Mean fluorescence of ThT measurements in samples of CsgA (A) or PSMα3 (B), with or without the D-peptides, at a 1:5 molar ratio. Error bars represent standard error of the mean calculated from triplicates. ANK6 and DB3DB3 delayed fibril formation of CsgA and reduced the fluorescence signal, while demonstrating no effect on PSMα3 fibrillation. D3 showed a minor effect on CsgA (A). (C) Electron micrographs of CsgA incubated overnight, showing the formation of fibrils. In the presence of ANK6 or DB3DB3 (1:10 molar ratio of CsgA to D-peptides), only amorphous aggregates were observed. Scale bars are provided for each micrograph.

Fig 3. The D-peptides stabilize soluble CsgA and inhibit fibrillation.

The effect of D-peptide inhibitors on CsgA fibrillation at 5:1 molar ratios, as quantified based on CsgA ability to migrate on a 15% SDS-PAGE (exemplified in S12 Fig). CsgA incubated with ANK6 and DB3DB3 showed a prolonged soluble state, suggesting inhibition of the rapid formation of SDS-insoluble fibrils. The graph represents three experimental repeats. The experiments were repeated at least three times on different days, all giving similar results.

Fig 4. D-peptides reduce formation of S. typhimurium static biofilm.

D Static S. typhimurium MAE52 biofilm production assessed after a 48-hour incubation at 30°C, in the presence or absence of increasing concentrations of D-peptides, quantified using crystal-violet staining measured by optical density at 600 nm (OD₆₀₀). Statistical significance was analyzed using the Mann-Whitney non-parametric test. Error bars represent standard deviations. * p<0.05 compared to control (no inhibitor added).

Fig 5. D-peptides reduce formation of surface-attached S. typhimurium static biofilm.

Confocal microscopy images showing surface-attached static S. typhimurium MAE52 biofilms formed after 48 hours at 30°C, in the absence (control) or presence of 10 μM D-peptides. Samples were stained with propidium iodide.

Fig 6. D-peptides reduce curli production in S. typhimurium biofilm.

Quantification of the effect of D-peptides on curli fibril formation in the bacteria. Shown are the levels of measured residual CR in the agar following the removal of the biofilm colony of the S. typhimurium MAE150 cellulose-deficient mutant. Control colonies that were not exposed to the D-peptides were assigned 100% CR residual stain remaining in the agar. A higher percentage of residual CR stain indicates less CR adsorption by the colony and hence, reduced curli fibril formation. Error bars represent standard deviations. * p<0.05 and ** p<0.005 compared to control (no inhibitor added). CR plates from one of the experiments are shown in S15B Fig.