Bacterial Chaperones CsgE and CsgC Differentially Modulate Human α-Synuclein Amyloid Formation via Transient Contacts

Amyloid formation is historically associated with cytotoxicity, but many organisms produce functional amyloid fibers (e.g., curli) as a normal part of cell biology. Two E. coli genes in the curli operon encode the chaperone-like proteins CsgC and CsgE that both can reduce in vitro amyloid formation by CsgA. CsgC was also found to arrest amyloid formation of the human amyloidogenic protein α-synuclein, which is involved in Parkinson’s disease. Here, we report that the inhibitory effects of CsgC arise due to transient interactions that promote the formation of spherical α-synuclein oligomers. We find that CsgE also modulates α-synuclein amyloid formation through transient contacts but, in contrast to CsgC, CsgE accelerates α-synuclein amyloid formation. Our results demonstrate the significance of transient protein interactions in amyloid regulation and emphasize that the same protein may inhibit one type of amyloid while accelerating another

Bacterial Chaperones CsgE and CsgC Differentially Modulate Human α-Synuclein Amyloid Formation via Transient Contacts

Amyloid formation is historically associated with cytotoxicity, but many organisms produce functional amyloid fibers (e.g., curli) as a normal part of cell biology. Two E. coli genes in the curli operon encode the chaperone-like proteins CsgC and CsgE that both can reduce in vitro amyloid formation by CsgA. CsgC was also found to arrest amyloid formation of the human amyloidogenic protein α-synuclein, which is involved in Parkinson’s disease. Here, we report that the inhibitory effects of CsgC arise due to transient interactions that promote the formation of spherical α-synuclein oligomers. We find that CsgE also modulates α-synuclein amyloid formation through transient contacts but, in contrast to CsgC, CsgE accelerates α-synuclein amyloid formation. Our results demonstrate the significance of transient protein interactions in amyloid regulation and emphasize that the same protein may inhibit one type of amyloid while accelerating another

The bacterial curli system possesses a potent and selective inhibitor of amyloid formation.

Curli are extracellular functional amyloids that are assembled by enteric bacteria during biofilm formation and host colonization. An efficient secretion system and chaperone network ensures that the major curli fiber subunit, CsgA, does not form intracellular amyloid aggregates. We discovered that the periplasmic protein CsgC was a highly effective inhibitor of CsgA amyloid formation. In the absence of CsgC, CsgA formed toxic intracellular aggregates. In vitro, CsgC inhibited CsgA amyloid formation at substoichiometric concentrations and maintained CsgA in a non-β-sheet-rich conformation. Interestingly, CsgC inhibited amyloid assembly of human α-synuclein, but not Aβ42, in vitro. We identified a common D-Q-Φ-X0,1-G-K-N-ζ-E motif in CsgC client proteins that is not found in Aβ42. CsgC is therefore both an efficient and selective amyloid inhibitor. Dedicated functional amyloid inhibitors may be a key feature that distinguishes functional amyloids from disease-associated amyloids

Aggregation of α-synuclein in the presence of CsgC and CsgE.

<p><b>A.</b> ThT assay for α-synuclein aggregation with and without 1-to-10 molar ratio of CsgE:synuclein (red) or 1-to-10 molar ratio of CsgC:synuclein (blue). <b>B.</b> Bar graph showing the lag time for α-synuclein aggregation at 3 different ratios of CsgE and CsgC (1-to-3, 1-to-10, 1-to-100; 70 μM α-synuclein in all cases). ‘*’ Denotes no rise in ThT emission after 85 hrs. The error bars represent three experimental replicates. <b>C.</b> Fluorescence microscopy of end products of ThT assay for α-synuclein alone and for 1-to-3, 1-to-10, and 1-to-100 molar ratio of CsgE/CsgC-to-synuclein mixtures. Scale bar 100 μm. <b>D-F.</b> AFM images of end products after ThT experiments for α-synuclein alone (<b>D</b>), and in the presence of CsgE (<b>E</b>) and CsgC (<b>F</b>), as indicated. Scale bar 1 μm.</p

Solution NMR of CsgC/CsgE interactions with ¹⁵N labeled α-synuclein.

<p>¹H-¹⁵N HSQC spectra at 10°C for 100 μM α-synuclein alone (red data in all panels) and upon addition of a 1-to-1 molar ratio of CsgC (<b>A</b>, blue) and CsgE (<b>B</b>, blue), and for a 1-to-5 molar ratio sample of CsgC and α-synuclein that had been shaken at 37°C for 48 h (<b>C,</b> blue). The data shown in <b>A</b> and <b>B</b> demonstrate that blue and red signals overlap except for His50 (<b>Insets</b> in <b>A</b> and <b>B</b>) and these spectra did not change over the course of three days. The visible chemical shifts in <b>C</b> were analyzed by NMR diffusion experiments to obtain an estimate of the molecular size. <b>D.</b> Analysis of perturbed residues in α-synuclein in the incubated CsgC-synuclein sample, based on the ¹H-¹⁵N HSQC peak intensities in <b>C</b> and reported assignments [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140194#pone.0140194.ref033" target="_blank">33</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140194#pone.0140194.ref034" target="_blank">34</a>]. Boxed residues represent chemical shifts assigned to specific residues (78 of the 140 residues were identified and used for the analysis). After shaking, 38 residues disappeared or broadened severely in the new species as judged from the peak intensities. Residues that broadened beyond detection are shown in red, and residues that lost > 90% of the original intensity are shown in yellow. Marked in bold are residues that show no apparent chemical shift change (Δω < 0.02 ppm, calculated as Δω = |0.2Δ¹⁵N+Δ¹H|).</p

Secondary structure of CsgC-induced oligomers.

<p>Far-UV CD spectra for 70 μM monomeric α-synuclein alone, and after shaking at 37°C for 48 h with and without 14 μM CsgC. The contribution from CsgC to the CD signal has been subtracted (CsgC shaken alone for 48 h did not result in any change of CD signal, data not shown). Whereas monomeric α-synuclein has random-coil like secondary structure, α-synuclein adopts β-sheet structure after incubation as a result of amyloid fiber formation. In the presence of CsgC, α-synuclein remains random-coil also after 48 h of shaking at 37°C.</p