The bacterial chaperone CsgC inhibits functional amyloid CsgA formation by promoting the intrinsically disordered pre-nuclear state.

E. coli assembles a functional amyloid called curli during biofilm formation. The major curlin subunit is the CsgA protein, which adopts a beta-sheet rich fold upon fibrillization. The chaperone protein CsgC inhibits CsgA amyloid formation. CsgA undergoes a 3-stage aggregation process: an initial lag phase where beta-rich nuclei form, an exponential elongation phase, and a plateau phase. It is currently not known if CsgC inhibits amyloid formation by inhibiting formation of a pre-fibril nucleus, or if CsgC inhibits a later stage of amyloid formation by blocking monomer addition. Here, CsgC homologs from C. youngae, C. davisae, and H. alvei were purified and characterized for their ability to interrogate CsgA amyloid formation. Each of the CsgC homologs prolonged the lag phase of E. coli CsgA amyloid formation similar to E. coli CsgC. Additionally, we found E. coli CsgC interacted transiently and weakly with a monomeric, pre-nucleus species of CsgA which delayed amyloid formation. A transient CsgC-CsgA heterodimer was observed using ion mobility-mass spectrometry. When CsgC was added to actively polymerizing CsgA, exponential growth commonly associated with nucleation-dependent amyloid formation was lost. Adding preformed CsgA seeds did not rescue exponential growth, indicating that CsgC also has inhibitory activity during fibril elongation. Indeed, CsgC interacted strongly with CsgA fibers, suggesting the interaction between CsgC and CsgA fibers can slow new fiber growth. CsgC displays unique inhibitory activity at multiple stages of amyloid formation and acts as an energy-independent chaperone that transiently interacts with prefibrillar CsgA and an amyloid fiber.