Abstract
E. coli secretes a functional amyloid called curli during biofilm formation. Curli fibers are composed of polymers of the CsgA protein, which adopts a beta-sheet rich fold upon fibrillization. A chaperone-like protein called CsgC inhibits CsgA amyloid formation. Like other amyloidogenic proteins, CsgA undergoes a 3-stage aggregation process: an initial lag phase where a beta-rich nucleus forms, an exponential elongation phase, and a plateau phase. It is currently not known if CsgC inhibits amyloid formation by inhibiting formation of the pre-fibril nucleus, or rather, if CsgC inhibits a later stage of amyloid formation by blocking monomer addition to a growing fiber. 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 in a similar fashion as E. coli CsgC. Additionally, we found that E. coli CsgC interacted transiently and weakly with a monomeric, pre-nucleus species of CsgA and that this interaction 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. However, the addition of 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 that the interaction between CsgC and CsgA fibers can slow new fiber growth. CsgC displays a unique inhibitory activity at multiple stages of amyloid formation. CsgC acts as an energy-independent chaperone that transiently interacts with prefibrillar CsgA as well as an amyloid fiber.