Cell surface-localized CsgF condensate is a gatekeeper in bacterial curli subunit secretion

Area of Science:

• Microbiology
• Biochemistry
• Structural Biology

Background:

• Curli are functional amyloids on the E. coli outer membrane, essential for biofilm formation.
• CsgF protein is a critical component required for the proper assembly of curli structures.

Purpose of the Study:

• To investigate the in vitro phase separation behavior of CsgF.
• To correlate CsgF phase separation with its function in curli biogenesis.
• To elucidate the role of CsgF in templating curli assembly on the E. coli cell surface.

Main Methods:

• In vitro phase separation assays of CsgF and its variants.
• Functional complementation assays using CsgF variants in csgF-deficient E. coli.
• Analysis of CsgA secretion and curli assembly in response to CsgF modulation.
• Investigation of CsgB aggregation within CsgF condensates.

Main Results:

• CsgF undergoes phase separation in vitro, and this property is directly linked to its function in curli biogenesis.
• Specific mutations, such as phenylalanine substitutions in the N-terminus, disrupt CsgF phase separation and impair curli assembly.
• Exogenous CsgF can complement the curli assembly defect in csgF-deficient cells.
• CsgF influences the cell surface secretion of CsgA, the major curli subunit.
• CsgB forms SDS-insoluble aggregates within dynamic CsgF condensates.

Conclusions:

• CsgF phase separation is a key mechanism regulating curli amyloid formation.
• Multicomponent CsgF-B condensates act as nucleation sites for templating CsgA amyloid assembly on the E. coli cell surface.
• Understanding CsgF's role in condensate formation provides insights into bacterial amyloid biogenesis and potential therapeutic targets.