The C-Terminal Repeating Units of CsgB Direct Bacterial Functional Amyloid Nucleation

Abstract

Curli are functional amyloids produced by enteric bacteria. The major curli fiber subunit, CsgA, self-assembles into an amyloid fiber in vitro. The minor curli subunit protein, CsgB, is required for CsgA polymerization on the cell surface. Both CsgA and CsgB are composed of five predicted β–strand-loop-β–strand-loop repeating units that feature conserved glutamine and asparagine residues. Because of this structural homology, we proposed that CsgB might form an amyloid template that initiates CsgA polymerization on the cell surface. To test this model, we purified wild-type CsgB, and found that it self-assembled into amyloid fibers in vitro. Preformed CsgB fibers seeded CsgA polymerization as did soluble CsgB added to the surface of cells secreting soluble CsgA. To define the molecular basis of CsgB nucleation, we generated a series of mutants that removed each of the five repeating units. Each of these CsgB deletion mutants was capable of self-assembly in vitro. In vivo, membrane-localized mutants lacking the 1st, 2nd or 3rd repeating units were able to convert CsgA into fibers. However, mutants missing either the 4th or 5th repeating units were unable to complement a csgB mutant. These mutant proteins were not localized to the outer membrane, but were instead secreted into the extracellular milieu. Synthetic CsgB peptides corresponding to repeating units 1, 2 and 4 self assembled into ordered amyloid polymers, while peptides corresponding to repeating units 3 and 5 did not, suggesting that there are redundant amyloidogenic domains in CsgB. Our results suggest a model where the rapid conversion of CsgB from unstructured protein to a β-sheet-rich amyloid template anchored to the cell surface is mediated by the C-terminal repeating units.

A comprehensive guide to pilus biogenesis in Gram-negative bacteria

“…Once at the surface, CsgB controls the nucleation and polymerisation of CsgA molecules into curli fibres in a CsgF-dependent manner 129,130 . It is still unclear whether the incorporation of curli subunits occurs from the proximal or distal end of the curli fibre.…”

Amyloid Structures as Biofilm Matrix Scaffolds

“…Curli assembly requires the products of at least two divergently transcribed operons, csgBAC and csgDEFG (13). CsgA and CsgB are the protein subunits that make up curli, whereas the rest of the proteins are involved in the regulation of the production, stabilization, and secretion of CsgA and CsgB subunits (1,(14)(15)(16) (Fig. 1B).…”

“…1B). CsgA has the propensity to self-aggregate; however, it requires the CsgB subunit for nucleation into fibers in vivo (14,17). CsgA and CsgB are translocated into the periplasm by the Sec translocon system and exported to the extracellular medium through a pore-like structure constituted by the CsgG protein formed in the outer membrane (18).…”

Solution NMR structure of CsgE: Structural insights into a chaperone and regulator protein important for functional amyloid formation

“…Curli fibers are primarily composed of the major structural subunit, CsgA (2-4), but also contain a small amount of the minor structural subunit, CsgB (25,26). Although CsgA is prone to aggregation and forms amyloid fibers in vitro (2,27), the formation of curli fibers in vivo is nucleated by CsgB (28)(29)(30). CsgC exists in periplasm and is an effective inhibitor of CsgA polymerization, suppressing fibrilization at substoichiometric ratios as low as 1:500 (CsgC:CsgA) (31), and perhaps preventing premature periplasmic amyloid formation and toxicity to the bacterium.…”