The curli nucleator protein, CsgB, contains an amyloidogenic domain that directs CsgA polymerization.
Curli are functional amyloid fibers assembled by enteric bacteria such as Escherichia coli and Salmonella spp. In E. coli, the polymerization of the major curli fiber subunit protein CsgA into an amyloid fiber depends on the minor curli subunit protein, CsgB. The outer membrane-localized CsgB protein shares approximately 30% sequence identity with the amyloid-forming protein CsgA, suggesting that CsgB might also have amyloidogenic properties. Here, we characterized the biochemical properties of CsgB and the molecular basis for CsgB-mediated nucleation of CsgA. Deletion analysis revealed that a CsgB molecule missing 19 amino acids from its C terminus (CsgB(trunc)) was not outer membrane-associated, but secreted away from the cell. CsgB(trunc) was overexpressed and purified from the extracellular milieu of cells as an SDS-soluble, nonaggregated protein. Soluble CsgB(trunc) assembled into fibers that bound to the amyloid-specific dyes Congo red and thioflavin-T. CsgB(trunc) fibers were able to seed soluble CsgA polymerization in vitro. CsgB(trunc) displayed modest nucleator activity in vivo, as demonstrated by its ability to convert extracellular CsgA into an amyloid fiber. Unlike WT CsgB, CsgB(trunc) was only able to act as a nucleator when cells were genetically manipulated to secrete higher concentrations of CsgA. This work represents a unique demonstration of functional amyloid nucleation and it suggests an elegant model for how E. coli guides efficient amyloid fiber formation on the cell surface.
Hammer
Neal D
ND
Department of Molecular, Cellular, and Developmental Biology, University of Michigan College of Literature, Science, and the Arts, 830 North University, Ann Arbor, MI 48109-0620, USA.
Schmidt
Jens C
JC
Chapman
Matthew R
MR
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D002942:Circular Dichroism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data
D000682:Amyloid; D001426:Bacterial Proteins; C109182:CsgB protein, E coli; D029968:Escherichia coli Proteins; C489688:csgA protein, E coli; C077481:Crl protein, Bacteria