The Determinants of Bacterial Amyloid Nucleation and Polymerization

Abstract

Amyloid fibers are filamentous protein structures commonly associated with neurodegenerative diseases. Unlike disease-associated amyloids, which are the products of protein misfolding, E. coli assembles functional amyloid fibers called curli on its surface using an elegant biogenesis machine. Functional amyloids promise to provide novel insight into controlled amyloid formation. Curli fibers are composed of two proteins, CsgA and CsgB. In vivo, the polymerization of the major curli subunit protein, CsgA, is dependent on the CsgB nucleator. I characterized the in vitro polymerization mechanism of CsgA and found that the conversion of soluble CsgA into an insoluble fiber involved the transient formation of an intermediate similar to that characterized for several disease-associated amyloids. CsgA is composed of 5 imperfect repeats that constitute its amyloid core (R1 to R5). I discovered that peptides representing three of these repeating units are amyloidogenic in vitro. I demonstrated that The N- and C-terminal repeats (R1 and R5) govern CsgA nucleation and polymerization in vivo, and that the conserved glutamine and asparagine residues in those two repeats were required for CsgB-mediated nucleation and CsgA self-polymerization. Moreover, I discovered that the specific aspartate and glycine residues in the middle repeats R2, R3 and R4 function as “gatekeepers” by inhibiting their intrinsic aggregation propensities and responsiveness to nucleation. Genetic alteration of gatekeeper residues in CsgA resulted in uncontrolled amyloid propagation, even in the absence of the nucleation machinery. I further showed that these gatekeeper residues not only modulated bacterial amyloid polymerization but also decreased toxicity associated with amyloid propagation.