Characterization of the Curli Nucleator CsgB.

Amyloid biogenesis is the underlying cause of many neurological disorders such as Alzheimer’s disease. During the progression of Alzheimer’s disease it is thought that normally soluble Aβ peptides self assemble into β-sheet rich, detergent resistant amyloid fibers via a template mediated-nucleation dependent polymerization reaction. Although the polymerization of Aβ is well characterized in vitro, the molecular events that govern nucleation in vivo are poorly described. Curli fibers are extracellular functional amyloid fibers utilized by enteric bacteria for the formation of biofilms. The fibers are composed of two proteins, CsgA, the major subunit, and CsgB, the minor subunit. CsgB is required for nucleating CsgA polymerization in vivo. CsgA and CsgB are 50% similar at the amino level and in silico structural predictions suggest both proteins are β-sheet rich. We hypothesized that CsgB drives the polymerization of CsgA by acting as a β-sheet rich folding template at the cell surface, and predicted that CsgB could self-polymerize into amyloid fibers in vitro. We found that CsgB and mutant variants polymerized into amyloid fibers in vitro, and that these fibers seeded CsgA polymerization. Perturbing the C-terminal portion of CsgB caused CsgB to be mislocalized in vivo and greatly reduced CsgB function. Our results are consistent with the hypothesis that CsgB templates CsgA polymerization by adopting a β-sheet rich fold, and that disruption of the CsgB-cell surface interaction leads to impaired CsgB function. Using the methods established in the course of this work, I have also begun to identify protein- and small molecule inhibitors of curli nucleation and polymerization. For example, CsgE, an accessory protein required for curli biogenesis, and rationally-designed small molecule inhibitors of Aβ polymerization inhibit CsgA polymerization in vitro. Together these results suggest curli biogenesis may be a powerful model for elucidating the initial molecular events involved in amyloid biogenesis.Ph.D.Microbiology & ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/63820/1/nhammer_1.pd