The Application of Genetic, In Silico and In Vitro Tools to Elucidate the Biology of a Functional Amyloid Fiber.

Curli are thin aggregative fimbriae produced by many Enterobacteriaceae as a structural component of biofilms. Curli share many biochemical and biophysical properties with amyloid fibers which are often associated with human neurological diseases including Alzheimer’s, mad cow, and Parkinson’s. However, curli are the product of a dedicated assembly system that consists of a complex gene regulatory network featuring CsgD; a secretion system including CsgG, CsgE, and CsgF; and the major and minor fiber subunits CsgA and CsgB. As a model system, many aspects of curli formation have been explored including subunit secretion, regulation, biological function, and amyloidogenesis. My work focused on the genetics of curli formation in Escherichia coli, the economic constraints on the evolution of CsgA and other extracellular proteins, and the in vitro amyloidogenesis of CsgA-His. I screened the Keio collection of single gene deletions to discover new genes that affect curli production. More than 300 genes modulate curli production including the sodium antiporter nhaA, a regulator of the glycine cleavage system gcvA, multiple LPS biosynthesis genes, and genes involved in many fundamental cellular processes. This analysis suggests that curli production is part of a highly regulated and complex developmental pathway. The regulation of glyA by CsgD and the curli phenotype of gcvA focused my attention on the amino acid composition of CsgA. CsgA is incredibly rich in glycine and serine. As simple amino acids, both are inexpensive to synthesize. Consequently, CsgA is relatively cheap to produce on a per unit basis. Strikingly, other extracellular proteins including those in Escherichia coli, Pseudomonas syringae, Mycobacterium tuberculosis, Saccharomyces cerevisiae, and other microbes are also inexpensive relative to intracellular proteins. Since extracellular proteins are often lost to the environment, evolution has in turn selected them for increased economy to counteract lost resources. Finally, we studied the in vitro amyloid formation of CsgA-His. Like disease-associated amyloids, CsgA-His bound Thioflavin T upon polymerizing into fibers, reacted with an amyloid specific antibody, self seeded, and displayed other aspects of amyloid formation. Collectively, this work sheds new light on the biology of the functional amyloid fiber curli and hopefully will beget novel directions of inquiry.Ph.D.Molecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/78834/1/dnlsmith_1.pd