Biogenesis and Function of Bacterial Amyloid fibers

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DESCRIPTION (provided by applicant): None. (adapted from application): Curli are extracellular organelles produced by Escherichia coli and certain Salmonella species. Dr. Chapman has demonstrated that these fibers are structurally and biochemically related to eukaryotic amyloid fibers that are involved in several mammalian ailments including Alzheimer's disease, systemic amyloidosis, and spongiform encephalopathies such as mad cow disease and Creutzfeldt-Jacob disease. Unlike mammalian amyloid fibers that appear to be formed by aberrant pathways of protein folding, curli assembly in bacteria involves a dedicated multistep pathway that requires the csgBA and csgDEFG operons. Polymerization of the major curli subunit protein CsgA is dependent on the CsgB nucleator, and transport of CsgA and CsgB to the cell surface is mediated by the assembly factors CsgE, CsgF and CsgG. Specific Aim 1 will determine the mechanism of the nucleation and polymerization reactions and test the hypothesis that CsgB adopts an amyloid-like structure that stimulates CsgA polymerization. The structural and tinctoral properties of purified CsgB will be determined by circular dichroism (CD) spectroscopy and by Congo red (CR) and thioflavin T (thT) binding assays. The importance of the conserved Asn and Gln residues in CsgB and CsgA will be ascertained using site-directed mutagenesis. The nucleating activity of CsgB mutants will be scored in vivo and using a recently developed in vitro CsgA polymerization assay. Specific Aim 2 will elucidate the molecular details of curli biogenesis with special emphasis on the mechanism of subunit secretion. Dr. Chapman will test the hypothesis that the outer membrane lipoprotein CsgG forms a curli specific pore that is responsible fro the secretion of curli subunits and that GsgG function is dependant on CsgE. CsgG will be purified and characterized by EM, CD, and Blue native gel electrophoresis, and its pore-forming ability will be assessed by antibiotic sensitivity assays in vivo and liposome swelling assays in vitro. Dr. Chapman will attempt to identify sequences on CsgA and CsgB that mediate their CsgG-dependent secretion and to demonstrate interactions of CsgE with CsgG by using co-purification and cell-fractionation methods. Specific Aim 3 will define the role of curli in stimulating the host inflammatory response. The ability of purified CsgA (both soluble and polymerized) to induce inflammatory cytokines from human macrophages in vitro will be assessed. CsgA mutants that cannot polymerize will be used to test the hypothesis that polymerization is required for stimulating cytokine production. Mice will also be challenged IP with purified curli or CsgA, and NO production, cytokine production, and elevation of serum creatinine and conjugated bilirubin will be measured to test the hypothesis that curli are a bacterial pathogen-associated microbial pattern (PAMP) that directly stimulated the ill-regulated innate inflammatory response that characterizes septic shock.