Escherichia coli (E.coli) and other enteric bacteria are a major cause of human diseases. Biofilm formation contributes greatly to bacterial persistence and antimicrobial resistance in the host. Many Enterobacteriaceae, such as E. coli, produce functional amyloid fibers called curli as a major proteinaceous component of their extracellular matrix. It is now clear that functional amyloids are widespread, with examples found throughout cellular life. The curli system in E. coli provides a rich and high throughput genetic and biochemical toolbox for the study of amyloid formation. The work has contributed to a curli assembly model where the main fiber component CsgA and the minor subunit CsgB are secreted through the outer membrane-located CsgG. CsgB attaches to the surface of the cell and templates the folding of CsgA into an amyloid fiber. CsgA subunits that inappropriately polymerize in the periplasm are inhibited from amyloid accumulation via CsgC. The exquisitely- controlled curli biogenesis system ensures that E. coli is not exposed to the potentially cytotoxic outcomes of amyloid formation. Uncontrolled or inappropriate amyloid formation results in several neurodegenerative diseases, including Parkinson?s. The hallmark of Parkinson?s disease is the amyloid aggregation of alpha-synuclein, although it is unknown how amyloid formation is initiated. Colonization of mice with curli amyloid producing bacteria results in alpha-synuclein amyloid formation and Parkinson?s like symptoms. Furthermore, purified CsgA protein can accelerate alpha-synuclein amyloid formation in vitro. Therefore, it is imperative to learn how E. coli controls curli amyloid formation and how CsgA can accelerate alpha-synuclein amyloid formation. Interestingly, CsgC from E. coli can inhibit CsgA and alpha-synuclein amyloid formation. Knowledge gained from the following experiments will have implications for microbial pathogenesis, general protein folding, and amyloid biogenesis, thus paving the way for new therapies that rationally target these critical biological processes.
In Aim 1 The mechanism and specificity of CsgC will be revealed, including how CsgC functions to inhibit CsgA and alpha- synuclein polymerization at low stoichiometric ratios.
In Aim 2 The relationship between the intrinsic ability of CsgA to form amyloid and its ability to accelerate alpha-synuclein amyloid formation will be assessed. Finally, in Aim 3 CsgA and CsgC-like proteins in the sequenced genomes of the human gut microbiota will be identified and biochemically characterized. Together the successful completion of these aims will give an overall understanding of the mechanism of amyloid inhibitory activity and the interactions between bacterial amyloid formation and neurodegenerative diseases.
Many Gram-negative bacteria encode the genes necessary for the formation of functional amyloid fibers called curli that contribute to biofilm formation. Curli amyloids produced by gut bacteria can accelerate Parkinson?s-like symptoms in mice. Work in this grant is focused on understanding curli biogenesis and how curli can interact with human proteins to initiate pathogenic pathways that ultimately lead to neurodegeneration.
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