Regulating amyloid formation: structural studies of the secretion and assembly of 'curli' fibres

Proteins are important building blocks for cells and organs, as well being important messengers that circulate throughout an organism. Occasionally, cells produce abnormal proteins that have tendency to form fibrous protein aggregates. The mis-folding of proteins and their subsequent accumulation into what are now termed ?amyloids? has a major detrimental effect on the health of a cell. Amyloids are therefore implicated in the causes of numerous human diseases, such as type-2 diabetes mellitus, Alzheimer?s and Parkinson?s. Despite the damaging effects of amyloid deposits, bacteria have devised a way of carefully controlling their formation, such that they are prevented from damaging the cell. Bacteria also utilise these fibrous structures, known as ?curli?, to their advantage, such as promoting biofilms and shielding themselves from extreme environmental changes. The curli assembly and secretion system includes a membrane pore that transports amyloid subunits to the outside of the cell and a number of accessory/chaperone molecules that assist in regulating the process. We propose to study the structures and interactions of the curli assembly and secretion system with a view to understanding its mode of action in atomic detail. Current treatments for amyloid diseases of humans are limited and truly effective remedies have yet to be developed, therefore they represent a major public health concern. Our studies will provide guidance for the rational design of new therapeutic approaches for preventing or treating slowing amyloid diseases in humans.

Curli represent a novel class of bacterial surface fibers that are produced by enteric bacteria such as Escherichia and Salmonella, including many pathogenic strains and species. Curli promote biofilm formation and host cell internalization as well as mediate binding to a variety of eukaryotic proteins including fibronectin and immune system components. A remarkable feature of curli is that they share biochemical and structural properties with disease-associated fibers called amyloids. Amyloids are characterized by unusually stable fibrous protein aggregates formed from a cross ?-sheet structure, and are implicated in the cause of major human diseases, such as type-2 diabetes mellitus, Alzheimer?s and Parkinson?s. Unlike disease-associated amyloid formation (amyloidogenesis), which is the product of protein misfolding, curli amyloid assembly is regulated by a highly specialised biogenesis pathway. Curli biogenesis requires specific accessory proteins, that include an outer-membrane translocator, CsgG, putative chaperone proteins CsgE and CsgF and an additional protein CsgC. The biological importance of CsgG as a secretion system is underlined by its conservation across the majority of known species within the bacterial kingdom. CsgG forms a membrane pore that recruits CsgE and CsgF and regulates the export and assembly of CsgA/B subunits. CsgC plays a role in the correct assembly of CsgA. In depth studies of curli biogenesis provide a unique opportunity to discover how cells are able to control amyloid formation. In this proposal we aim to build on a robust platform of excellent preliminary data and develop sophisticated structural and functional analyses of the curli translocation complex. We will be addressing the following research questions:

o How do curli accessory proteins prevent the premature polymerisation of subunits?
o How is amyloid assembly propagated on the cell surface?
o How is curli the translocation machinery gated for secretion?

These timely studies will not only reveal new functional and mechanistic insights, but also suggest strategies for therapeutic control of human amyloidogenesis.