Structural biology of bacterial functional amyloid formation

Amyloids are proteinaceous aggregates best known for their role in degenerative diseases involving protein misfolding. Research into amyloid has intensified in recent times due to its prominence in many debilitating human diseases and limited understanding of the causes. The discovery of functional amyloids in a broad range of species has enhanced our understanding of amyloid, of these the curli system of E. coli has been extensively studied, in this system CsgC was identified as a potent inhibitor of amyloid. An additional protein was discovered in some curli operons in other species termed CsgH and warrants further study. A morphologically similar but genetically distinct bacterial functional amyloid system was identified in Pseudomonas encoded by the fapABCDEF operon and termed amyloid-like fibres (Alf). The study of functional amyloid has the potential to provide insights into how amyloid can be controlled. The aims of this thesis were to investigate the novel functional amyloid system of Pseudomonas with a view to structural and functional characterisation of the individual components. The structure and function of the CsgH protein were also studied by nuclear magnetic resonance (NMR) and the ThioflavinT (ThT) amyloid fibrillation assay. Constructs were produced for all the Alf proteins and the more structured components, FapD and FapF, were optimised to produce constructs for structural study. The structure of CsgH was solved successfully using NMR and showed that the protein shared a similar tertiary structure to CsgC. The function of the CsgH was shown to be similar to CsgC inhibiting amyloid formation by CsgA at substoichiometric concentrations. Mutagenesis, ThT assay and NMR were used to show that CsgH and CsgA interact and that several charged residues have an important role in function. It was also interesting to note that CsgH was capable of inhibiting amyloid formation by the FapC amyloid protein of Pseudomonas.Open Acces