Biophysical and structural characterisation of functional bacterial amyloid secretion systems

Abstract

Amyloids are characterised by their innate capacity to aggregate into insoluble fibrils, which are commonly recognised for their cytotoxicity and association to neurodegenerative diseases. Their unique physicochemical properties are exploited by bacteria for various functional roles, including the formation of extracellular matrix that is linked to biofilm construction and antimicrobial resistance. The curli fimbriae of Escherichia coli was the first functional bacterial amyloid (FuBA) to be discovered. Two operons encode a curli secretion system that is comprised of several distinct proteins including curli-forming subunits, chaperones, amyloid inhibitors, and an outer-membrane (OM) transporter. Amyloid secretion systems enable FuBA fibril formation, whilst minimising their cytotoxicity to the host-cell. A functional amyloid in Pseudomonas (Fap) operon was recently identified in the Pseudomonas genus encoding a novel FuBA secretion system. Unlike the curli system, detailed insight into the Fap system is lacking. The curli and Fap secretion systems export biochemically similar amyloid forming subunits; however, their FuBA secretion systems are genetically distinct. The biophysical studies of this thesis sought to provide further insight into the structure and function of FapF, a uniquely structured OM transporter, as well as FapD, a periplasm-residing protein that is predicted to serve a proteolytic and chaperoning role within the Fap system. Previous reports demonstrate the evolutionary co-conservation of FapF and FapD, suggesting their functional co- dependence. In this thesis, a combination of biophysical techniques, including nuclear magnetic resonance (NMR) spectroscopy, are used to demonstrate the presence of a unique, asymmetric, parallel trimeric coiled-coil domain within the periplasmic N- terminus of FapF. Furthermore, a transient interaction between FapF and FapD was identified under solution conditions. Protocols that enable the near-native study of the FapF OM domain by solution-state NMR spectroscopy were also optimised. These studies pave the way for future research to enhance our mechanistic understanding of FuBA secretion systems, with aim to increase our capacity to modulate FuBA formation.