Unravelling the molecular basis of subunit specificity in bacterial pilus assembly mechanisms

This is a joint grant application PI: Professor Sheena Radford Co-PIs: Dr. Alison Ashcroft and Professor Gabriel Waksman Reference: G52950X Objectives, Summaries, Technical Summary, Beneficiaries, and Case for Support for this application can be found under Professor Sheena Radford's submission

Many pathogenic Gram-negative bacteria, including E. coli and Salmonella enterica, produce proteinaceous fibres, known as adhesive pili, on their surfaces that initiate the invasion of the host and are crucial for pathogenesis. These highly ordered protein assemblies are fascinating biopolymers, being comprised of hundreds of contiguous copies of immunoglobulin domains, assembled non-covalently in a precise order. Each pilus subunit folds, in a chaperone-dependent manner, to a structure based on an incomplete immunoglobulin fold, which is subsequently completed by the donation intermolecularly of a single ?/strand from an adjacent subunit formed by its N-terminal extension (Nte). Despite the importance of this biological self-assembly process from both fundamental and applied viewpoints, the structural molecular mechanism by which proteins assemble into pili is not understood. Key questions concern how the precise order of subunits within the pilus is determined and the role of the chaperone and membrane-bound usher in controlling or adapting the assembly mechanism in vivo. Here we propose to use non-covalent mass spectrometry combined with structural analysis, kinetic and thermodynamic measurements, protein engineering and peptide design, to address these questions. Specifically, we will focus on P pili of uropathogenic E. coli associated with pyelonephritis (Pap) pili as a model, a system we have shown to be highly tractable for the experiments proposed, to determine the roles of the N-terminal extension (Nte), the pilin:chaperone complex and the soluble N-terminal domain of the usher protein and in defining and controlling the order of subunit-assembly. Finally, using our ability to purify intact functional usher protein we aim to develop an assay capable of providing the first insights into pilus assembly at a membrane surface in vitro.