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

It has been known for more than half a century that some pathogenic bacteria (e.g. Escherichia coli and Salmonella enterica) produce hair-like structures on their surfaces that promote haemagglutination. These 'pili' or 'fimbriae' contain at their tip a special protein molecule, called an adhesin, which allows the bacteria to attach to the host cell surface, thereby initiating the process of infection. These hair-like structures are formed from the assembly of hundreds of copies of protein subunits with similar structure into a long polymer chain linked by non-covalent interactions between each subunit. A complex folding and assembly machinery known as the chaperone-usher pathway is responsible for the assembly of these pili, typified by the P pili of uropathogenic E. coli. Key components of this pathway include (i) a specific chaperone which is needed to fold the pilus subunit into an assembly-competent conformation and to prevent premature subunit assembly in the periplasmic space; (ii) the pilus subunits themselves which, in the case of P pili, involves six different subunit types; and (iii) an outer membrane-embedded usher protein, which acts as the assembly platform where chaperone:pilus subunit complexes are brought to the basal end of the growing pilus for subsequent incorporation into the growing fibre. In a manner that is currently not understood, and is entirely independent of ATP, the usher catalyzes pilus assembly, adds a defined, specially chosen subunit to the base of the growing pilus, and extrudes it to the outer surface of the bacterium. The usher also serves as an anchor, tethering the pilus to the bacterial surface, arming the bacterium for attack. In recent years the X-ray structures of several pilus chaperones have been elucidated, along with many pilus subunits (known as pilins). Fascinatingly, these studies have shown that pilins have a common structure, based on an immunoglobulin (Ig) fold. However, whilst the canonical Ig fold contains seven ?-strands, the pilins have only six strands and their structure is thus incomplete and unstable. One role of the chaperone is to donate a ?-strand to the pilus subunit, temporarily completing its content of ?-strands. During pilus assembly the chaperone's ?-stand is then displaced from its binding site by the incoming pilin subunit, which forms a new ?-strand from its initially disordered N-terminal region (known as the N-terminal extension (Nte)), resulting in a very stable, intermolecular chain of Ig molecules. The ordered assembly of bacterial pili provides a fascinating problem in structural biology and molecular recognition that has far-reaching impact. First, it poses important fundamental questions about molecular self-assembly mechanisms and asks to what extent these are dictated by the biophysical properties of the amino acid chain (its sequence, or the kinetics or thermodynamics of the interactions) and how these are controlled, modulated and/or coordinated in vivo. Secondly, and equally importantly, elucidation of the molecular mechanism of pilus formation has immense importance for the possible development of new anti-microbial agents against bacterial infection mediated by pili. In this proposal we describe a series of experiments involving three applicants with complementary expertise that aim to reveal how proteins assemble into pili in unprecedented detail. Specifically, our aims are to determine the role of the N-terminal extension (Nte), the chaperone:subunit complex, and the soluble N-terminal domain of the membrane-bound usher 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.

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.