Biophysical studies of the pilus biogenesis in UPEC

Bacterial pili are essential virulence factors involved in recognition of and attachment to the host by pathogenic bacteria. Among bacterial pili, the most abundant are those assembled by the so-called "chaperone-usher (CU)" pathway. The current project is based on the investigation of the mechanism for subunit polymerization in the CU pilus biogenesis of type 1 pili which are responsible for the recognition of and attachment to the bladder by uropathogenic Escherichia coli (UPEC) and hence responsible for the onset of cystitis. An alternating recruitment model has been proposed and will be tested using a relatively recently developed methodology, namely site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy. This combination of methodologies is a powerful tool for the analysis of structures and conformational dynamics of proteins. Both continuous wave (cw) and pulsed EPR techniques will be used. Cw- EPR provides information on the local structure, the solvent accessibility, the polarity of the environment, and dynamic conformational changes in response to a perturbation. Furthermore, and importantly for this project, precise distances between spin labels up to 8 nm may be determined by pulsed EPR techniques. The Fim system which is responsible for assembling type 1 pili has been chosen as model system. Previous studies led to the assumption that both protomers of the usher (a dimer of two FimD molecules), the "secretion" and the "assisting" protomer, are alternatively involved in the chaperone-subunit recruitment. This model will be tested using nitroxide spin labels judiciously placed on the chaperone-subunit complexes and the N-terminals of the usher protomers in order to measure distances between nitroxide spin-labels. In Silico models of the interacting proteins will then be constructed using distance data derived from pulsed EPR to constrain docking simulations, and thus providing 3-dimensional structural models of complexes at different periods of pili growth. This work is expected to support the elucidation of the type 1 pilus biogenesis mechanism which can then serve as basis for the development of new therapeutic strategies.