A systems-based and whole cell tomography study of Type IV pilus biogenesis in Pseudomonas aeruginosa. (360G-Wellcome-093388_Z_10_Z)

This proposal aims to develop a novel approach to the study of the biogenesis of type IV pili in Pseudomonas aeruginosa (Pa). It is driven by the identification of small molecule inhibitors of the process, which will then be employed to generate a quantitative, systems-based model for pilus formation and to trap the biogenesis complex in particular structural states which can be observed in vivo by cryoelectron tomography. Inhibitor screening will be carried out by i) high throughput screening o f Pa ii) structure-based drug design, by experimental location of chemical fragments using X-ray crystallography applied to recombinant pilus biogenesis proteins, followed by seaches of chemical databases to identify suitable inhibitors. The effect of these inhibitors on pilus production and surface pilus expression will then be evaluated, the data quantified and used to construct a theoretical model for pilus biogenesis. Finally, we will link the effect of specific inhibitors to structural ch anges in vivo using cryoelectron tomography to calculate 3D reconstructions of Pa, from which we can draw inferences concerning the spatial disposition of pilus fibres and the formation of transient biogenesis complexes.