Final Report Summary - P-USHER (Structural analysis of the newly described hybrid secretion system of the pathogen Pseudomonas aeruginosa involving the outer membrane transporter CupB3 (‘P-usher’) and the adhesin CupB5) The P-usher project deals with bacterial pathogens causing chronic infections like pneumonia, Urinary Tract Infections (UTI) or meningitis in humans. Their ‘bacterial hairs’ (pili) are large polymers of protein subunits (pilus subunits or pilins) that are displayed at the surface of the bacterial cell envelope and play essential roles in the process of host recognition and bacterial attachment, and thus are major virulence factors. Pili are assembled and secreted by sophisticated membrane embedded machines made of large macromolecular protein complexes.There are 3 major classes of specialized pilus assembly machines in Gram-negative bacteria. These are: i- the chaperone:usher (CU) pilus assembly system; ii- the Type 4 pilus (TFP) assembly machinery; and iii- the curli assembly machinery. CU pili and TFPs are the most abundant classes of pili. We proposed here to investigate the complex macromolecular systems that assemble these pili. More specifically, we proposed to work on the CupB CU pilus assembly system operating in pathogenic Pseudomonas aeruginosa and decided to expand to another assembly system, that of TFPs in Neisseria meningitidis, a Gram-negative bacterium known as the main causative agent of meningitis. Both systems are of immense biomedical importance. Knowing the high-resolution structure of any of these systems will allow designing small molecules able to bind to crucial parts of the proteins involved in the assembly process and therefore impairing pilus formation. This rational and knowledge-based approach is expected to be highly relevant to the public (research and healthcare) and private sector (pharmaceutical industry). The main objectives of this structural biology project were i) to determine the high resolution structure of the inactive form of the outer membrane pilus assembly platform termed P-usher (CupB3 protein) or homologue proteins in the same pathogenic bacterial strain (ushers CupA3, CupC3 or CupE5), and ii) determine the high resolution structure of a membrane protein complex (PilM, PilN, PilO, PilP) of the TFP assembly machinery in Neisseria meningitidis.Genes from both the CU and the TFP pathway have been cloned either individually or together on vectors for heterologous expression in E. coli. Different types of vectors, tagging, expression and purification strategies have been used to obtain a stable usher and a stable TFP protein complex suitable for crystallization trials with the ultimate aim of crystal diffraction and structure resolution. Via pull-down assays CupB4 could be identified as the chaperone of the adhesin CupB6, being probably responsible for the stabilization of CupB6 in the periplasm and targeting thereof to the outer membrane usher (as described for homologues proteins in the Fim and Pap chaperone-usher pathways).The P-usher (CupB3) is at the stage of crystallization trails, requiring further optimization of conditions suitable for crystal growth. Currently, a broad-range screen involving not only detergents but also commercially available lipids is being carried out.In the TFP part of the project a stable complex consisting of the proteins PilM, PilN, PilO and PilP could be purified and crystallized. Crystals of the native complex diffracted to 4Å and the Molecular Replacement method is currently applied with good progress for structure resolution. In parallel selenomethionin-labled crystals were grown with the aim of solving the structure via the Phasing method, the anomalous signal reaching currently to 6.8Å. In both strategies improvement in the diffraction quality of the crystals is aimed for. In addition, initial crystallization hits have been identified for a complex involving PilN, PilO and PilP. Currently, the crystallization conditions a being optimized. Last, a complex involving PilF, PilM, PilN, PilO and PilP could be purified and yields and purity are being improved.The expected final results are a high-resolution structure of an apo/inactive form of a full-length usher protein of a CU pathway and a high resolution structure of a inner membrane protein complex of the TFP assembly machinery. Both projects are in the productive phase and consolidation will be possible since they profit from further long-term funding from public bodies thanks to excellent progress for the duration of the present grant.
