Specialised macromolecule secretion systems are essential pathogenicity determinants. Type IV secretion systems and the effector molecules that they transport, are potentially ideal targets for new classes of anti-infective molecules. Fundamental research will increase our understanding of the structure and function of Type IV secretion systems. This data will allow us to both design novel strategies for anti-infectious intervention and to design screening methods for the identification of inhibitors from the Drug Discovery program of our industrial partner. The molecules identified will allow the development of strategies for the optimisation of the use of anti-infective agents, since the aim of these inhibitors is to disarm the pathogen by blocking the export of essential virulence factors, and thus make it sensitive to the host defences.
DESCRIPTION OF WORK
We have defined aspects of the biology of Type IV secretion which we will explore in depth to identify targets for new molecules, concentrating on the systems in three pathogenic bacteria, Brucella, H. pylori, and B. pertussis but also use the tools developed in the participating laboratories based on the IncP, IncF and IncW conjugation systems and the Agrobacterium VirB systems. We will develop a three pronged approach to: 1. Address the question of specificity of each system for the transported substrate which will be of the prime importance when designing inhibitors- will an inhibitor of one system be capable of inhibiting another? Saturation mutagenesis will be used to identify key residues in the protein substrates of the Helicobacter Cag and Agrobacterium VirB systems. 2. Examine several structural and functional aspects of the transport apparatus defining its role in pathogen/cell interactions. Four components of the transport machinery, the VirB2/VirB5 pilus, VirB11/VirB4 NTPases, the VirB7 lipoprotein, and VirB1 lytic transglycosylases have been chosen for investigation as specific targets. Analysis will be at different levels including the construction and analysis of mutants in appropriate virulence models, a study of the physical and functional role of the transport complex in pathogen host interactions by microscopy, biochemical and cell biology approaches, and by structural analysis by NMR and X-ray crystallography. 3. Develop a simple screening assay for general Type IV secretion inhibitors using a bacterial conjugation model. Data from 1 and 2 above will allow us to develop screens for specific inhibitors of VifB1 lytic transglycosylases, VirB7 lipoprotein processing, and for VirB4 and VirB11 NTPases. Assays for inhibitors of substrate transport will be developed based on enzyme-fusion proteins with the recently identified protein substrates of the Gaf and VirB systems. The inhibitors identified will be validated in in vitro and in vivo models.