Partner 1 (Pansegrau)
Scientific contribution to the project
Molecular cloning of VirB11-like NTPases from H. pylori, B. suis and A. actinomycetemcomitans. Purification of VirB11-like NTPases following overexpression in E. coli. Molecular cloning of the NTPase subdomain of CagE from H. pylori. Purification of the subdomain following overexpression in E. coli. Characterisation of type IV system-related NTPases and development of screening procedures for inhibitors. X-ray crystallisation studies and structural analysis of the purified NTPases. Development of specific inhibitors by molecular modelling and high-throughput screening procedures.
Partner 1 has a long experience in the field of horizontal gene transfer. He has extensive knowledge on heterologous protein overexpression, purification, setting-up of enzyme assays and characterisation of enzymatic activities. X-ray crystallisation-studies as well as high-throughput screening studies will be done in collaboration with specialised laboratories located at the Chiron main site, Emeryville.
The Immunobiological Research Institute of Siena (IRIS) is well prepared for the proposed workpackages. Facilities for protein purification and enzymological characterisation are available. Work with human pathogenic bacteria and genetically modified microorganisms is performed routinely and according to Italian and European laws and regulations.

Scientific contribution to the project
Cloning, expression and generation of isogenic mutants of the individual cag genes, including selection of clinical isolates. Assay for translocation of substrate in eukaryotic cells in culture. Functional complementation using a recently developed shuttle vector. In vivo animal model of infection and pathogenesis. Cellular proteins interacting with the substrate identified by a two-hybrid system. Functional dissection of cag Type IV secretion system: isolation of new substrates for translocation and Type IV-specific chaperonins. Purification of the cag complex and determination of the structure by confocal, scanning, transmission microscopy and x-ray crystallography. Developing screening procedures. Providing B. pertussis mutants and DNA with immunological reactives to other partners.
Prof. Covacci has a long term experience with molecular biology of pathogenic bacteria and with the cellular aspects of bacterial infections. He is a recognised world expert on Type IV secretion systems. Other selected areas of expertise include: genetic manipulation of micro-organisms and genomics (computation, global expression, proteomics). Identification and purification of subcellular structures and resolution of the complexes at biochemical level. Early events in the signalling cascade. Bacterial mechanisms of pathogenesis using unicellular and multicellular host (including transgenic mice). IRIS is at forefront of research on new generation vaccines. Facilities for DNA, protein, mass production, animal care are already existing. The Immunology department complements most of the activities of the molecular biology department. Structures for development and production of therapeutics are based in Siena, Marburg and Emeryville (USA).

Partner 2 (O’Callaghan)
Scientific contribution to the project
Cloning and mutating of the Brucella virB2 and virB5. Purification and analysis of the Brucella pilus. Construction and analysis of Brucella/Bordetella cross complementation systems. Analysis of the virulence of mutants and genetic constructs in cell culture models. Analysis of the effects of anti-infective molecules in cell culture models. Providing Brucella and A. actinomycetemcomitans strains mutants and DNA to other partners.
INSERM U431 studies principally the facultative intracellular bacterial pathogen Brucella. All aspects of the biology of Brucella are investigated, including genome structure and organisation, the identification of virulence factors, the mechanisms of invasion and intracellular survival, the effects on the biology of infected cells, the host immune response to infection and vaccine development. Recently, Partner 2 has identified a Type IV secretion system playing a key role in the virulence of Brucella.
INSERM U431 is specialised in the study of pathogens at the molecular level. The Unité has one BL3 and three BL2 containment laboratories. Facilities for cell biology, protein engineering and protein purification are available. Pr M Ramuz is head of Microbiology at the Montpellier/Nimes University teaching hospital and is in daily contact with the realities of treatment of infectious diseases and the spread of antibiotic resistance.

Partner 3 (García Lobo)
Scientific contribution to the project:
Assay of the conjugative ability of Brucella virB system. Construction of Brucella mutants.
Complementation of the system with missing components from other loci in the Brucella genome or with heterologous systems.
Study of molecular events leading to DNA transfer with emphasis on inhibition.
Study of mutants and inhibitors in virulence assays in cultured cells and in mice.
Qualification and experience of the team
The team in the Department of Molecular Biology of the University of Cantabria has been working the last 15 years in the areas of plasmid molecular biology and bacterial pathogenesis. This has produced relevant results in the areas of transposition, site-specific recombination, plasmid conjugation, pathogenicity of B. abortus and characterisation and improvement of the B. abortus B19 vaccine. In the last three years the team participated in a CEE project in which vectors have been constructed for gene integration into the BCG genome. According, with this record we consider we have both the technical experience and the relationship to other groups in the field to reach the objectives proposed.
The University of Cantabria has all the facilities for molecular biology, protein chemistry and cell biology. Partner 3 has a BL3 confinment laboratory. A BL3 animal facility is under construction and will be functional for the predicted start of the project.

Partner 4 (Koraimann)
Scientific contribution to the project
Purification, and structural analysis of VirB1 lytic transglocylases.
Development and validation of a screening proceedure for lytic transglocylase inhibitors.
Providing purified proteins and raised antisera to the partners.
Partner 4 is a recognised expert in the field of horizontal gene transfer and bacterial conjugation. He and his group have studied the role of lytic transglycosylase like enzymes in both bacterial conjugation and virulence. Participating scientists have expertise in the fields of protein purification, and structural analysis with CD-spectrophotometry, NMR, X-ray crystallography.
The Institute of Microbiology is well suited for the proposed research project. All necessary equipment for conducting gene cloning, protein expression and protein purification is present at the research institution. Work with micro-organisms, especially with E. coli laboratory strains is performed according to GSP/GLP standards. Work with GM micro-organisms is performed routinely and according to the Austrian and European regulations and laws.

Partner 5 (Baron)
Scientific contribution to the project
Development of a screening system for inhibitors of type IV secretion-mediated plasmid transfer. Dissection of the molecular target of type IV secretion inhibitors isolated in different work packages in A. tumefaciens and pKM101-carrying bacteria. Cross-complementation of virB2- and virB5-deficient mutants of A. tumefaciens, and of similar insertion mutants of pKM101, with pilus components from homologous systems. Providing mutant A. tumefaciens strains, pKM101-derivatives and plasmids to other partners.
Partner 5 is an expert in the genetic and biochemical analysis of the mechanism of type IV secretion in A. tumefaciens and IncN plasmid pKM101-carrying bacteria. Focus of recent work is the analysis of surface structures, which are probably involved in cell-to-cell contact formation necessary for transfer of virulence factors to host cells. His expertise in the analysis of components of type IV secretion systems will be applied in different work packages of the project.
The Department of Microbiology at the University of Munich offers state-of-the-art facilities for the characterisation of type IV secretion systems using tools of molecular biology, biochemistry (protein purification, immunochemical techniques), cell biology (immunofluorescence microscopy, image processing) and genetic manipulation of plants (growth chambers).

Partner 6 (Hooykaas)
Scientific contribution to the project
Determination of the transport signal mediating transport of Vir-proteins (VirF,VirE2) through the VirB-pilus by deletion mutagenesis and alanine scanning, using a recently developed genetic test. In silicio analysis of the available Ti-plasmid sequence for other proteins sharing this "consensus" sequence, as well as comparison with related Brucella and Helicobacter and B. pertussis systems. Possible identificaton of transported proteins in these systems and test for transport in the heterologous Agrobacterium test system. Genetic (yeast two hybrid system) and biochemical analysis with the transport consensus for retrieval of the interaction partner in the pilus (VirD4?) and determination of the interaction domain. Development of screening procedures for inhibitors of substrate secretion using substrate-enzyme fusion proteins.
Prof. Paul Hooykaas occupies the chair of genetics at Leiden University. He has been involved for many years in the studies of Agrobacterium-mediated plant transformation and the development of plant vector systems and is considered as a world expert in the field. The laboratory of Paul Hooykaas at the Institute of Molecular Plant Sciences (IMP) at Leiden University, has all the modern facilities required for the proposed research. Funds are requested to hire a postdoc and support from a technician.