Tyrosine phosphorylation networks in bacterial physiology and virulence – TYR-PHOS.NET

Top down: two-hybrid interactomics, phosphoproteomics
Bottom up: structural studies, biochemistry, genetics, bacterial physiology

We managed to establish the interaction network of BY-kinases using yeast two hybrid. In the following months, we are expecting the results from quantitative phosphoproteomics (in collaboration with UNI Tuebingen, Germany). As expected, our functional analysis revealed new modulators of BY-kinases.

The discovery of new modulators for BY-kinases is a major breaktrough, and will spark new research, trying to establish the distinct roles for multiple modulators in cell physiology. In S. pneumoniae, new vistas have been open regarding the role of BY-kinases in cell cycle control.

The ANR funding has allowed us to date to publish 7 peer reviewed papers and deliver 3 oral communications with the results of this project.

TyrPhos.net is a project focused on investigating bacterial tyrosine kinases (BY-kinases) and their involvement in bacterial virulence and other aspects of cellular regulation. The project is established by four research groups with an outstanding collaboration record, and a clear leadership status in the domain. The partners comprise the microbiologists laboratories of Prof. Ivan Mijakovic and Dr. Christophe Grangeasse, both with significant contributions in the functional characterization of BY-kinases, the structural biology group of Prof. Sylvie Nessler who has solved the first BY-kinase structure, and the interactomics group of Dr. Marie-Francoise Noirot-Gros. The rationale behind this project is to take a global approach, and decipher the network of interactions in which BY-kinases partake in the cell, including the kinase substrates, modulators and other putative interactants. The fundamental/applied duality of our project is reflected in the choice of microorganisms. For the applied aspect of the project we have chosen to work on one of the most important human pathogens causing life-threatening disease, Streptococcus pneumoniae, whereas for the more fundamental part of our research we have chosen to focus on the related Gram-positive model organism Bacillus subtilis, where the BY-kinase interaction network has already been characterized to some extent. In fact, this duality will allow us to apply the same analytical pipeline on both organisms, and carry the momentum already existing in B. subtilis research to make rapid advances in S. pneumoniae. Our analytical strategies will strongly rely on specific competencies and preliminary results of our research consortium: available phosphoproteomes (indicating BY-kinase substrates), genomic libraries for yeast two-hybrid, resolved BY-kinase structures, etc. We will use the synergy created between our teams to apply molecular biology, interactomics and structural biology approaches to identifying BY-kinase interactants, characterizing new substrates and modulators, investigating their biological role, and explaining the regulatory mechanisms underlying kinase/substrate and kinase/modulator interactions in molecular detail. We will enjoy strong support from fully committed international collaborators (work agreements in annex) for key inputs in this project, such as quantitative phosphoproteomics and infection assays on murine models. On the fundamental aspect, our project will provide interaction networks that will add a new regulatory layer to existing systems biology models and thus contribute to the ultimate understanding of the inner workings of a bacterial cell. On the applied side, this structure-function characterization of BY-kinases is expected to open new venues towards specific inhibition of these idiosyncratic bacterial enzymes and thus pave a way towards new antibacterial drugs.