PeptidoNMR

Essential component of the bacterial cell wall, peptidoglycan is the major target of most antibiotics. This biopolymer also contains key chemical features signaling the innate immunity systems for microbial invasion. Both arguments are plain justification for the actual focus of fundamental research projects towards the determination of the structure and function of this gigantic (3 × 106 kDa) polymer. The ultimate goal of such studies is a better understanding of host-pathogens interactions and development of new efficient therapeutic strategies. To-date the chemical structure and inter-species diversity of peptidoglycan are well-known. Macroscopic organization of the peptidoglycan network on the contrary remains a field of active debates and questions, while pivotal to assess and understand the mechanisms of interaction with protein partners at the molecular level. It is thus crucial to manage an experimental approach able to yield the peptidoglycan structure alone as well as in interaction with other biomolecules. The obtained preliminary results establish solid-state NMR (coupled to efficient isotope-labeling techniques of the bacterial cell-wall) as a method of choice to reach this objective. In fact spectra collected on isolated sacculi (or even on entire bacteria) show that information can be retrieved at the atomic scale. Facing these new perspectives, we propose to develop in a first step a strategy that will allow the analysis and determination of E. coli peptidoglycan structure and dynamics. Once worked-out this strategy will be applied on peptidoglycans issued from diverse bacterial sources in order to understand the relationships between chemical modifications and physico-chemical/mechanical properties of the bacterial cell-wall (thickness, reticulation, antibiotics resistance…). In a second part of the project, the interaction between model proteins (implicated in recognition and morphogenesis of the polymer) and peptidoglycan will be studied by liquid-state and solid-state NMR. These two complementary approaches will lead to the differentiation of molecular processes implying only isolated specific chemical fragments from macroscopic phenomena involving the extended polymer network. In order to turn the identified molecular interactions into a physiological and integrated view at the cellular level, similar studies will be directed on fully complete bacteria (instead of isolated sacculi). In addition to address specific questions on the morphogenesis or growth of the bacterial cell-wall, the issue of this last part is to tackle biological problems of fundamental importance such as bacterial resistance and virulence mechanisms or molecular determinants implied in host-pathogens interactions.