Le métabolisme lipidique comme senseur du stress nutritionnel bactérien ? mécanisme moléculaire de régulation des enzymes de synthèse du (p)ppGpp – LipidStress

Bacteria adapt constantly their growth rate to the nutritional status of their environment. In case of a sudden starvation stress, they start the stringent response characterized by growth arrest and activation of stress response genes. Both growth rate control and the stringent response are mediated primarily by the (p)ppGpp nucleotides playing the role of second messenger. This function is highly conserved in all bacteria and is central in the adaptation of pathogens to their host environment and in the different phases of infection cycles. The synthesis of (p)ppGpp nucleotides is performed by specialized enzymes able to detect and respond to various starvation events. However, the molecular mechanisms involved are poorly understood. We have shown that in beta- and gamma-proteobacteria, the Acyl Carrier Protein (ACP), a central cofactor of fatty acid biosynthesis, interacts with and regulates the (p)ppGpp control enzyme SpoT. We have also shown that stringent response is linked physically and genetically with lipid metabolism by other ways than the ACP/SpoT interaction. Finally, it has been shown independently that membrane stress modifies (p)ppGpp levels in Pseudomonas and that fatty acid intermediates trigger the transitions between the different phases of Legionella infection cycle. Therefore, stringent response and growth rate control seem to be closely linked to lipid metabolism through a complex network of physical and genetic interactions. Our project is to decipher the mechanism of the regulation of (p)ppGpp synthesis enzymes by signals in the lipid metabolism and to understand the integration between lipid metabolism and growth rate control by a network of protein-protein interactions. These questions will be answered at two levels, a reductionist one aiming at reconstituting the events in vitro and a more global one, dealing with the integration at the network level. (I) We will first dissect the molecular mechanism of SpoT regulation by ACP. We have to identify the lipidic signal carried by ACP that is recognized by SpoT and explain how the interaction triggers conformational change in SpoT resulting in (p)ppGpp synthesis activation. Other ligands may play a role in SpoT regulation and will be studied altogether (the other (p)ppGpp synthesis enzyme in E. coli RelA and the small GTPase Obg/CgtA to start with). Indeed, we have preliminary data showing that RelA is involved in the SpoT-dependent response, which is against the accepted model that RelA and SpoT proteins function independently of each other in order to respond to separate starvation events (amino acid for RelA, all the others for SpoT). For CgtA, we have to take into account the recent literature suggesting that CgtA binds SpoT and maintains it in a (p)ppGpp degradation state. Because CgtA may be linked to the membrane, it is of interest for us. (II) We will characterize the network of protein interactions linking (p)ppGpp synthesis enzymes, their regulators, and lipid metabolism enzymes. We have developed the tools and acquired the required equipment in order to be able to follow such a network from the regulation of the gene expression of its constituents, to the protein-protein interactions. When the network is fully described and cleaned for high confidence interactions, we will study the dynamic of this network in response to stress. The classical RelA-dependent stringent response will be triggered by amino acid starvation, the SpoT-dependent stringent response will be triggered by cerulenin addition inhibiting fatty acid. Finally, we will also test stresses affecting the membrane homeostasis that have also been described to trigger a stringent response. In reverse, studying the dynamic of this network will help understanding how the lipid metabolism is shut down during stringent response, and in particular what is the role of direct interactions (microcompartimentalization) between (p)ppGpp synthesis enzymes and the enzymes targeted by (p)ppGpp such as PlsB. Our results will be of fundamental interest for the understanding of protein interaction network dynamic and bacterial growth control. Through this, they will also give input into the role of stringent response in infection and dormancy. Finally, fatty acid synthesis being targeted by many antibiotics, understanding the link between lipid metabolism and bacterial growth control may be of biomedical interest.