Mechanism for carrier lipid recycling at work during peptidoglycan biogenesis – BacWall

The peptidoglycan is an essential and specific component of the bacterial cell wall. It consists of long glycan chains crosslinked by short peptide motifs. This semi-rigid polymer surrounds the cytoplasmic membrane, allowing the cell to resist internal osmotic pressure and providing each bacterium a specific shape. Peptidoglycan is a real Achilles' heel for bacteria, as evidenced by the existence of a very large number of antibacterial agents that degrade it or interfere with its biosynthesis. The majority of peptidoglycan biosynthesis enzymes are gathered within multiprotein complexes that coordinate and regulate their activities both spatially and temporally. These complexes, called elongasome and divisome, present a precise organization and dynamics during the cell cycle. Their formation and progression are mainly controlled from inside the cell by filamentous proteins forming the bacterial cytoskeleton. The constitutive elements of peptidoglycan are synthesised in the cytoplasm and then linked to the undecaprenyl-phosphate, a specific lipid that allows the translocation of the disaccharide-peptide subunits across the membrane with the aid of a flippase. The polymerisation of the peptidoglycan is then ensured by different enzymes, which catalyse transglycosylase and transpeptidase reactions and release the lipid carrier in the undecaprenyl-pyrophosphate form on the outer side of the membrane. The recycling of undecaprenyl-phosphate is essential for peptidoglycan biosynthesis because this lipid is present in a low copy number per cell and is used for the synthesis of other cell wall polymers. It is recycled by the action of phosphatases of BacA or PAP2 type, which act immediately after the action of peptidoglycan polymerases and whose plurality in the same bacterium raises the question of their respective roles. The lipid is then translocated to the inner side of the membrane by an unknown mechanism in order to enter a new cycle of biosynthesis. The recycling of the lipid carrier represents a critical point in the control of the biogenesis of the cell wall, which has only been partially elucidated. In particular, the interplay between lipid recycling enzymes and the elongasome and divisome peptidoglycan biosynthesis machineries is not yet established. The BacWall project aims to understand the overall recycling of undecaprenyl-phosphate in the model bacterium Escherichia coli. The role of BacA and PAP2 proteins in cell wall biogenesis will be addressed by complementary and multidisciplinary approaches (genetics, biophysical, biochemical and microscopic). We will study their potential flippase activity, analyse their localisation and dynamics during the cell cycle and identify their protein partners. Due to its essential role, this metabolic pathway opens the way to new antibacterial strategies. Consequently, the recycling of undecaprenyl-phosphate will also be studied in the multidrug resistant pathogen Enterococcus faecalis. We will measure the impact of an impairment of this metabolism, by antibacterial agents or by gene inactivation, on the fitness and virulence of this pathogen using mouse models of infection and colonisation. We will also elucidate the mechanism of action of a recently identified bacteriocin that targets the BacA protein of E. faecalis. This study will pave the way for the exploitation of this bacteriocin as an antibacterial agent directed against enterococci, as well as the development of engineering experiments aiming at extending or specifying its spectrum of action against other pathogenic species.