PneumoPG

Reconstituting pneumococcal peptidoglycan synthesis Context and objectives After five decades of antibiotic use, resistance is now major concern. A case in point is the pneumococcus, which kills over a million people per year. In France, half of clinical isolates have a reduced susceptibility to beta-lactams, still the most widely used antibiotics. Beta-lactams inhibit the enzymes responsible for the assembly of the peptidoglycan, the polymer surrounding and protecting bacteria. Streptococcus pneumoniae resists beta-lactams by expressing altered targets, the penicillin-binding proteins (PBPs), with a lower affinity for the drugs. Although beta-lactam resistance in pneumococcus is relatively well understood, the physiological reactions performed by the PBPs remain largely unstudied. Understanding these reactions should help to combat resistance by resolving the following paradox. How altered 'low-affinity' PBPs retain their physiological function, while they don't react with b-lactams which are mimics of the natural substrates? To address this question, our aim is to reconstitute in vitro the final steps of the peptidoglycan assembly. Is it wise to pursue the study of old antibiotic targets, such as the PBPs? Yes, more than ever, as concluded after a decade of postgenomic quest for new antibiotics. Indeed, after submitting 67 new targets to half a million compounds and failing to uncover promising drug candidates, D. L. Pompliano from GSK decided that 'discovering novel strategies of inhibiting pharmacologically validated targets is a more rational strategy'. Corroborating this hard earned empirical conclusion, a study of gene essentiality in Salmonella found that remarkably few metabolic pathways are essential, and that these pathways are in fact targets of the known antibiotic classes. The peptidoglycan, is a giant macromolecule composed of glycan chains of repeated disaccharides cross-linked by peptide bridges. The assembly is catalyzed by penicillin-binding proteins (PBPs) from a lipid-linked disaccharide pentapeptide (lipid II). Class B PBPs are monofunctional enzymes thought to perform the peptide cross-bridging of glycan strands only, whereas class A PBPs are bifunctional and have been shown to perform both glycan strand polymerization from the precursor (lipid II) and peptide bridge cross-linking. The evidence for the activity of class B PBPs is limited and indirect, and the reaction has never been achieved in vitro with susbstrates closely resembling the actual peptidoglycan. SEDS proteins (for shape, elongation, division and sporulation), are integral membrane proteins that very likely participate with class B PBPs to peptidoglycan assembly, as suggested y genetic arguments. We hypothesize that class B PBP activity requires the presence of an associated SEDS protein. The class B PBPs from S. pneumoniae, PBP2x and PBP2b will be produced recombinantly, together with their respective SEDS protein, FtsW and RodA. The activity will be assayed either using prepolymerized glycan chains, or by including a class A PBP and the precursor substrate. Assays will be set up, with our collaborators, with detergent solubilized proteins, with reconstituted vesicles and on tethered lipid bilayers. The role of possible regulatory proteins, such as the complexes DivIB/FtsL/DivIC or MreC/MreD will be investigated as well. The project is particularly challenging in that all the proteins involved are membrane proteins, so that a major hurdle will be the preparation appropriate amounts of material. However, taking the risk of tackling the muliple membrane proteins participating to cell wall synthesis is now necessary to make significant biochemical progress in the field. Reconstituting in vitro peptidoglycan assembly catalyzed in part by class B PBPs will open the way to investigations of the functions of the different class A and B PBPs, the SEDS proteins (FtsW, RodA), into the enzymatic mechanisms and into the role of possible accessory proteins (DivIB/FtsL/DivIC and MreC/MreD). We wish to identify the elements that maintain the enzymatic activity of altered PBPs with low reactivity for beta-lactams, whether they are features of the substrates or the presence of accessory proteins. The role of alternative substrates in the expression of the resistance to beta-lactams mediated by altered PBPs will be investigated as well.