Comparative flux control through the cytoplasmic phase of cell wall biosynthesis

Abstract

The introduction of antibacterial drugs in the middle of the last century heralded a new era in the treatment of infectious disease. However the parallel emergence of antibiotic resistance and decline in new drug discovery threatens these advances. The development of new antibacterials must therefore be a high priority. The biosynthesis of the bacterial cell wall is the target for several clinically important antibacterials. This extracellular structure is essential for bacterial viability due to its role in the prevention of cell lysis under osmotic pressure. Its principal structural component, peptidoglycan, is a polymer of alternating N-acetyl-glucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc) residues crosslinked by peptide bridges anchored by pentapeptide stems attached to the MurNAc moieties. The biosynthesis of peptidoglycan proceeds in three phases. The first, cytoplasmic, phase is catalysed by six enzymes. It forms a uridine diphosphate (UDP) bound MurNAc residue from UDP-GlcNAc and attaches the pentapeptide stem. This phase is a relatively unexploited target for antibacterials, being targeted by a single clinically relevant antibacterial, and is the subject of this thesis. The Streptococcus pneumoniae enzymes were kinetically characterised and in silico models of this pathway were developed for this species and Escherichia coli. These models were used to identify potential drug targets within each species. In addition the potentially clinically relevant interaction between an inhibitor of and feedback loops within this pathway was investigated. The use of direct parameter estimation instead of more traditional approaches to kinetic characterisation of enzymes was found to have significant advantages where it could be successfully applied. This approach required the theoretical analysis of the models used to determine whether unique parameter vectors could be determined. Such an analysis has been completed for a broad range of biologically relevant enzymes. In addition a relatively new approach to such analysis has been developed