The enzymology of Streptococcus pneumoniae peptidoglycan polymerisation

Abstract

Bacterial cell survival depends on intact peptidoglycan, an extensive cell wall polymer of alternating N-acetylglucosamine and N-acetylmuramic acid residues, cross-linked by short peptides. Peptidoglycan biosynthesis is a viable and validated antimicrobial target; the intracellular, membrane-bound and extracellular synthetic stages provide a multitude of enzymes for interception with inhibitors. The ultimate phase of peptidoglycan biosynthesis occurs on the extracellular face of the cytoplasmic membrane and involves the polymerisation of Lipid II (the monomeric peptidoglycan precursor) by the transglycosylase and transpeptidase activities of the Penicillin-Binding Proteins (PBPs). The work presented in this thesis primarily focused on the biochemical characterisation of the integral membrane proteins Streptococcus pneumoniae PBP1a, PBP2b and PBP2x. These enzymes are clinically relevant; they are essential targets of !-lactam antibiotics and also mediate resistance against this important antimicrobial class. Full-length and truncated forms of the PBPs were cloned, expressed and purified to high levels. Two novel spectrophotometric assays were designed and developed to study the enzymology of the individual transglycosylase and transpeptidase activities of the PBPs with their natural substrate, Lipid II. Preliminary kinetic characterisations of the bifunctional PBP1a transglycosylase activity were performed and assay conditions were optimised to recreate an in vivo environment. PBP1a active site mutants revealed that transglycosylase activity was elevated in the absence of a functional transpeptidase domain. PBP1a and PBP2x exhibited transpeptidase activity with an apparent substrate preference for glycan polymers over Lipid II. PBP2x transpeptidase activity was not detected. The recorded rates of PBP activity were insufficient to support bacterial cell integrity, highlighting a gap in the understanding of PBP requirements. Finally, the PBPs were subjected to crystallisation trials for structural characterisations. This work provides an excellent foundation for the analysis and elucidation of PBP specificities. Future information attained could contribute to the design of novel inhibitors, alleviating the global threat of antibiotic resistance