The role of species-specific modifications in peptidoglycan biosynthesis

Penicillin binding proteins (PBPs) are responsible for the final extracellular steps transglycosylation and transpeptidation) in the biosynthesis of peptidoglycan, the essential cell-wall carbohydrate polymer, from its Lipid II precursor. They are excellent targets for antibiotics due to their essentiality for cell viability in most bacteria. Genus- and species-specific variation in the chemical structure of Lipid II can have significant consequences for the formation and metabolism of the peptidoglycan sacculus by the PBPs. Characterisation of these enzymes from Grampositive bacteria, including the substrate-enzyme interactions involved, is essential in both understanding the mechanisms of peptidoglycan biosynthesis and contributing to the development of new antimicrobials. The work presented in this thesis focuses primarily on the substrate specificity of the Streptococcus pneumoniae bifunctional PBPs; PBP1a and PBP2a; both recombinantly expressed and purified to a high level. The preference, by both enzymes, for amidated Lipid II as a transglycosylase substrate was identified by two complementary assay systems. A novel spectrophotometric assay was used to observe transpeptidation by S. pneumoniae PBP1a in a continuous manner; the first time this has been achieved for a Gram-positive PBP, and with potentially farreaching implications in the future of antibiotic discovery. Attempts were made to synthesise dipeptide branched Lipid II, implicated in β-lactam resistance, as substrates for the bifunctional PBPs. The role of amidation in Staphylococcus aureus peptidoglycan biosynthesis was also investigated, and variation in the requirement for this modification between the monofunctional transglycosylase MGT and bifunctional PBP2 identified. Two novel monosaccharide compounds were identified as inhibitors of transglycosylation. This thesis provides an important basis for understanding the peptidoglycan biosynthesis mechanisms of two globally important pathogens. This insight, and future work leading from it, could contribute to the development of new antibiotics, helping to reduce the global threat of antimicrobial resistance

Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function

Substrate and Stereochemical Control of Peptidoglycan Cross-Linking by Transpeptidation by Escherichia coli PBP1B

Penicillin binding proteins (PBPs) catalyzing transpeptidation reactions that stabilize the peptidoglycan component of the bacterial cell wall are the targets of β-lactams, the most clinically successful antibiotics to date. However, PBP-transpeptidation enzymology has evaded detailed analysis, because of the historical unavailability of kinetically competent assays with physiologically relevant substrates and the previously unappreciated contribution of protein cofactors to PBP activity. By re-engineering peptidoglycan synthesis, we have constructed a continuous spectrophotometric assay for transpeptidation of native or near native peptidoglycan precursors and fragments by Escherichia coli PBP1B, allowing us to (a) identify recognition elements of transpeptidase substrates, (b) reveal a novel mechanism of stereochemical editing within peptidoglycan transpeptidation, (c) assess the impact of peptidoglycan substrates on β-lactam targeting of transpeptidation, and (d) demonstrate that both substrates have to be bound before transpeptidation occurs. The results allow characterization of high molecular weight PBPs as enzymes and not merely the targets of β-lactam acylation

Carbohydrate scaffolds as glycosyltransferase inhibitors with in vivo antibacterial activity

The rapid rise of multi-drug-resistant bacteria is a global healthcare crisis, and new antibiotics are urgently required, especially those with modes of action that have low-resistance potential. One promising lead is the liposaccharide antibiotic moenomycin that inhibits bacterial glycosyltransferases, which are essential for peptidoglycan polymerization, while displaying a low rate of resistance. Unfortunately, the lipophilicity of moenomycin leads to unfavourable pharmacokinetic properties that render it unsuitable for systemic administration. In this study, we show that using moenomycin and other glycosyltransferase inhibitors as templates, we were able to synthesize compound libraries based on novel pyranose scaffold chemistry, with moenomycin-like activity, but with improved drug-like properties. The novel compounds exhibit in vitro inhibition comparable to moenomycin, with low toxicity and good efficacy in several in vivo models of infection. This approach based on non-planar carbohydrate scaffolds provides a new opportunity to develop new antibiotics with low propensity for resistance induction

Prospects for novel inhibitors of peptidoglycan transglycosylase

The lack of novel antimicrobial drugs under development coupled with the increasing occurrence of resistance to existing drugs by community and hospital acquired infections is of grave concern. The biosynthesis of the bacterial cell wall has provided a rich vein of antimicrobial targets in the past but relatively little development has been directed toward the transglycosylase step of this process. In this article, we review the assay methods developed for the key enzymes involved and review recent novel chemical inhibitors discovered in relation to both the lipidic substrates and natural product inhibitors of the transglycosylase step

In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA)

The O-acetylation of the essential cell wall polymer peptidoglycan occurs in most Gram-positive bacterial pathogens, including species of Staphylococcus, Streptococcus and Enterococcus. This modification to peptidoglycan protects these pathogens from the lytic action of the lysozymes of innate immunity systems and, as such, is recognized as a virulence factor. The key enzyme involved, peptidoglycan O-acetyltransferase A (OatA) represents a particular challenge to biochemical study since it is a membrane associated protein whose substrate is the insoluble peptidoglycan cell wall polymer. OatA is predicted to be bimodular, being comprised of an N-terminal integral membrane domain linked to a C-terminal extracytoplasmic domain. We present herein the first biochemical and kinetic characterization of the C-terminal catalytic domain of OatA from two important human pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Using both pseudosubstrates and novel biosynthetically-prepared peptidoglycan polymers, we characterized distinct substrate specificities for the two enzymes. In addition, the high resolution crystal structure of the C-terminal domain reveals an SGNH/GDSL-like hydrolase fold with a catalytic triad of amino acids but with a non-canonical oxyanion hole structure. Site-specific replacements confirmed the identity of the catalytic and oxyanion hole residues. A model is presented for the O-acetylation of peptidoglycan whereby the translocation of acetyl groups from a cytoplasmic source across the cytoplasmic membrane is catalyzed by the N-terminal domain of OatA for their transfer to peptidoglycan by its C-terminal domain. This study on the structure-function relationship of OatA provides a molecular and mechanistic understanding of this bacterial resistance mechanism opening the prospect for novel chemotherapeutic exploration to enhance innate immunity protection against Gram-positive pathogens

Activity and domain structure of OatA.

<p><b>A</b>. PG is comprised of alternating GlcNAc (G) and MurNAc (M) residues with stem peptides (small circles). The lysozymes of innate immunity systems (LYZ) hydrolyze the linkage between M and G residues which results in cell rupture and death. OatA O-acetylates the C-6 hydroxyl group of MurNAc residues (red triangles) in PG of pathogenic Gram-positive bacteria which sterically inhibits the action of the lysozymes, thereby conferring resistance to this first line of the innate immune response. <b>B</b>. Domain organization of OatA. This bimodular protein is comprised of two domains, a predicted N-terminal Acyl_transferase_3 (Pfam PF01757) transmembrane domain and a C-terminal SGNH/GDSL extracytoplasmic domain. The genes encoding OatA from <i>S</i>. <i>aureus</i> and <i>S</i>. <i>pneumoniae</i> were engineered to produce the 25 kDa C-terminal SGNH/GDSL domains (OatA_C) as shown.</p

Active site structure of <i>Sp</i>OatA_C.

<p>The H-bonding network of catalytic and oxyanion hole residues in <b>A</b>, resting <i>Sp</i>OatA_C and <b>B</b>, <i>Sp</i>OatA_C in complex with MeS (<i>Sp</i>OatA_C-MeS). The water molecule w1 and the potential inter-residue interactions are depicted as a red sphere and black dashed lines, respectively. <b>C</b>. The <i>2F</i>_<i>o</i><i>-F</i>_<i>c</i> electron density map of the MeS-Ser438 adduct contoured at 1.0 σ. <b>D</b>. Superposition of the <i>Sp</i>OatA_C and <i>Sp</i>OatA_C-MeS active sites.</p

Specific activities of <i>Sp</i>OatA_C and <i>Sa</i>OatA_C variants.

<p>Specific activities of <i>Sp</i>OatA_C and <i>Sa</i>OatA_C variants.</p

Structural comparison of <i>Sp</i>OatA_C with representative members of the SGNH/GDSL and AlgX-N/DHHW families of enzymes.

<p><b>A</b>. The cartoon representation of <i>Sp</i>OatA_C (gray) is superposed with <i>Bos taurus</i> platelet-activating factor acetylhydrolase (PAF-AH) (blue) and the N-terminal catalytic domain of <i>P</i>. <i>aeruginosa</i> AlgX (green). Right inset: Cartoons depicting the respective peptide backbones of the Block II-loop in the three enzymes. <b>B</b>. Sequence alignments of residues comprising the signature sequence Blocks of the SGNH/GDSL and AlgX-N/DHHW families of enzymes. Red lettering denotes invariant residues in the respective families.</p