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

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

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

<i>Sp</i>OatA_C and <i>Sa</i>OatA_C-catalyzed <i>O</i>-acetyltransferase reactions.

<p>ESI-MS analysis of reaction products of 2 mM chitotetraose (G₄) in 50 mM sodium phosphate buffer pH 6.5 incubated at 37 ^oC for 1 h in the absence (control) and presence of enzymes (5 μM, final concentration) with 1 mM concentrations of <b>A,</b> acetyl-CoA; <b>B,</b> 4MU-Ac; or <b>C,</b> <i>p</i>NP-Ac as potential donor acetyl substrates.</p

Stem peptide specificity of <i>Sp</i>OatA_C and <i>Sa</i>OatA_C.

<p><b>A</b>. Stacked and offset ESI-mass spectra of mutanolysin-treated products from reactions of 10 μg·mL^-1 of (left to right) muroglyan-5P, muroglycan-4P, and muroglycan-3P in 50 mM sodium phosphate buffer pH 6.5 incubated with 0.5 mM <i>p</i>NP-Ac in the absence (control) and presence of the respective enzyme (10 μM). The major O-acetylated products are labeled in blue which are 42.01 m/z units larger than the respective unmodified PG monomer. <b>B</b>. MS/MS analysis of the major product ions identified in the respective panels above and <b>C</b>, interpretation of the corresponding fragment ions.</p