Peptidoglycan at its peaks: how chromatographic analyses can reveal bacterial cell wall structure and assembly

Abstract

The peptidoglycan (PG) cell wall is a unique macromolecule responsible for both shape determination and cellular integrity under osmotic stress in virtually all bacteria. A quantitative understanding of the relationships between PG architecture, morphogenesis, immune system activation, and pathogenesis can provide molecular-scale insights into the function of proteins involved in cell-wall synthesis and cell growth. High Performance Liquid Chromatography (HPLC) has played an important role in our understanding of the structural and chemical complexity of the cell wall by providing an analytical method to quantify differences in chemical composition. Here, we present a primer on the basic chemical features of wall structure that can be revealed through HPLC, along with a description of the applications of HPLC PG analyses for interpreting the effects of genetic and chemical perturbations to a variety of bacterial species in different environments. We describe the physical consequences of different PG compositions on cell shape, and review complementary experimental and computational methodologies for PG analysis. Finally, we present a partial list of future targets of development for HPLC and related techniques.

A cell wall damage response mediated by a sensor kinase/response regulator pair enables beta-lactam tolerance

“…PG Quantification. For PG quantification and analysis, cultures were normalized to the same cfu/mL, and muropeptides were isolated following previously described methods (34,35).…”

High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography

“…The disaccharide subunits composing the glycan strands contain one N-acetyl-glucosamine (GlcNAc) and one N-acetylmuramic acid (MurNAc), to which a peptide stem with five amino acids is appended through a D-lactic acid linkage (1). The disaccharide subunit and the peptide stem are collectively referred to as a muropeptide.…”

“…In bacteria, the cell wall is an important antibiotic target (4), with treatment often disrupting the integrity of the cell wall and eventually leading to cell lysis (5). The cell wall plays an important role in pathogenesis, in part due to the uncommon stereochemistry of the three D-isomers of amino acids that defend against most proteases that otherwise would degrade the cell wall (1,6). Furthermore, many surface proteins anchored to the cell wall are involved in pathogenic processes such as host-cell invasion and immune system interactions (7).…”

“…The bacterial cell wall (also known as the murein sacculus) is a single macromolecular polymer network composed of peptidoglycan, which consists of long strands of glycans that are bound together through cross-links between short strings of peptides (1). The disaccharide subunits composing the glycan strands contain one N-acetyl-glucosamine (GlcNAc) and one N-acetylmuramic acid (MurNAc), to which a peptide stem with five amino acids is appended through a D-lactic acid linkage (1).…”

“…HPLC techniques have been tailored to analyze microbial biomolecules associated with cell wall growth, including muropeptides, cell wall digestion products, glycan strands, stem peptides, and D-amino acid incorporation (1,22). For soluble muropeptides isolated from bacterial cell walls, typical HPLC runs require 1-2 h and 100 -200 l of sample injected to resolve individual peaks (1). Typical HPLC preparations result in ϳ0.5 mg of muropeptides, which produces roughly 1 absorbance unit for the highest peaks (M4 or D44, Fig.…”