Abstract
Many software solutions are available for proteomics and glycomics studies, but none are ideal for the structural analysis of peptidoglycan (PG), the essential and major component of bacterial cell envelopes. It icomprises glycan chains and peptide stems, both containing unusual amino acids and sugars. This has forced the field to rely on manual analysis approaches, which are time-consuming, labour-intensive, and prone to error. The lack of automated tools has hampered the ability to perform high-throughput analyses and prevented the adoption of a standard methodology. Here, we describe a novel tool called PGFinder for the analysis of PG structure and demonstrate that it represents a powerful tool to quantify PG fragments and discover novel structural features. Our analysis workflow, which relies on open-access tools, is a breakthrough towards a consistent and reproducible analysis of bacterial PGs. It represents a significant advance towards peptidoglycomics as a full-fledged discipline.
Highlights
The characterisation of bacterial cell walls started with the development of electron microscopy techniques (Mudd, et al, 1941), and it has ever since been the focus of countless studies
We sought to replicate a shotgun proteomics approach to create an analysis pipeline dedicated to PG analysis, referred to as “peptidoglycomics” (Wheeler, et al, 2014)
Any observed mass within 10 ppm tolerance was considered as a match and the corresponding inferred structure and theoretical mass were added to a list of matched structures (Figure 2, library 1)
Summary
The characterisation of bacterial cell walls started with the development of electron microscopy techniques (Mudd, et al, 1941), and it has ever since been the focus of countless studies. It confers cell shape and resistance to osmotic stress and represents an unmatched target for antibiotics (Mainardi, et al, 2008, Vollmer, et al, 2008). PG (murein; originally known as mucopeptide) is a giant, insoluble, bag-shaped molecule, and its composition was characterised soon after its discovery (Cummins, et al, 1956, Rogers, et al, 1959, Weidel, et al, 1964). It is composed of glycan chains containing alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues linked by β,1-4 bonds. The lactyl group of MurNAc residues is substituted by pentapeptide stems which often has the L-Ala1-γ-D-Glu2-L-DAA3-
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