From cells to muropeptide structures in 24 h: peptidoglycan mapping by UPLC-MS

Abstract

Peptidoglycan (PGN) is ubiquitous in nearly all bacterial species. The PGN sacculus protects the cells against their own internal turgor making PGN one of the most important targets for antibacterial treatment. Within the last sixty years PGN composition has been intensively studied by various methods. The breakthrough was the application of HPLC technology on the analysis of muropeptides. However, preparation of pure PGN relied on a very time consuming method of about one week. We established a purification protocol for both Gram-positive and Gram-negative bacteria which can be completely performed in plastic reaction tubes yielding pure muropeptides within 24 hours. The muropeptides can be analyzed by UPLC-MS, allowing their immediate determination. This new rapid method provides the feasibility to screen PGN composition even in high throughput, making it a highly useful tool for basic research as well as for the pharmaceutical industry.

Figure 1. Muropeptide profile of S. aureus SA113 obtained by UPLC and UPLC/MS.

(a) Muropeptide profile of S. aureus SA113 obtained by reversed phase UPLC. (b) TIC of UPLC/MS analysis of S. aureus SA113 obtained by reversed-phase UPLC coupled to MS. Masses of indicated peaks are shown in table 1 and table S1 including molecule composition and proposed sum formula.

Figure 2. Muropeptide profile of E. coli Nissle 1917 obtained by UPLC and UPLC/MS.

(a)Muropeptide profile of E. coli Nissle 1917 obtained by reversed phase UPLC. (b) TIC of UPLC/MS analysis of E. coli Nissle 1917 obtained by reversed-phase UPLC coupled to MS. Masses of indicated peaks are shown in table 2 and table S2 including molecule composition and proposed sum formula.

Figure 3. Schematic structure of muropeptides of S. aureus and E. coli.

The basic structure consists of the disaccharide (GlcNAc-MurNAc) with an adjacent stem peptide (L-Ala – D-iGlx – L-Lys – D-Ala – D-Ala) with Glx being either Gln or Glu. Our model organism S. aureus, harbors also a five-glycine interpeptide bridge branching from the L-Lys, which constitutes indirect cross-links between two adjacent stem peptides, as it is depicted in the multimeric muropeptide. The C at position 6 of the MurNAc can be O-acetylated.