Towards an automated analysis of bacterial peptidoglycan structure

Abstract

Peptidoglycan (PG) is an essential component of the bacterial cell envelope. This macromolecule consists of glycan chains alternating N-acetylglucosamine and N-acetylmuramic acid, cross-linked by short peptides containing nonstandard amino acids. Structural analysis of PG usually involves enzymatic digestion of glycan strands and separation of disaccharide peptides by reversed-phase HPLC followed by collection of individual peaks for MALDI-TOF and/or tandem mass spectrometry. Here, we report a novel strategy using shotgun proteomics techniques for a systematic and unbiased structural analysis of PG using high-resolution mass spectrometry and automated analysis of HCD and ETD fragmentation spectra with the Byonic software. Using the PG of the nosocomial pathogen Clostridium difficile as a proof of concept, we show that this high-throughput approach allows the identification of all PG monomers and dimers previously described, leaving only disambiguation of 3-3 and 4-3 cross-linking as a manual step. Our analysis confirms previous findings that C. difficile peptidoglycans include mainly deacetylated N-acetylglucosamine residues and 3-3 cross-links. The analysis also revealed a number of low abundance muropeptides with peptide sequences not previously reported. Graphical Abstract The bacterial cell envelope includes plasma membrane, peptidoglycan, and surface layer. Peptidoglycan is unique to bacteria and the target of the most important antibiotics; here it is analyzed by mass spectrometry.

Keywords: Cell wall; Cross-link; Glycoproteomics; Muropeptides; Peptidoglycan; Proteomics; Tandem mass spectrometry.

Graphical Abstract

The bacterial cell envelope includes plasma membrane, peptidoglycan, and surface layer. Peptidoglycan is unique to bacteria and the target of the most important antibiotics; here it is analyzed by mass spectrometry.

Fig. 1

Composition and polymerization of bacterial peptidoglycan (PG). PG building blocks, cut from larger PG chains with mutanolysin cleavages indicated by (M), correspond to the disaccharide-pentapeptide GlcN-MurNAc-l-Ala-iD-Glu-mDAP-d-Ala-d-Ala. PG components can be cross-linked by two distinct mechanisms: (a) d,d-transpeptidation results in the formation of a 4–3 bond between a donor and an acceptor stem; (b) l,d-transpeptidation follows cleavage of d-Ala in position 5 and results in the formation of a 3–3 bond between a donor and an acceptor stem

Fig. 2

(a) Total ion chromatogram shows PG components ordered by hydrophobicity, monomers generally eluting before dimers and trimers. (b) Major component for each elution peak. Most peaks also contain several minor components. Peaks 11 and 12 and also 16 and 17, contain isomers, possibly epimers, indistinguishable by precursor mass and MS/MS scans

Fig. 3

Collisional dissociation (HCD) spectrum of an unanticipated sequence variant, recognized by high-accuracy (5 ppm precursor and 20 ppm fragment) mass spectrometry. Other fourth residue variants include: Phe, Ile/Leu, Ser, Met, Val, Arg, Lys and His. The small peptide fragmentation diagram on the right side of the tandem mass spectrum shows Byonic’s automatic assignment of spectrum peaks

Fig. 4

(a) HCD and (b) ETD spectra of a dimer with that could be either GlcN-MurNAc-A-E-mDAP 3–3 linked to GlcN-MurNAc-A-E-mDAP-A-A or GlcN-MurNAc-A-E-mDAP-A 4–3 linked to GlcN-MurNAc-A-E-mDAP-A. The HCD spectrum contains no peaks (such as an A-A y-ion for 3–3) that can distinguish the two possibilities, but the ETD spectrum contains peaks at 899.45 and 884.42 matching the theoretical masses (given on the fragmentation diagram) of 899.32 and 884.39 for c- and z-ions splitting donor and acceptor peptides. The ETD spectrum therefore shows that the dimer is 4–3 linked. Byonic gives a fragmentation diagram and annotates c2, y2, y3, z2 and z3 for [+438]AEM[+41][+881]A crosslinked to [+438]AEM[+41]A, where [+438] represents the disaccharide, M[+41] represents mDAP, and [+881] the cross-link “modification” on M[+41], but Byonic does not annotate internal fragments nor the disambiguating peaks at 899.45 and 884.42

Fig. 5

ETD spectrum of GlcN-MurNAc-A-E-mDAP 3–3 linked to GlcN-MurNAc-A-E-mDAP-A. The peaks at 828.45 and 884.42 match the theoretical masses of 828.38 and 884.39 for c- and z-ions splitting donor and acceptor peptides. ETD fragments PG components at both peptide and glycosidic bonds, including the C–O bond between the lactyl group and MurNAc, shown in the fragmentation diagram with a line across MurNAc