Characterization of structural variations in the peptidoglycan of vancomycin-susceptible Enterococcus faecium: understanding glycopeptide-antibiotic binding sites using mass spectrometry

Abstract

Enterococcus faecium, an opportunistic pathogen that causes a significant number of hospital-acquired infections each year, presents a serious clinical challenge because an increasing number of infections are resistant to the so-called antibiotic of last resort, vancomycin. Vancomycin and other new glycopeptide derivatives target the bacterial cell wall, thereby perturbing its biosynthesis. To help determine the modes of action of glycopeptide antibiotics, we have developed a bottom-up mass spectrometry approach complemented by solid-state nuclear magnetic resonance (NMR) to elucidate important structural characteristics of vancomycin-susceptible E. faecium peptidoglycan. Using accurate-mass measurements and integrating ion-current chromatographic peaks of digested peptidoglycan, we identified individual muropeptide species and approximated the relative amount of each. Even though the organism investigated is susceptible to vancomycin, only 3% of the digested peptidoglycan has the well-known D-Ala-D-Ala vancomycin-binding site. The data are consistent with a previously proposed template model of cell-wall biosynthesis where D-Ala-D-Ala stems that are not cross-linked are cleaved in mature peptidoglycan. Additionally, our mass-spectrometry approach allowed differentiation and quantification of muropeptide species seen as unresolved chromatographic peaks. Our method provides an estimate of the extent of muropeptides containing O-acetylation, amidation, hydroxylation, and the number of species forming cyclic imides. The varieties of muropeptides on which the modifications are detected suggest that significant processing occurs in mature peptidoglycan where several enzymes are active in editing cell-wall structure.

Figure 1

Total ion chromatogram for digested E. faecium muropeptides. The relative intensity is represented by total ion current as a function of retention time.

Figure 2

Mass spectra and assigned structures for variations of the muropeptide of m/z = 939.4150. The most likely structure for an m/z of 939.4150 (top) is that with a D-aspartic acid bridge. An m/z of 938.4310 (second from top) corresponds to the muropeptide with double amidation, D-asparagine and D-iso-glutamine. A mass increase of 42.011 is consistent with O-acetylation of N-acetylmuramic acid (second from bottom). A mass decrease of 18.010 suggests the loss of water, and could be indicative of two types of cyclic imides (bottom). The structure shown is consistent with NMR data (see Figure 4).

Figure 3

Selected ion chromatograms for singly charged species with m/z of (a) 824.4 and 825.4; (b) 938.4 and 939.4; (c) 1009.5 and 1010.5; and (d) 1080.5 and 1081.5. The relative intensities of 1080.5 and 1081.5 are small, consistent with there being few D-Ala-D-Ala stems. Muropeptides with bridges elute as two distinct peaks with different retention times in the chromatogram. The selected ion chromatogram for m/z 824.4 shows only one peak.

Figure 4

¹⁵N CPMAS echo-spectrum of E. faecium cell-wall isolates enriched with L-[6-¹⁵N]lysine. The amide peak at 95 ppm occurs when lysine has a D-Asx bridge attached, and the amine peak at 5 ppm occurs when lysine does not have an attached bridge. We assign the 151 ppm peak to lysyl succinimides.

Figure 5

Two-dimensional schematic representation of the template model of peptidoglycan biosynthesis for E. faecium. Chain extension occurs from right to left and is synchronized with cross-linking at lipid II. The few peptidoglycan stems terminating in D-Ala-D-Ala (red) occur in nascent and template peptidoglycan where they are cross-linked. We suspect that L,D-carboxypeptidase acts on stems in mature peptidoglycan that are not cross linked. We propose that other cell-wall editing enzymatic processes occur in mature peptidoglycan as well, creating muropeptides with O-acetylation (green), D-asparagine (blue), and cyclic imides (purple) as shown.

Scheme 1

Representative chemical structure of E. faecium peptidoglycan. The β-1,4 linkages between N-acetylmuramic acid and N-acetylglucosamine are cleaved by N-acetylmuramidase as indicated for the repeat unit on the left. The cross-links remain intact. D-iso-glutamine and D-aspartic acid may be substituted by either D-iso-glutamic acid or D-asparagine, respectively.

Scheme 2

Structures of assigned muropeptides. The relative proportion of each structure is shown in Table 1.