Forming cross-linked peptidoglycan from synthetic gram-negative Lipid II

Abstract

The bacterial cell wall precursor, Lipid II, has a highly conserved structure among different organisms except for differences in the amino acid sequence of the peptide side chain. Here, we report an efficient and flexible synthesis of the canonical Lipid II precursor required for the assembly of Gram-negative peptidoglycan (PG). We use a rapid LC/MS assay to analyze PG glycosyltransfer (PGT) and transpeptidase (TP) activities of Escherichia coli penicillin binding proteins PBP1A and PBP1B and show that the native m-DAP residue in the peptide side chain of Lipid II is required in order for TP-catalyzed peptide cross-linking to occur in vitro. Comparison of PG produced from synthetic canonical E. coli Lipid II with PG isolated from E. coli cells demonstrates that we can produce PG in vitro that resembles native structure. This work provides the tools necessary for reconstituting cell wall synthesis, an essential cellular process and major antibiotic target, in a purified system.

Figure 1

Bacterial cell wall is assembled from the precursor Lipid II. (a) Schematic of an E. coli cell and the chemical structure of its cell wall, composed of alternating GlcNAc (blue) and Mur-NAc (green) residues with attached peptide side chains that can be crosslinked. Purified E. coli cell wall typically lacks terminal D-Ala residues (red). (b) Chemical structure of Gram-positive L-Lys Lipid II (1a) and Gram-negative m-DAP Lipid II (1b), which differ at the third residue of the peptide side chain. Because R varies depending on the organism, a flexible route to 1 is needed to study the PG synthesis of various organisms.

Figure 2

The m-DAP residue in Lipid II is essential for trans-peptidase-catalyzed crosslinking. (a) Schematic of method for analyzing PG synthesis by PBPs. (b) LC/MS extracted chromatograms of PBP1A and L-Lys Lipid II (1a) reactions produce only A′, representing unmodified polymer, in the presence of the inhibitor penG (i), but both A′ and B′, representing hydrolyzed peptide side chain, without penG (ii). PBP1A and m-DAP-Lipid II (1b) reactions reveal fragment C in addition to fragments A and B, indicating the formation of crosslinked PG (iv); no crosslinks are formed in the presence of penG (iii). The PGT inhibitor moenomycin prevented formation of all fragment peaks (Figure S1). For chromatograms (i, ii): (M+2H)/2 ions corresponding to fragments A′ and B′ were extracted: A′: 485.2; B′: 449.7; masses corresponding to predicted crosslinked fragments were not observed. For chromatograms (iii, vi): (M+2H)/2 ions corresponding to fragments A–D were extracted: A: 507.2; B: 471.7; C: 968.9; D: 933.4.

Figure 3

The composition of PG produced in vitro resembles PG isolated from E. coli cells. (a) Schematic of experimental procedure for PG analysis shows that fragments A and C would result from degradation of PG synthesized in vitro, while B and D would result from prior hydrolysis of PG by E. coli PBP5, which liberates terminal D-Ala residues. (b) Treatment of PBP1A and 1b reactions with PBP5 produces hydrolysis fragments B and D (i) in proportions similar to those found in treated in vivo PG sample (ii). In vitro samples were prepared as before, except quenched PBP1A reactions were treated with PBP5 (0.8 μM) for 2 hr prior to analysis. See SI for details on cell wall isolation. (M+2H)/2 ions corresponding to fragments A–D were extracted from each chromatogram: A: 507.2; B: 471.7; C: 968.9; D: 933.4.

Scheme 1

Synthesis of m-DAP Lipid II (1b)^{a a}Reagents and conditions: (a) i. Grubbs 2^nd Gen. catalyst, DCM, 12h, reflux (50%); ii. H₂/Pd, MeOH, 1 h, RT (90%); (b) i. NH₂-D-Ala-D-Ala-OMe, EDC, HOBt, DIEA, DCM, 2h, RT; ii. 4M HCl in dioxane, 15 min., RT; iii. Boc-L-Ala-γ-D-Glu(OH)-OMe, EDC, HOBt, DIEA, DCM, 2h, RT; iv. 4M HCl in dioxane, 15 min., RT (54%); (c) i. 3, DMTNMM, DIEPA, MeOH, RT, 2h; ii. NaOH, H₂O, dioxane, 45 min., RT (48%); (d) UDP-GlcNAc, MurG, buffer, 1h, RT.