Rotational-Echo Double Resonance Characterization of the Effects of Vancomycin on Cell Wall Synthesis in Staphylococcus aureus

Abstract

Cross-polarization magic-angle spinning and rotational-echo double resonance 13C and 15N NMR experiments have been performed on intact cells of Staphylococcus aureus labeled with D-[1-13C]alanine and [15N]glycine or with [1-13C]glycine and L-[epsilon-15N]lysine. The cells were harvested during stationary or exponential growth conditions, the latter in media with and without the addition of vancomycin. The results of these experiments allowed the in situ determination of the relative concentrations of peptidoglycan cross-links (the number of peptide-stem D-alanines covalently linked to a pentaglycyl bridge) and bridge-links (the number of peptide-stem lysines covalently linked to a pentaglycyl bridge). The concentration of cross-links remained constant in the presence of vancomycin, whereas the number of bridge-links decreased. These changes suggest that vancomycin (at therapeutic levels) interrupts peptidoglycan synthesis in S. aureus by interference with transglycosylation.

Vancomycin analogues active against vanA-resistant strains inhibit bacterial transglycosylase without binding substrate

“…There is considerable biophysical data to support the hypothesis that lipid-substituted glycopeptides have enhanced avidity for both D-Ala-D-Ala-and D-Ala-D-Lacmodified surfaces (41), and it has also been shown that covalent dimers and trimers of vancomycin bind covalent multimers of D-Ala-D-Ala many orders of magnitude better than the monomers bind (42). Nevertheless, there is no evidence that chlorobiphenyl vancomycin derivatives kill vanA strains by dimerizing near cell surfaces and binding D-Ala-D-Lac (43,44). Furthermore, it is virtually impossible to design biophysical experiments that report on the extent to which enhanced D-Ala-D-Lac binding contributes to killing resistant microorganisms.…”

Structures of Staphylococcus aureus Cell-Wall Complexes with Vancomycin, Eremomycin, and Chloroeremomycin Derivatives by 13C{19F} and 15N{19F} Rotational-Echo Double Resonance

“…aureus cells were grown in 300 ml of defined media (20) containing [1][2][3][4][5][6][7][8][9][10][11][12][13] C]glycine and L-[ε -15 N]lysine. The cells were harvested at OD 660 = 1.0 and were complexed with 6.7 mg (4.0 μmoles) of FPBV, resulting in 66% cell-wall binding-site occupancy (15).…”

“…The REDOR dephasing function for CF 3 was approximated by treating the three fluorines as a single super spin (with two spin quantum numbers, I = 1/2 and 3/2) placed at the center of the plane defined by the positions of the three fluorines. This approximation is valid for C-F distances of 6 Å or more, and C-N distances of 5 Å or more (27).…”

“…peptidoglycan biosynthesis of cell walls (2,3) by binding to the D-Ala-D-Ala carboxyl termini of pentapeptide stems of nascent (immature) peptidoglycan (Figure 1), and peptidoglycan pentapeptide precursors, including lipid II (2,4).…”

Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy

“…Thus, despite the prevalent roles that biofilms play in human infection, there has been little reliable quantitative information available regarding biofilm matrix composition. Solid-state NMR spectroscopy is uniquely suited to study complex and insoluble biological materials (12) such as whole cells (13)(14)(15)(16)(17)(18), biofilms, and the extracellular matrix (19)(20)(21)(22). Solidstate NMR approaches can be used to quantify composition and to measure internuclear distances to determine parameters of architecture in such macromolecular assemblies and does not require soluble or crystalline samples (15).…”