FIG 2.

Inhibition of membrane-associated cell wall biosynthesis steps by oritavancin. (A) Synthesis and analysis of the individual biosynthesis steps were performed as described in Materials and Methods. The amount of reaction products synthesized in the absence of antibiotics was taken as 100%. The bactoprenol-containing products were analyzed by TLC. Radiolabeling was based on ³H-labeled C₅₅P (for lipid I), [¹⁴C]GlcNAc for lipid II, and [¹⁴C]glycine for lipid II-Gly₁. Antibiotics were added in molar ratios with respect to the bactoprenol-bound substrates as indicated. The lantibiotic nisin was used as a control. (B) Impact of oritavancin on the staphylococcal pentaglycine and enterococcal d-aspartate crossbridge formation. The addition of [¹⁴C]glycine by purified FemXAB-His₆ (S. aureus) or the formation of the [¹⁴C]aspartate crossbridge by Asl-His₆ (E. faecium) was assayed in the presence of increasing oritavancin concentrations (0.5 to 2 molar ratio with respect to lipid II). The reaction products synthesized in the absence of oritavancin were taken as the 100% level. The lipid intermediates were extracted from the reaction mixture and analyzed by TLC and subsequent phosphorimaging. The mean values from three independent experiments are shown. All experiments were performed at least in triplicate, and data are presented as means ± standard deviations (SD). ORI, oritavancin.