Vancomycin and Oritavancin Have Different Modes of Action in Enterococcus faecium

Abstract

The increasing frequency of Enterococcus faecium isolates with multidrug resistance is a serious clinical problem given the severely limited number of therapeutic options available to treat these infections. Oritavancin is a promising new alternative in clinical development that has potent antimicrobial activity against both staphylococcal and enterococcal vancomycin-resistant pathogens. Using solid-state NMR to detect changes in the cell-wall structure and peptidoglycan precursors of whole cells after antibiotic-induced stress, we report that vancomycin and oritavancin have different modes of action in E. faecium. Our results show the accumulation of peptidoglycan precursors after vancomycin treatment, consistent with transglycosylase inhibition, but no measurable difference in cross-linking. In contrast, after oritavancin exposure, we do not observe the accumulation of peptidoglycan precursors. Instead, the number of cross-links is significantly reduced, showing that oritavancin primarily inhibits transpeptidation. We propose that the activity of oritavancin is the result of a secondary-binding interaction with the E. faecium peptidoglycan. The hypothesis is supported by results from 13 C{ 19 F} REDOR experiments on whole cells enriched with L-[1-13 C] lysine and complexed with desleucyl [ 19 F]oritavancin. These experiments establish that an oritavancin derivative with a damaged D-Ala-D-Ala binding pocket still binds to E. faecium peptidoglycan. The 13 C{ 19 F} REDOR dephasing maximum indicates that the secondary-binding site of oritavancin is specific to nascent and template peptidoglycan. We conclude that the inhibition of transpeptidation by oritavancin in E. faecium is the result of the large number of secondary-binding sites relative to the number of primary-binding sites.

Structural Variations of the Cell Wall Precursor Lipid II and Their Influence on Binding and Activity of the Lipoglycopeptide Antibiotic Oritavancin

“…Like vancomycin, the lipoglycopeptide antibiotics such as oritavancin and telavancin inhibit transglycosylation via binding to the D-Ala-D-Ala terminus of lipid II. In addition, structural modifications, like the 4-epi-vancosamine and the 4=-chlorobiphenyl methyl side chain of oritavancin, account for improved antibacterial activity against MRSA, VISA, VRSA, and VRE strains (22,40,54). Here, we provide evidence at the molecular level that these structural modifications enable oritavancin, in contrast to vancomycin, to bind to further target sites of the lipid II peptidoglycan precursor other than the D-Ala-D-Ala terminus.…”

“…Here, we provide evidence at the molecular level that these structural modifications enable oritavancin, in contrast to vancomycin, to bind to further target sites of the lipid II peptidoglycan precursor other than the D-Ala-D-Ala terminus. With use of solidstate nuclear magnetic resonance (NMR) and rotational-echo double resonance (REDOR), a model for the oritavancin-PG complex has been proposed in which the crossbridge is bound in a cleft between the epi-vancosamine residue and the core of the oritavancin molecule (36,39,40). We now provide biochemical evidence that the crossbridge and the D-iso-glutamine in position 2 of the lipid II stem peptide represent crucial binding sites for oritavancin.…”

“…In the presence of increasing concentrations of oritavancin, inhibition of all enzymatic steps (MurG, FemX, PBP2) using lipid I or lipid II as a substrate was observed. Since the crossbridge has been proposed to be an additional binding site for oritavancin (36,40), we further analyzed the impact of oritavancin on both pentaglycine (S. aureus) and D-aspartate (E. faecium) crossbridge formation in vitro, as catalyzed by the purified enzymes FemXAB and Asl (37,38,45), respectively (Fig. 2B).…”

“…Recent studies suggested that binding of oritavancin involves multiple interactions with its molecular target lipid II. Solid-state nuclear magnetic resonance (NMR) and computational modeling predicted secondary interactions of the 4=-chlorobiphenyl methyl side chain with the S. aureus pentaglycine crossbridge and the D-aspartate/D-asparagine (D-Asx) crossbridge in Enterococcus faecium (36)(37)(38)(39)(40). To better understand the subtleties of the mechanism of action of oritavancin, we characterized the binding to its target lipid II in vivo and in vitro using purified lipid II variants.…”

Lipoglycopeptide Antibacterial Agents in Gram-Positive Infections: A Comparative Review

“…This allows it to inhibit not only transglycosylase activity, and thus precursor chain extension, but also transpeptidase activity, and thus precursor cross-linking [38]. In Enterococcus faecium, inhibition of transpeptidase becomes even more prominent due to the preferential interaction of oritavancin with multiple sites on the peptidic bridge [39]. In this case, interaction with DAla-D-ala termini becomes marginal, explaining why transglycosylase activity is no longer the primary target of the drug.…”