Vancomycin and oritavancin have different modes of action in Enterococcus faecium


Journal of Molecular Biology Volume 392, Issue 5, 9 October 2009, Pages 1178-1191

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 did 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 13C{19F} rotational-echo double-resonance (REDOR) experiments on whole cells enriched with l-[1-13C]lysine and complexed with desleucyl [19F]oritavancin. These experiments establish that an oritavancin derivative with a damaged d-Ala--d-Ala binding pocket still binds to E. faecium peptidoglycan. The 13C{19F} 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.

Complete experimental deconvolution of the carbonyl-carbon 13C CPMAS echo spectra of E. faecium whole cells enriched with d-[1-13C]alanine and d-[15N]aspartic acid grown in the presence of alaphosphin and either with no vancomycin (bottom), or with 25 ug/ml vancomycin (top). The REDOR difference was scaled by the isotopic enrichment of d-[15N]Asp. The concentration of d-Ala-d-Ala stems increases in cells treated with 25 ug/ml of vancomycin as a result of the accumulation of cytoplasmic pentapeptide precursors, a signature of transglycosylase inhibition..

13C CPMAS echo spectra of E. faecium whole cells enriched with d-[1-13C]alanine grown in the presence of alaphosphin and either no oritavancin (black), or with 25 ug/ml oritavancin (red). The spectra are normalized with respect to the natural-abundance, aliphatic-carbon signal intensities between 0 and 35 ppm. The line shape of the peak centered at 175 ppm does not change significantly with the addition of oritavancin (inset). The absence of increased signal at 178 ppm corresponding to cytoplasmic cell-wall precursors, as observed for cells treated with vancomycin (see Fig. 1), shows that oritavancin does not significantly affect transglycosylation.

Complete experimental deconvolution of the carbonyl-carbon 13C spectra of E. faecium whole cells enriched with d-[1-13C]alanine and d-[15N]aspartic acid grown in the presence of alaphosphin and either with no oritavancin (bottom), or with 25 ug/ml oritavancin (top). The REDOR difference was scaled by the isotopic enrichment of d-[15N]Asp. The concentration of stems terminating in d-Ala-d-Ala does not increase in cells treated with oritavancin. In contrast to vancomycin treatment, oritavancin treatment does not result in the accumulation of pentapeptide cytoplasmic precursors characteristic of transglycosylase inhibition.