Abstract
Amphomycin and MX-2401 are cyclic lipopeptides exhibiting bactericidal activities against Gram-positive pathogens. Amphomycin and MX-2401 share structural similarities with daptomycin, but unlike daptomycin they do not target bacterial membrane. In this study, we investigate in vivo modes of action for amphomycin and MX-2401 in intact whole cells of Staphylococcus aureus by measuring the changes of peptidoglycan and wall teichoic acid compositions using solid-state NMR. S. aureus were grown in a defined media containing isotope labels [1-13C]glycine and L-[ε-15N]lysin, L-[1-13C]lysine and D-[15N]alanine, or D-[1-13C]alanine and [15N]glycine, to selectively 13C-15N pair label peptidoglycan bridge-link, stem-link, and cross-link, respectively. 13C{15N} and 15N{13C} rotational-echo double resonance NMR measurements determined that cyclic lipopeptide-treated S. aureus exhibited thinning of the cell wall, accumulation of Park’s nucleotide, inhibition of glycine utilization for purine biosynthesis, reduction of ester-linked D-Ala in teichoic acids, and reduction of peptidoglycan cross-linking. Whole cell NMR analysis also revealed that S. aureus, in presence of amphomycin and MX-2401, maintained the incorporation of D-Ala during peptidoglycan biosynthesis while the incorporation of D-Ala into teichoic acids was inhibited. These effects are consistent with amphomycin’s dual inhibition of both peptidoglycan and wall teichoic acid biosyntheses in S. aureus.
Highlights
(3) superimposable D-amino acids at positions 2, 5, 7, and 8 of the cyclic-peptide core, and (4) a hydrophobic tail of similar lengths
We investigated in vivo mode of action of amphomycin and MX-2401 by measuring the changes in cell wall composition of intact whole-cells of S. aureus using solid-state NMR
It was found that amphomycin-treated S. aureus exhibit cell wall thinning (Figs 1b, 2b and 3b) concomitant with the accumulation of Park’s nucleotide (Figs 1a and 3d), which indicates that amphomycin inhibits cell wall biosynthesis by targeting steps at or prior to PG transglycosylation
Summary
(3) superimposable D-amino acids (or glycine) at positions 2, 5, 7, and 8 of the cyclic-peptide core, and (4) a hydrophobic tail of similar lengths. One key difference is that daptomycin is a depsipeptide with a lactone linkage instead of a peptide bond between the amino acid positions 1 and 10, (Fig. 1a, yellow highlight). Despite their structural similarities, daptomycin and amphomycin-class of antibiotics exhibit different modes of action. The structure of amphomycin bound to C55–P is unknown, but the x-ray crystal structure of tsushimycin suggests that a binding cleft for the phosphate of C55–P may be formed at the dimer interface between the cyclic peptide core structures[19]. Cyclic lipopeptides were added to S. aureus at mid-exponential growth phase at sub-MIC, the changes of peptidoglycan (PG) and wall teichoic acid (WTA) compositions were determined by 13C{15N} and 15N{13C} rotational-echo double resonance (REDOR) NMR20
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