Solid-state NMR characterization of amphomycin effects on peptidoglycan and wall teichoic acid biosyntheses in Staphylococcus aureus

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 [1-13 C]glycine and L-[μ-15 N]lysin, L-[1-13 C]lysine and D-[ 15 N]alanine, or D-[1-13 C]alanine and [ 15 N]glycine, to selectively 13 C-15 N pair label peptidoglycan bridge-link, stem-link, and cross-link, respectively. 13 C{ 15 N} and 15 N{ 13 C} 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.