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. 2016 Aug 19:6:31757.
doi: 10.1038/srep31757.

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

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

Manmilan Singh et al. Sci Rep. .

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-[ε-(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.

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Figures

Figure 1
Figure 1. Daptomycin and amphomycin have different modes of action.
(a) Chemical structures of cyclic decapeptide antimicrobial agents: friulimicin, amphomycin, tsushimycin, MX-2401, and daptomycin. All of these cyclic lipopeptides share a ten-membered peptide core structure with hydrophobic tail. The hydrophobic tail group in amphomycin is anteiso-tridecenonyl, and in tsushimycin and friulimicin B it is iso-tetradecenonyl. All cyclic decapeptides exhibit calcium dependent bactericidal activity against Gram-positive bacteria and have a conserved calcium-binding motif sequence Asp4-Asp6-Gly7. Daptomycin is a cyclic-depsipeptide with a lacton linkage highlighted in yellow. (b) ATP-leakage assay performed on overnight culture of S. aureus. Daptomycin induces leaking of ATP from S. aureus, but only at a high drug concentration (100 μg/mL). MX-2401 did not cause the leakage in S. aureus at any concentrations. Thus, MX-2401 does not disrupt the membrane despite sharing structural similarities with daptomycin. (c) Growth curves of S. aureus treated with amphomycin (left), and MX-2401 (right) as monitored by the optical density (OD) at 660 nm. Cyclic lipopeptides were added to final concentrations of 20 or 40 μg/mL at mid-exponential growth (OD660nm 0.7). Addition of cyclic lipopeptides at these concentrations did not significantly perturb the growth. The cells were harvested for NMR analysis after one doubling time (60 minutes) of incubation with drugs at the approximate OD660nm of 0.9.
Figure 2
Figure 2. Amphomycin-treated S. aureus exhibit cell wall thinning and Park’s nucleotide accumulation.
(a) Incorporation of [1-13C]Gly and L-[ε-15N]Lys to peptidoglycan (PG). 15N-CPMAS NMR spectra from amphomycin-treated whole cells of S. aureus (40 μg/mL, red line) and untreated whole cells (black line). Amphomycin-treated S. aureus show reduced lysyl-ε-amide peak at 95 ppm due to cell-wall thinning, and increased lysyl-ε-amine peak at 10 ppm due to accumulation of Park’s nucleotide. These effects are consistent with amphomycin inhibition of cell wall biosynthesis by targeting steps at or prior to PG transglycosylation. Each spectrum is the result of 20,480 accumulated scans. (b) 13C{15N} REDOR spectra from whole cells of S. aureus at 1.6 ms of dipolar evolution. S0 spectra are at the bottom, and ΔS spectra at the top. Spectra are normalized to natural abundance peaks from 10 to 30 ppm range. The 170-ppm glycyl peak intensities in S0 are directly proportional to the amount of [1-13C]Gly in S. aureus. The decrease in 170-ppm peak intensity in the S0 spectrum of amphomycin (40 μg/mL) treated S. aureus is consistent with thinning of the cell wall. Amphomycin-treated S. aureus also show decrease in the 149-ppm peak intensity in the S0 indicating reduction of [1-13C]Gly into purine biosynthesis. In ΔS spectra (top) only covalently bonded 13Cs from 13C-15N spin pairs unique to PG bridge-links are detected at 1.6 ms dipolar evolution. 170-ppm dephasing (ΔS/S0) decreases from 11.5% to 8.9% with addition of amphomycin, which indicates the reduction in the proportion of L-[ε-15N]Lys due to incorporation of unlabeled L-Lys into PG stem structure. Each spectrum is the result of 10,240 accumulated scans.
Figure 3
Figure 3. Amphomycin-treated S. aureus exhibit reduced peptidoglycan crosslinking.
(a) Incorporation of D-[1-13C]Ala (red dots) and [15N]Gly (blue dot) and the 13C-15N pair labeled in the PG cross-link. (b) 15N{13C} REDOR spectra after dipolar evolution of 1.6 ms from S. aureus labeled with D-[1-13C]Ala and [15N]Gly. Full-echo spectra (S0) are shown in black and the REDOR dephased spectra (S) are in red, while spectra from the sample with amphomycin (40 μg/mL) are on the right and without on the left. (c) The difference in peak intensities (S0-S) measures the relative number of cross-links per PG pentaglycyl bridging segment. Amphomycin reduced cross-linking, which is consistent with mild inhibition of transpeptidation. (d) Enlarged carbonyl-carbon regions of the 13C{15N} REDOR full-echo spectra of amphomycin treated (red) and untreated (black) S. aureus. The lineshape of spectra from amphomycin treated cells shows increased 178 ppm contribution from the carboxyl-carbonyl carbon of D-[1-13C]Ala from Park’s nucleotide unincorporated into PG by cross-linking. The decreased 172 ppm contribution is due to the reduced amount of ester-linked D-[1-13C]Ala in WTA.
Figure 4
Figure 4. Amphomycin and MX-2401 inhibit D-Ala incorporation into wall teichoic acid.
(a) Incorporation of D-[15N]Ala into PG and wall teichoic acid (WTA) cell wall monitored using 15N-CPMAS NMR. D-[15N]Ala in PG has an alanyl amide at 95 ppm in the 15N-CPMAS spectrum, while in WTA ester-linked D-[15N]Ala is an amine at 10 ppm (lower right). The effect of cyclic lipopeptides on WTA biosynthesis is monitored by measuring the ratio between PG to WTA. (b) 15N{13C} REDOR spectra of S. aureus treated with amphomycin (0, 20, and 40 μg/mL) at 1.6 ms dipolar evolution. (c) 15N{13C} REDOR spectra of whole cells of S. aureus treated with MX-2401 (0, 20, and 40 μg/mL) at 1.6 ms dipolar evolution. The full-echo spectrum (S0) is at the bottom and the REDOR difference (ΔS) at the top. S0 spectra are normalized to equal alanyl-amide peak at 95 ppm of ΔS spectra. Addition of amphomycin and MX-2401 resulted in the decrease in alanyl-amine peak at 10 ppm in S0 spectra, indicating reduction in the amount of ester-linked D-Ala in teichoic acid.

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