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. 2017 Apr 27;121(16):3925-3932.
doi: 10.1021/acs.jpcb.7b00324. Epub 2017 Apr 14.

Hidden Mode of Action of Glycopeptide Antibiotics: Inhibition of Wall Teichoic Acid Biosynthesis

Manmilan Singh  1 , James Chang  2 , Lauryn Coffman  2 , Sung Joon Kim  2
Affiliations

Hidden Mode of Action of Glycopeptide Antibiotics: Inhibition of Wall Teichoic Acid Biosynthesis

Manmilan Singh et al. J Phys Chem B. .

Abstract

Glycopeptide antibiotics inhibit the peptidoglycan biosynthesis in Gram-positive bacteria by targeting lipid II. This prevents the recycling of bactoprenol phosphate, the lipid transporter that is shared by peptidoglycan and wall teichoic acid biosyntheses. In this study, we investigate the effects of glycopeptide antibiotics on peptidoglycan and wall teichoic acid biosynthesis. The incorporation of d-[1-13C]alanine, d-[15N]alanine, and l-[1-13C]lysine into peptidoglycan and wall teichoic acid in intact whole cells of Staphylococcus aureus was measured using 13C{15N} and 15N{13C} rotational-echo double resonance NMR. S. aureus treated with oritavancin and vancomycin at subminimal inhibitory concentrations exhibit a large reduction in d-Ala incorporation into wall teichoic acid, but without changes to the peptidoglycan cross-links or the stem-links. Thus, sequestration of bactoprenol phosphate by glycopeptide antibiotics resulted in inhibition of d-Ala incorporation into the wall teichoic acid prior to the inhibition of peptidoglycan biosynthesis. Our finding shows that S. aureus responds to glycopeptide-induced cell wall stress by routing all available d-Ala to the peptidoglycan biosynthesis, at the cost of reducing the wall teichoic acid biosynthesis.

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Figures

Figure 1
Figure 1. S. aureus treated with disaccharide-modified glycopeptide antibiotics at sub-MICs do not readily induce ATP leakage
a, Chemical structures of disaccharide-modified glycopeptide antibiotics with increasing aliphatic side chain lengths (from left to right): vancomycin (no modification), LY309687 (trifluoromethoxybenzyl side chain), oritavancin (chlorophenyl-benzyl side chain), FNCE (N-9-fluorononyl side chain), and FBBCE (N-9-fluorobiphenylbenzyl side chain). b, ATP leakage was attempted in S. aureus harvested at OD660nm 1.5 by addition of glycopeptide antibiotics to final concentrations of 0, 1, 2, 5, 10, 50, and 100 μg/mL, and daptomycin (D) at 100 μg/mL. All glycopeptide antibiotics did not induce appreciable ATP leakage attributable to membrane depolarization at the concentrations tested. In comparison, daptomycin induced ATP leakage consistent with the membrane disruption. All error bars represent 95% confidence interval.
Figure 2
Figure 2. Glycopeptide antibiotics inhibit both PG transglycosylation and WTA biosynthesis in S. aureus
a, PG and WTA labeling by L,D-[1-13C]Ala in S. aureus. b, Deconvolution of 13C-CPMAS spectrum of intact whole cells of S. aureus with D-alanyl carboxyls at 178 ppm (blue), D-alanyl peptide carbonyls at 174 ppm (green), and ester carbonyls of WTA at 171 ppm (red). c, 13C-CPMAS spectrum of untreated S. aureus (middle, red) is subtracted from the spectrum of antibiotic treated whole cells (top, red). The difference spectra (bottom) show all antibiotic-treated S. aureus have a negative 171 ppm peak which corresponds to decreased ester-linked D-Ala incorporation to WTA. Park’s nucleotide accumulation (positive 178 ppm peak) is observed for all glycopeptide-treated S. aureus.
Figure 3
Figure 3. Oritavancin is a potent WTA inhibitor
a, L,D-[15N]Ala and L-[1-13C]Lys labeling of S. aureus PG and WTA. Isotope labeled 13C-15N spin pair is predominantly found at the stem-link position of PG. 15N-CPMAS spectrum from intact whole cells of S. aureus shows D-[15N]Ala incorporated into WTA appearing at 16 ppm, and L,D-[15N]Ala into proteins and PG at 93 ppm. b, 13C{15N} REDOR spectra from intact whole cells of S. aureus grown with and without antibiotics: vancomycin (5 μg/mL), and oritavancin (5 μg/mL) following 1.6 ms dipolar evolution. The L-[1-13C]Lys incorporation to S. aureus is visible as a lysyl-carbonyl carbon at 175 ppm in the S0 spectra (bottom). In the ΔS spectra (top), only the 13C-lysyl-carbonyl carbons of L-[1-13C]Lys peptide bonded to the 15N of L,D-[15N]Ala are dephased at 1.6 ms dipolar evolution. Hence the ΔS 175-ppm intensity is directly proportional to the in situ stem-link density of the cell wall. The stem-link density of S. aureus is unaffected by glycopeptide antibiotic treatments, which indicates that D-[15N]Ala and L-[1-13C]Lys incorporations to PG are unaffected at the glycopeptide antibiotic concentration of 5 μg/mL. c, 15N{13C} REDOR spectra of intact whole cells of S. aureus at 1.6 ms dipolar evolution. In the ΔS spectra (top) 93-ppm intensity directly proportional to the stem-links in cell wall is unaffected by the glycopeptide antibiotic treatment. However, the S0 spectra (bottom) of vancomycin and oritavancin treated S. aureus show reductions in the alanyl-amine peak at 16 ppm. The 16-ppm intensity corresponds to ester-linked D-Ala in WTA, and therefore glycopeptide antibiotics inhibited D-Ala incorporation into WTA in S. aureus suggesting inhibition of the WTA biosynthesis. d, The D-alanyl-amine peak at 16 ppm from the S0 spectra are overlaid. Oritavancin shows maximum inhibition of D-Ala incorporation into WTA. Each spectra were the result of 10,000 accumulated scans.
Figure 4
Figure 4. Vancomycin-treated S. aureus show inhibition of WTA biosynthesis preceding PG biosynthesis
a, D-[15N]Ala and L-[1-13C]Lys labeling of S. aureus PG and WTA in presence of alanine racemase inhibitor alaphosphin (5 ug/ml). 93-ppm peak intensities in the ΔS spectra (top) of 15N{13C} REDOR at 1.6 ms show that alaphosphin improves D-[15N]Ala incorporation into PG. The addition of vancomycin did not affect the PG stem-link density; however, the S0 spectra show a large reduction in the D-[15N]Ala incorporation into WTA (16 ppm). b, Schematic representation of WTA and PG biosyntheses with bactoprenol-phosphate (C55-P) as the central lipid transporter. Glycopeptide antibiotics prevent regeneration of the lipid transporter and thereby inhibit both PG and WTA biosyntheses. However, as shown in Fig. 4a D-[15N]Ala incorporation into WTA (red arrow) was inhibited in vancomycin-treated S. aureus by rerouting all available D-[15N]Ala into maintaining the PG biosynthesis (blue arrow). Hence, the vancomycin inhibition of D-[15N]Ala incorporation into the WTA biosynthesis precedes interference of the PG biosynthesis.
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