REDOR constraints on the peptidoglycan lattice architecture of Staphylococcus aureus and its FemA mutant

Singh, Manmilan; Kim, Sung Joon; Sharif, Shasad; Preobrazhenskaya, M. N.; Schaefer, Jacob

doi:10.1016/j.bbamem.2014.05.025

Cited by 14 publications

(19 citation statements)

References 14 publications

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“…However, the tertiary structure of bacterial cell walls is poorly understood due to its heterogeneous and insoluble nature, making it unsuitable for X-ray crystallography and solution NMR studies. Here we use solid-state NMR [30–33] to investigate the tertiary structure of the peptidoglycan of the E. faecalis cell wall [20, 21]. Two E. faecalis whole-cell and two cell-wall samples were prepared for NMR by culturing E. faecalis in media containing combinations of 13 C and 15 N specific labels in D- and L-alanine and L-lysine (in the presence of the alanine racemase inhibitor, alaphosphin).…”

Section: Introductionmentioning

confidence: 99%

Characterization of the tertiary structure of the peptidoglycan of Enterococcus faecalis

Yang

Singh

Kim

et al. 2017

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Solid-state NMR spectra of whole cells and isolated cell walls of Enterococcus faecalis grown in media containing combinations of 13C and 15N specific labels in D- and L-alanine and L-lysine (in the presence of an alanine racemase inhibitor alaphosphin) have been used to determine the composition and architecture of the cell-wall peptidoglycan. The compositional variables include the concentrations of (i) peptidoglycan stems without bridges, (ii) D-alanylated wall teichoic acid, (iii) cross-links, and (iv) uncross-linked tripeptide and tetra/pentapeptide stems. Connectivities of L-alanyl carbonyl-carbon bridge labels to D-[3-13C]alanyl and L-[ε-15N] lysyl stem labels prove that the peptidoglycan of E. faecalis has the same hybrid short-bridge architecture (with a mix of parallel and perpendicular stems) as the FemA mutant of Staphylococcus aureus, in which the cross-linked stems are perpendicular to one another and the cross-linking is close to the ideal 50% value. This is the first determination of the cell-wall chemical and geometrical architecture of whole cells of E. faecalis, a major source of nosocomial infections worldwide.

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Section: Introductionmentioning

confidence: 99%

Characterization of the tertiary structure of the peptidoglycan of Enterococcus faecalis

Yang

Singh

Kim

et al. 2017

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…In related approaches, particularly in selectively labeled samples, frequency-selective inversions followed by spin diffusion have also been employed to extract connectivity and proximity information in complex assemblies such as plant leaves and bacterial whole cells. 27–29 In this study we implemented a three-part sequence relay in which we begin with fsREDOR to observe carbons bonded to amine nitrogens of interest, spread magnetization through dipolar-assisted rotational resonance (DARR) 30 , and then probe whether the observable carbonyls are near to amide nitrogens. As described below, this allowed us to identify whole-cell NMR contributions that we ascribe to teichoic acid D-alanine.…”

Section: Introductionmentioning

confidence: 99%

Frequency-selective REDOR and spin-diffusion relays in uniformly labeled whole cells

Rice

Romaniuk

Cegelski

2015

Solid State Nuclear Magnetic Resonance

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Solid-state NMR is a powerful and non-perturbative method to measure and define chemical composition and architecture in bacterial cell walls, even in the context of whole cells. Most NMR studies on whole cells have used selectively labeled samples. Here, we introduce an NMR sequence relay using frequency-selective REDOR (fsREDOR) and spin diffusion elements to probe a unique amine contribution in uniformly 13 C-and 15 N-labeled Staphylococcus aureus whole cells that we attribute to the D-alanine of teichoic acid. In addition to the primary peptidoglycan structural scaffold, cell walls can contain significant amounts of teichoic acid that contribute to cell-wall function. When incorporated into teichoic acid, D-alanine is present as an ester, connected via its carbonyl to a ribitol carbon, and thus has a free amine. Teichoic acid D-Ala is removed during cell-wall isolations and can only be detected in the context of whole cells. The sequence presented here begins with fsREDOR and a chemical shift evolution period for 2D data acquisition, followed by DARR spin diffusion and then an additional fsREDOR period. fsREDOR elements were used for 13 C observation to avoid complications from 13 C-13 C couplings due to uniform labeling and for 15 N dephasing to achieve selectivity in the nitrogens serving as dephasers. The results show that the selected amine nitrogen of interest is near to teichoic acid ribitol carbons and also the methyl group carbon associated with alanine. In addition, its carbonyl is not significantly dephased by amide nitrogens, consistent with the expected microenvironment around teichoic acid.

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“…For disaccharide-modified lipoglycopeptides, the drug's hydrophobic side chain forms a secondary binding site that enables targeting of the cross-linked bridge structure of PG (Fig. 1d) (25)(26)(27). Unlike the Edman degradation product of vancomycin, which is devoid of any activity, des-Ori retains potent antimicrobial activities against both vancomycin-susceptible and -resistant pathogens despite the damaged D-Ala-D-Ala binding site (28,29).…”

Section: Discussionmentioning

confidence: 99%

“…We attribute these changes to des-Ori binding to the PG template at the inner layer of mature cell wall close to the cell membrane where the nascent PG is incorporated. This interaction with the PG template interferes with both the alignment and orientation of nascent PG stem and bridge structures, which are necessary for efficient incorporation of these new PGs into mature cell wall as mediated by the transpeptidase (26,(32)(33)(34). Thus, subsequent PG biosynthesis based on these defective templates results in the ensuing disorder of the cell wall, with reduced PG cross-linking, altered PG acetylation, and immature PG (improper PG stem modifications), which leaves bacteria highly susceptible to lysis.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Staphylococcus aureus Cell Wall Biosynthesis by Desleucyl-Oritavancin: a Quantitative Peptidoglycan Composition Analysis by Mass Spectrometry

et al. 2017

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Oritavancin is a lipoglycopeptide antibiotic that exhibits potent activities against vancomycin-resistant Gram-positive pathogens. Oritavancin differs from vancomycin by a hydrophobic side chain attached to the drug disaccharide, which forms a secondary binding site to enable oritavancin binding to the cross-linked peptidoglycan in the cell wall. The mode of action of secondary binding site was investigated by measuring the changes in the peptidoglycan composition of Staphylococcus aureus grown in the presence of desleucyl-oritavancin at subinhibitory concentration using liquid chromatography-mass spectrometry (LC-MS). Desleucyl-oritavancin is an Edman degradation product of oritavancin that exhibits potent antibacterial activities despite the damaged D-Ala-D-Ala binding site due to its functional secondary binding site. Accurate quantitative peptidoglycan composition analysis based on 83 muropeptide ions determined that cell walls of S. aureus grown in the presence of desleucyl-oritavancin showed a reduction of peptidoglycan cross-linking, increased muropeptides with a tetrapeptide-stem structure, decreased O-acetylation of MurNAc, and increased N-deacetylation of GlcNAc. The changes in peptidoglycan composition suggest that desleucyl-oritavancin targets the peptidoglycan template to induce cell wall disorder and interferes with cell wall maturation.IMPORTANCE Oritavancin is a lipoglycopeptide antibiotic with a secondary binding site that targets the cross-linked peptidoglycan bridge structure in the cell wall. Even after the loss of its primary D-Ala-D-Ala binding site through Edman degradation, desleucyl-oritavancin exhibits potent antimicrobial activities through its still-functioning secondary binding site. In this study, we characterized the mode of action for desleucyl-oritavancin's secondary binding site using LC-MS. Peptidoglycan composition analysis of desleucyl-oritavancin-treated S. aureus was performed by determining the relative abundances of 83 muropeptide ions matched from a precalculated library through integrating extracted ion chromatograms. Our work highlights the use of quantitative peptidoglycan composition analysis by LC-MS to provide insights into the mode of action of glycopeptide antibiotics.

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REDOR constraints on the peptidoglycan lattice architecture of Staphylococcus aureus and its FemA mutant

Cited by 14 publications

References 14 publications

Characterization of the tertiary structure of the peptidoglycan of Enterococcus faecalis

Characterization of the tertiary structure of the peptidoglycan of Enterococcus faecalis

Frequency-selective REDOR and spin-diffusion relays in uniformly labeled whole cells

Inhibition of Staphylococcus aureus Cell Wall Biosynthesis by Desleucyl-Oritavancin: a Quantitative Peptidoglycan Composition Analysis by Mass Spectrometry

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