Spectral Snapshots of Bacterial Cell-Wall Composition and the Influence of Antibiotics by Whole-Cell NMR

Abstract

Gram-positive bacteria surround themselves with a thick cell wall that is essential to cell survival and is a major target of antibiotics. Quantifying alterations in cell-wall composition are crucial to evaluating drug modes of action, particularly important for human pathogens that are now resistant to multiple antibiotics such as Staphylococcus aureus. Macromolecular and whole-cell NMR spectroscopy allowed us to observe the full panel of carbon and nitrogen pools in S. aureus cell walls and intact whole cells. We discovered that one-dimensional (13)C and (15)N NMR spectra, together with spectroscopic selections based on dipolar couplings as well as two-dimensional spin-diffusion measurements, revealed the dramatic compositional differences between intact cells and cell walls and allowed the identification of cell-wall signatures in whole-cell samples. Furthermore, the whole-cell NMR approach exhibited the sensitivity to detect distinct compositional changes due to treatment with the antibiotics fosfomycin (a cell-wall biosynthesis inhibitor) and chloramphenicol (a protein synthesis inhibitor). Whole cells treated with fosfomycin exhibited decreased peptidoglycan contributions while those treated with chloramphenicol contained a higher percentage of peptidoglycan as cytoplasmic protein content was reduced. Thus, general antibiotic modes of action can be identified by profiling the total carbon pools in intact whole cells.

Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy

“…Both NG8 (black) and PC3370 (red) cell wall spectra exhibit resonances for protein and peptidoglycan species with expected relative intensities (Reichhardt et al 2015). Specifically, our observation of protein chemical shifts (resonances for carbonyl carbons at 170–180 ppm, α-carbons at 50–60 ppm, aliphatic carbons at 10–40 ppm) as well as polysaccharide chemical shifts (acetyl carbons at 175–180ppm, anomeric carbons at 95–100 ppm, sugar-ring carbons at 70–80 ppm, methyl acetylation carbons at 10–20 ppm) agree well with previously published studies of Gram-positive cell walls (Nygaard et al 2015). A difference spectrum (NG8-PC3370, in blue) shows no substantial differences between the two cell wall preps.…”

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

“…14 We were even able to determine that the polysaccharide contributions to whole cells primarily arise from the cell wall by examining protoplasts in which much of the cell wall is removed. Furthermore, the polysaccharide signatures in whole cells were able to reflect the influence of two antibiotics with different modes of action.…”

“…Whole-cell 13 C spectra revealed an altered balance of 13 C contributions, where cell-wall polysaccharides were preferentially increased as protein synthesis was inhibited and protein contributions were reduced. 14 We have been working to extend this approach to other bacterial organisms and to explore the extent of atomic-level compositional detail that can be extracted from uniformly labeled samples, with one of the goals being to examine the full repertoire of cellular changes that accompany treatment with old and new antibiotics. Uniform labeling is typically simpler to implement than specific-labeling strategies.…”

Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

“…Changes in chemical shifts have also been noted to occur when proteins bind to the cell wall, inferring that the protein may be binding to those residues that experienced a chemical shift change [41]. A representative set of cell wall and whole cell two-dimensional 13 C spin diffusion-based spectra obtained in our laboratory is shown in figure 4 and is useful for qualitative insight and comparisons [53]. The carbohydrate spin system, reflected by C correlations, is comparable between cell walls and whole cells.…”

“…In this first example, a general approach to profile cell-wall composition was reported which can be performed with natural abundance (with no isotopic labelling required) or on uniformly 13 C-labelled samples (which makes possible other correlation experiments that require higher enrichment levels) [53]. One-dimensional NMR spectra of isolated cell walls reveal the unique compositional signatures of cell-wall material (figure 2b).…”