Dissecting the Bacterial Cell Wall with Solid-State NMR

Abstract

Gram-positive bacteria surround themselves with a macromolecular cell wall that is essential to cell survival and growth and serves as a major target for antibiotics. While the cell wall of S. aureus is comprised of only two major structural components, peptidoglycan and teichoic acids, it is a thick and heterogeneous matrix that is difficult to characterize with traditional biophysical techniques like x-ray crystallography and mass spectrometry. We use solid-state NMR spectroscopy to study the composition of isolated, but intact cell walls and also whole cells to measure and quantitatively compare chemical and atomic-level structural parameters across different strains and conditions, particularly to examine the modes of action of old and new antibiotics. We have introduced a new approach to measure the relative balance of teichoic acids and peptidoglycan within the cell wall structure. We extend this analysis to whole cells, where using spectral signatures of the cell wall, we identify distinct resonances to measure teichoic acid levels in intact bacterial cells. The distinguishing chemical shifts of the sugars and peptides in peptidoglycan, along with the phosphoribitol backbone and D-alanine ester decorations of teichoic acids provide insight into peptidoglycan and teichoic acid levels, respectively, in whole cell NMR samples. This analysis did not require isotopic labeling and can be performed with any bacterial strain. We do often couple such measurements with isotopic labeling and selective NMR detection schemes to fully dissect the atomic-level structure of the cell wall. We will also report on a focused set of results regarding the influence of growth state and the influence of inhibitors of teichoic acid synthesis on overall cell wall composition.