The peptidoglycan sacculus is an essential component of the bacterial cell wall. Surrounding the cytoplasmic membrane, it plays a crucial role in allowing cells to withstand osmotic pressure and in defining cellular shape. Unique to the bacterial world, this large polymer network is also the principal target of many antibiotics, and one of the main microbial products recognized by the immune system. Consequently, great efforts have been invested in the last decade to determine its architecture and biosynthesis. However, the large molecular weight (about 3×106 kDa for E. coli) and non-crystallinity of the sacculus makes the non-fragmented molecule inaccessible to most analytical methods. In collaboration with IBS, LRM has shown for the first time that solid-state NMR spectroscopy can successfully be applied to intact uniformly labeled peptidoglycan. Using this technique, different aspects of the bacterial cell wall were investigated, as e.g. protein-peptidoglycan interaction, dynamics of the different biopolymers involved in the cell wall and metal-ion complexation.
Fig. 1 : Comparison of peak intensities found in correlation spectra and T1 time constants for the different carbon sites in E. Coli alone (blue) or in interaction with YajG (red).
Peptidoglycan is a hetero-polymer made of linear glycan strands of two alternating b-1,4-linked carbohydrates, that are cross-linked by short peptides (Fig. 1). The peptides are covalently linked to the glycan chains, and can vary between different bacteria in the nature of the amino acids involved, and in the number and the structure of the peptide cross-links. For the solid-state NMR study, uniformly 13C-15N-isotopically enriched cells of different bacterial strains were grown, the sacculi purified and directly centrifuged into the NMR rotor. Considering the size and non-crystallinity of the sample, the obtained NMR spectra were of astonishing good resolution, allowing thus atom-resolved investigation of the peptidoglycan structure and dynamics.
Using 2D 13C correlation NMR spectra, an almost complete assignment of the monomeric unit of the peptidoglycan could be obtained, allowing thus the identification of each separate carbon atom in the spectra. Based on this knowledge, the interaction of E. coli sacculi with a recdently discovered peptidoglycan-interacting protein (YajG protein with a yet unknown role in peptidoglycan metabolism) has been investigated. The comparison of correlation spectra acquired on sacculi incubated alone or in the presence of the protein revealed a significant non-uniform modification of the cross-peak intensities. This could be related to changes in the 13C longitudinal relaxation time constants (see Fig. 1), implying that the interaction of the protein takes place primarily on the glycan chains, inducing a rigidification of the sugar network.
Fig. 2 : Schematic representation of the cation-mediated interaction between peptidoglycan and teichoic acids in Gram-positive cell walls.
In Gram-positive bacteria such as B. Subtilis or S. Aureus a network of anionic cell-wall polymers, called wall teichoic acids (WTAs), are covalently bound to the peptidoglycan. In such bacteria, the peptidoglycan and WTAs are directly at the interface between the cell and its environment. The polyionic network formed by the TAs has many roles, such as the regulation of metal cation homeostasis and ion trafficking across the cell. We have shown that solid-state 31P NMR is particularly well adapted to rapidly characterize WTA of different bacterial species, and this both on isolated cell walls or intact bacterial cells. The sensitivity of the WTA 31P chemical shifts to the presence of metal ions was used to measure the dissociation constant of Mg2+ in intact cell walls, showing a much higher affinity as found in previous studies performed on isolated TAs. The changes in 31P and 13C spectra upon complexation with the paramagnetic ion Mn2+, clearly demonstrated that the peptidoglycan itself is involved in the interaction of metal ions with WTAs. This allowed us to propose a new interaction model suggesting a cation-mediated interaction between the peptidoglycan and WTAs in Gram-positive cell walls (Fig. 2).
Selected publication(s): T. Kern et al., J. Am. Chem. Soc. 130 (2008) 5618.
Maj : 17/01/2017 (560)