An In-Cell Solid-State NMR Portrayal of the Action Mechanism of Antimicrobial Peptides with Intact Bacteria

Abstract

Antimicrobial peptides (AMPs) are promising candidates to act against drug-resistant bacteria since they can disrupt the bacterial lipid barrier, leading to cell death. 2H solid-state NMR is a valuable tool to study at a molecular level the action of AMPs on the lipid organization and dynamics within the membrane bilayer. While these studies are usually performed with model phospholipid membranes, the complexity of the bacterial cell wall prompts the development of in-cell NMR techniques to take into account interactions of all constituents. In particular, Gram(−) bacteria are protected by lipopolysaccharides in their outer membrane while a thick peptidoglycan covers the lipid bilayer of Gram(+) bacteria. We have thus developed novel 2H solid-state NMR tools to study in vivo the bactericidal action mechanism of aurein 1.2 and caerin 1.1, AMPs excreted by frog skin. Using deuterated palmitic acid, we have established a protocol to 2H-label phospholipids in Gram(+) Bacillus subtilis, and optimized the deuteration protocol of Gram(−) Escherichia coli. Via a combination of static and magic-angle spinning (MAS) experiments, our results support the interaction of the AMPs with membrane lipids leading to decrease in lipid chain order. This effect is, however, observed at higher peptide concentration with B. subtilis and most likely attributable to an interaction with negatively-charged components in its cell walls, such as teichoic acids. Our study shows that the nature of the cell wall plays a role upon the actual concentration of AMPs on the bacterial membrane and highlights the importance of studying membrane interactions with intact bacteria.