2H NMR Studies of Living Bacteria Interacting with Antimicrobial Peptides

Due to their activity against different pathogens and low toxicity to host cells, antimicrobial peptides (AMPs) are a promising approach to address the bacterial antimicrobial resistance. Biophysical methods have been used to observe the permeabilizing effect of AMPs on model membranes with the same lipid composition as the bacteria cell wall (BCW). In order to fully understand the antimicrobial properties of these peptides, it also necessary to investigate how AMPs interact with other relevant bacterial cell wall components including peptidoglycan and lipopolysaccharide. One way to better understand AMP interactions with non-lipidic components is to observe their effects on the complete bacterial cell wall. 2H Nuclear Magnetic Resonance (2H NMR) can be used to measure the orientational order parameters of lipid acyl chain segments. In this work, we have used 2H NMR to study the effect of AMPs on lipids in the membranes of whole bacteria. We use two techniques to prepare 2H labeled bacteria with isotope-labeled lipids introduced into the bacterial cell wall. These preparation techniques allow us to use 2H NMR to compare the orientational order parameter in the bacteria cell wall lipid bilayer of living wild type cells with and without AMP present. The effects two AMPs, either MSI-78 or CAME (Cepropin A (1-8) and melittin (1-10)), on both gram-negative (Escherichia coli) and gram-positive (Bacillus Subtilis) bacteria have been studied. In the presence of both MSI-78 and CAME, the 2H NMR spectral shape changes in ways that correspond to a decrease in the lipid acyl chain order parameter.