In Vivo 2H-NMR Study of the Action of Antibacterial Agents on Escherichia Coli Membranes

Abstract

Contrary to eukaryote membranes, bacterial membranes are negatively charged owing to the presence of phosphatidylglycerol (PG) or cardiolipin in their inner membranes. One of the action mechanisms of antibiotics such as antimicrobial peptides is to selectively interact with negatively charged lipids to create a breach in the bacterial membrane. The characterization of the cellular membrane integrity is, thus, a key element in understanding the mechanism of action of antimicrobial agents. Solid-state nuclear magnetic resonance spectroscopy (SS-NMR) is a useful tool that allows probing the organization and dynamics of phospholipids in a bilayer. In this work, we have performed an in vivo 2H-NMRstudy of Escherichia coli membranes labeled with deuterated palmitic acid. More specifically, we have studied the effect of nanoparticles (NPs) and antibacterial molecules on the bacteria membrane. The 2H-NMR spectra obtained on intact cells show that the E. coli membranes were successfully labeled according to previous work. A 10-hour exposure to the cationic detergent cetyltrimetylammonium chloride (CTAC) shows increased membrane fluidity at high detergent concentration (320µM). The study of the effect of fullerenol reveals that these NPs increase the lipid mobility in the membrane especially after 8 hours of exposure. The 2H-NMR spectra obtained in the presence of polymyxin B show no fast-tumbling membrane fragments, although UV analysis indicates the leakage of the cell content. These results suggest that this antibiotic would perforate E. coli membranes without their complete disruption. Our work illustrates the use of in vivo2H NMR studies to understand the specific action of different substances on labeled biological membranes.