Whole Cell 2H Solid-State NMR of Antimicrobial Peptides Interacting with Cell Envelopes: Role of Lipopolysaccharide

Abstract

Antimicrobial Peptides (AMPs) have been studied for more than two decades because of their promise to help overcome the problem of resistance to conventional antibiotics. However, AMPs have not been as successful as hoped, likely because we lack a detailed understanding of their mechanisms of action. Many biophysical studies of AMPs are performed in model membrane systems, composed of just one or two lipid components. Such studies have shown that membrane permeabilization is a common mechanism of action. Yet we don’t fully understand their connection between the behavior of AMPs in model lipids, where membrane permeabilization is seen and the behavior in real bacteria, where AMPs are observed to inhibit cell growth. Bacteria have an extra non-lipid component in their cell envelopes. It is thus possible that interactions between AMPs and non-lipid components of the cell envelope are important to their mechanisms of action. Specifically, the focus of this study is to find out if the lipopolysaccharide (LPS) outer membrane layer of Gram-negative bacteria promotes or inhibits AMP-induced membrane disruption. This work employed the magainin analog, MSI-78. We disrupt the lipopolysaccharide layer of Gram-negative bacteria (E.coli) via chelation of the stabilizing divalent cations. Then, we use deuterium NMR of deuterated intact bacteria to observe how AMP-induced acyl chain disruption is affected by LPS layer destabilization. In addition to 2H NMR, we do light microscopy and flow cytometry to confirm the EDTA disruption of LPS.