A novel approach to characterize the membrane disrupting activity of antimicrobial peptides using ²H solid-state NMR of whole Escherichia coli

Pius, James (2012) A novel approach to characterize the membrane disrupting activity of antimicrobial peptides using ²H solid-state NMR of whole Escherichia coli. Masters thesis, Memorial University of Newfoundland.

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Abstract

Antimicrobial peptides (AMPs) are ubiquitous molecules that can display antimicrobial activity against bacteria, viruses, protozoa and various other pathogens. They are commonly short, cationic amphipathic peptides that may offer valuable bases for the development of new therapeutic agents. AMPs can have diverse mechanisms of antibacterial activity; all these mechanisms involve interacting with the bacterial membrane to some degree. In order to better understand these mechanisms, a variety of biophysical techniques including solid-state NMR (SSNMR) have been used to study AMPs in model lipid systems. However, these simple model systems do not reflect the complexity of whole cells with which AMPs interact in vivo. In order to bridge the gap between SSNMR studies of AMPs in model membranes and the true biological context of AMPs, a procedure was developed which incorporates high levels of ²H NMR labels specifically into the cell membrane of Escherichia coli. Using these labeled cells, ²H NMR spectra were acquired both in the absence and presence of the AMP MSI-78. The effects of MSI-78 on bacterial membrane disorder were quantified in terms of the first moment of the spectrum, which directly reflects the activity of MSI-78 in disrupting the membranes of whole bacteria. Data obtained from NMR measurements show an increased disorder in the orientation of lipids in the cell with increasing levels of MSI-78. These effects are observed at molar peptide:lipid (P:L) ratios 30 times greater than that used for model lipid systems and 25 times less than the ratios needed to inhibit cell growth. Based on the observed results, MSI-78 can disrupt the lipid order in the cell envelope even at sublethal concentrations.

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