A Novel Approach To Characterize The Membrane Disrupting Activity Of Antimicrobial Peptides Using 2h Solid State Nmr Of Whole Escherichia Coli PDF Download

Antimicrobial peptides (AMPs) are small chains of amino acids with the ability to cause bacterial death. AMPs are usually amphiphilic but diverse in charge and structure. The mechanism or mechanisms that AMPs implement to kill bacteria are still under discussion. Studies in model membranes have widely demonstrated the capacity of AMPs to interact with and disrupt the bacterial cell membrane. There are however, good reasons to suspect that AMP interactions with nonmembrane components of bacteria are important. For instance, there is an enormous discrepancy between the peptide to lipid molar ratio (P:L) necessary to generate membrane disruption in model systems (~ 1:100), and the P:L necessary to stop bacterial growth, i.e. the minimal inhibitory concentration (MIC) (~ 10- 100:1). One potential explanation for this discrepancy is that AMPs may interact with non-membrane components of the bacterial cell envelope. The peptidoglycan (PGN) layer is one of the primary non-membrane components of Gram-positive bacterial cell envelopes and is responsible for cell shape and stability. Currently, there is an open discussion about whether PGN can entrap AMPs and prevent them from reaching the cell membrane or, instead promote the accumulation of AMPs on the cell membrane. The primary experimental approach employed in this thesis was 2H NMR spectroscopy of intact bacteria with 2H-labeled membranes. Specifically, since the quadrupolar splittings obtained from the spectra are proportional to the order parameter of the lipid acyl chains, the spectra reflect the structure and dynamics of the membrane. In particular, 2H NMR spectroscopy allows us to characterize the disruption of lipid bilayers caused by AMPs. In this study, different methods to grow 2H-membrane-enriched bacteria were optimized to obtain 2H NMR spectra of E. coli LA8, E. coli JM109 and B. subtilis. Additionally, 2H NMR was used to observe the level of disruption of the cell membrane of B. subtilis caused by the presence of different AMPs, MSI-78 and BP100. Both MSI-78 and BP100 caused the same level of membrane disruption at similar concentrations. Separately, comparison of the 2H NMR spectra of B. subtilis with intact and compromised PGN layers showed no differences. The lack of change in the spectra of PGN-compromised and PGN intact bacteria indicates that there is no change in the dynamics or structure of the lipid bilayer even with a weakened PGN layer. Finally, the disruption caused by MSI-78 and BP100 was measured in B. subtilis with a compromised PGN layer. The results indicate that there is no change in the level of membrane disruption caused by MSI-78 and BP100 when the PGN layer is partially removed.