Role of lipopolysaccharide in membrane-active-peptide interactions with cells as probed by whole-cell deuterium NMR

Kumari, Sarika (2022) Role of lipopolysaccharide in membrane-active-peptide interactions with cells as probed by whole-cell deuterium NMR. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

The increasing appearance of multidrug-resistant pathogens has created an urgent need for suitable alternatives to current antibiotics. Antimicrobial peptides (AMPs), which act as defensive weapons against microbes, have shown great promise because bacteria develop no or low resistance to AMPs. However, only a few antimicrobial peptides are clinically available for clinical use. Understanding how non-phospholipid components of bacteria affect antimicrobial peptide-induced membrane disruption is important for a comprehensive understanding of AMP mechanisms and informing AMP-based drug development. Therefore, the main aim of this thesis was to investigate how lipopolysaccharide (LPS) affects membrane disruption by the AMP MSI-78 and compare the results to the effect of TP2, a cell-penetrating peptide that crosses membrane bilayers without permeabilizing them. We destabilize the LPS layer of Escherichia coli (E. coli) cells via chelation of the stabilizing divalent cations. ²H NMR observations of membrane-deuterated E. coli demonstrate that an ethylenediaminetetraacetic acid (EDTA) concentration of 9.0 mM alone has a minor effect on lipid acyl chain order. Interestingly, we find that E. coli pretreated with 9.0 mM EDTA are more sensitive to AMP-induced acyl chain disruption resulting from subsequent treatment with the AMP MSI-78. This indicates that LPS protects E. coli from membrane disruption caused by MSI-78. Surprisingly, we also found that at the level of 2H-NMR, the peptide-induced acyl chain disruptions are similar for MSI-78 and CPP-TP2, although MSI-78 permeabilizes the bilayer and TP2 does not. Furthermore, having intact LPS appears to sensitize the bacteria to TP2, in contrast to intact LPS’ ability to protect bacteria from MSI-78. I also provide some information about AMP selectivity by examining whether non-bacterial cells, i.e., mammalian cells, can compete with bacteria for AMP binding in a mixture of bacteria and mammalian cells. Interestingly, our preliminary data shows that when the MSI-78 was added to the mixture of cells, i.e., membrane-deuterated E. coli and Ramos cells, the presence of Ramos cells slightly reduced the amount of MSI-78 available to interact with the E. coli. Presumably due to some binding of MSI-78 by the Ramos cells. Overall, we show here that LPS, present in bacteria but not model membranes, protects bacteria to some extent from the AMP MSI-78. LPS protection from AMP membrane permeabilization would explain why model lipid bilayers are more prone to permeabilization by AMP than are bilayers in whole bacteria. In addition, since efforts to optimize AMPs as drugs often rely solely on optimizing the AMP’s lipid-permeabilizing activities, consideration of other interactions like AMP-LPS interactions may prove helpful in AMP-based drug design.

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