Hydrophobic moment drives penetration of bacterial membranes by transmembrane peptides

With antimicrobial resistance (AMR) remaining a persistent and growing threat to human health worldwide, membrane-active peptides are gaining traction as an alternative strategy to overcome the issue. Membrane-embedded multi-drug resistant (MDR) efflux pumps are a prime target for membrane-active peptides, as they are a well-established contributor to clinically relevant AMR infections. Here, we describe a series of transmembrane peptides (TMs) to target the oligomerization motif of the AcrB component of the AcrAB-TolC MDR efflux pump from Escherichia coli. These peptides contain an N-terminal acetyl-A-(Sar)(3) (sarcosine; N-methylglycine) tag and a C-terminal lysine tag—a design strategy our lab has utilized to improve the solubility and specificity of targeting for TMs previously. While these peptides have proven useful in preventing AcrB-mediated substrate efflux, the mechanisms by which these peptides associate with and penetrate the bacterial membrane remained unknown. In this study, we have shown peptide hydrophobic moment ([Formula: see text] H)—the measure of concentrated hydrophobicity on one face of a lipopathic [Formula: see text]-helix—drives bacterial membrane permeabilization and depolarization, likely through lateral-phase separation of negatively-charged POPG lipids and the disruption of lipid packing. Our results show peptide [Formula: see text] H is an important consideration when designing membrane-active peptides and may be the determining factor in whether a TM will function in a permeabilizing or non-permeabilizing manner when embedded in the bacterial membrane.