The study looks at how two lab‑made versions of a natural human peptide (LL‑37) kill E. coli bacteria. It finds that killing isn’t just about poking holes in the bacterial membrane; instead, the peptides disturb the membrane’s electric charge and how its fats are packed, which leads to death. This effect works the same way regardless of the bacteria’s surface sugar coat.

OP-145 and SAAP-148, two 24-mer antimicrobial peptides derived from human cathelicidin LL-37, exhibit killing efficacy against both Gram-positive and Gram-negative bacteria at comparable peptide concentrations. However, when it comes to the killing activity against <i>Escherichia coli</i>, the extent of membrane permeabilization does not align with the observed bactericidal activity. This is the case in living bacteria as well as in model membranes mimicking the <i>E. coli</i> cytoplasmic membrane (CM). In order to understand the killing activity of both peptides on a molecular basis, here we studied their mode of action, employing a combination of microbiological and biophysical techniques including differential scanning calorimetry (DSC), zeta potential measurements, and spectroscopic analyses. Various membrane dyes were utilized to monitor the impact of the peptides on bacterial and model membranes. Our findings unveiled distinct binding patterns of the peptides to the bacterial surface and differential permeabilization of the <i>E. coli</i> CM, depending on the smooth or rough/deep-rough lipopolysaccharide (LPS) phenotypes of <i>E. coli</i> strains. Interestingly, the antimicrobial activity and membrane depolarization were not significantly different in the different LPS phenotypes investigated, suggesting a general mechanism that is independent of LPS. Although the peptides exhibited limited permeabilization of <i>E. coli</i> membranes, DSC studies conducted on a mixture of synthetic phosphatidylglycerol/phosphatidylethanolamine/cardiolipin, which mimics the CM of Gram-negative bacteria, clearly demonstrated disruption of lipid chain packing. From these experiments, we conclude that depolarization of the CM and alterations in lipid packing plays a crucial role in the peptides' bactericidal activity.