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A promising alternative to counter bacterial infections includes the antimicrobial peptides (AMPs), which have emerged as model molecules for rational design strategies. Here we focused on the study of<jats:italic>Pa<\/jats:italic>-MAP 1.9, a rationally designed AMP derived from the polar fish<jats:italic>Pleuronectes americanus<\/jats:italic>.<jats:italic>Pa<\/jats:italic>-MAP 1.9 was active against Gram-negative planktonic bacteria and biofilms, without being cytotoxic to mammalian cells. By using AFM, leakage assays, CD spectroscopy and<jats:italic>in silico<\/jats:italic>tools, we found that<jats:italic>Pa<\/jats:italic>-MAP 1.9 may be acting both on intracellular targets and on the bacterial surface, also being more efficient at interacting with anionic LUVs mimicking Gram-negative bacterial surface, where this peptide adopts \u03b1-helical conformations, than cholesterol-enriched LUVs mimicking mammalian cells. 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