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Antibiotic treatment for microbial infections is under scrutiny due to increasing resistance to conventional antibiotics, warranting discovery of new classes of antibiotic agents. Antimicrobial peptides are part of the innate defense system found in nearly all organisms and possess bactericidal mechanisms that make it more difficult for bacteria to develop resistance. Porcine lactoferricin (LFP-20) is an antimicrobial peptide located in the N terminus of lactoferrin (LF). To develop novel cell-selective antimicrobial peptides with improved antimicrobial specificity compared with LFP-20, analogs LF2A LF-2, LF-4, and LF-6 were substituted with Ala, Ser, or Trp residues at different positions in the molecule. Analogs displayed a 2- to 16-fold higher antimicrobial activity than LFP-20, but were hemolytic at 64 μg/mL. Additionally, LFP-20, LF2A, LF-2, and LF-4 exhibited lower cytotoxicity against human peripheral blood mononuclear cells than LF-6 at concentrations of 25 to 100 μg/mL. To better understand the antibacterial mechanisms of LFP-20 and its analogs we examined their effect on the cytoplasmic membrane of Escherichia coli. The LFP-20 was not effective in depolarizing cytoplasmic membranes, whereas the other 3 analogs gradually dissipated the membrane potential of E. coli. Membrane potential increased with minimal inhibitory concentrations changes, demonstrating a correlation between bactericidal activity and membrane depolarization. Analogs were more efficient than LFP-20 in displacing lipopolysaccharide-bound dansyl-polymyxin B, which also rapidly increased 1-N-phenyl-naphthylamine uptake and release of cytoplasmic β-galactosidase by increasing the permeability of the outer and inner membranes of E. coli. The 3 analogs caused an increased potential for calcein leakage from negatively charged lipid vesicles at high concentrations. Collectively, these results suggest that the first targets of LF-2, LF-4, and LF-6 in E. coli are cytoplasmic membranes. The 3 analogs exhibited lethal effects based on their abilities to disrupt membranes and permit transit of large intracellular components, such as calcein.

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