The antimicrobial peptides lactoferricin B and magainin 2 cross over the bacterial cytoplasmic membrane and reside in the cytoplasm.

The localization of immunolabelled antimicrobial peptides was studied using transmission electron microscopy. Staphylococcus aureus and Escherichia coli were exposed to lactoferricin B (17-41), lactoferricin B (17-31) and D-lactoferricin B (17-31). E. coli was also exposed to cecropin P1 and magainin 2. The lactoferricins were found in the cytoplasm of both bacteria. In S. aureus the amount of cytoplasmic lactoferricin B (17-41) was time- and concentration-dependent, reaching a maximum within 30 min. Cecropin P1 was confined to the cell wall, while magainin 2 was found in the cytoplasm of E. coli. The finding of intracellularly localized magainin is not reported previously.

Bactericidal kinetics of 3 lactoferricins against Staphylococcus aureus and Escherichia coli.

Bovine lactoferricin is an antimicrobial, cationic peptide generated upon gastric pepsin cleavage of bovine lactoferrin. We investigated the bactericidal effects of native lactoferricin [Lfcin B(17-41)], a shortened derivative [Lfcin B(17-31)] and the all-D-amino acid counterpart of Lfcin B(17-31) against Escherichia coli and Staphylococcus aureus. The results revealed different activities for the peptides against Gram-positive and -negative bacteria. D-Lfcin B(17-31) was the most efficient peptide against E. coli. The same peptide showed improved activity against S. aureus, D-Lfcin B(17-31) showed a significant better efficacy when compared to the L-form, but not when compared to Lfcin B(17-41). There was no correlation between the bactericidal concentrations and the time needed to achieve maximum effect. This indicates the importance of structural differences between the peptides and/or bacteria and implies that the simple thesis of I antibacterial target is not valid for lactoferricin.

Short antibacterial peptides and erythromycin act synergically against Escherichia coli.

Five different peptides (6-18 residues) with chain lengths shorter than the required minimum to span the bacterial cell membrane as monomeric helices were designed in order to elucidate whether variation in chain length exerted differences in their mode of action. To gain a better understanding of the possible mode of action of these peptides, they were studied in combination with clinically used antibiotics with different targets. Antibiotic-peptide combinations were tested against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. No synergy was observed between the peptides and antibiotics when tested against S. aureus. Synergic interactions between all peptides and erythromycin were observed when tested against E. coli. Synergy was also observed with rifampicin and two peptides against E. coli. There was no clear-cut correlation between the ability to interact synergically or antagonistically and the number of residues. We further investigated the combined action of our peptides and PGLa, to elucidate peptide-peptide interactions. In contrast to previously reported synergy between magainin 2 and PGLa, our peptides did not show any synergy when combined with PGLa. Thus, our results indicate an alternative mode of action of these antibacterial peptides as compared with peptides such as magainin 2.

Lactoferricin B causes depolarization of the cytoplasmic membrane of Escherichia coli ATCC 25922 and fusion of negatively charged liposomes.

Antimicrobial peptides have been extensively studied in order to elucidate their mode of action. Most of these peptides have been shown to exert a bactericidal effect on the cytoplasmic membrane of bacteria. Lactoferricin is an antimicrobial peptide with a net positive charge and an amphipatic structure. In this study we examine the effect of bovine lactoferricin (lactoferricin B; Lfcin B) on bacterial membranes. We show that Lfcin B neither lyses bacteria, nor causes a major leakage from liposomes. Lfcin B depolarizes the membrane of susceptible bacteria, and induces fusion of negatively charged liposomes. Hence, Lfcin B may have additional targets responsible for the antibacterial effect.

Initial binding sites of antimicrobial peptides in Staphylococcus aureus and Escherichia coli.

We examined the initial binding sites of magainin 1, cecropin P1 and lactoferricin B in Staphylococcus aureus and Escherichia coli. All 3 peptides were active against E. coli, whereas only lactoferricin B exerted any activity against S. aureus. Soluble lipoteichoic acid and lipopolysaccharide both interacted with all 3 peptides, whereas soluble teichoic acid interacted with lactoferricin B only. Antibodies against teichoic acid diminished the activity of lactoferricin B, while antibodies against lipoteichoic acid had no influence on the activity of lactoferricin B. Antibodies against lipopolysaccharide diminished the activity of lactoferricin B and magainin 1, but had no effect on the activity of cecropin P1 against E. coli. We conclude that the initial binding sites of lactoferricin B in S. aureus, and of lactoferricin B and magainin 1 in E. coli, are teichoic acid and lipopolysaccharide, respectively. Cecropin P1 seems to interact with a different binding site than those of magainin 1 and lactoferricin B in E. coli.

Interference of the antimicrobial peptide lactoferricin B with the action of various antibiotics against Escherichia coli and Staphylococcus aureus.

The antimicrobial peptide, lactoferricin, can be generated upon gastric pepsin cleavage of lactoferrin. We have examined the interaction of lactoferricin of bovine origin, Lf-cin B, with the antibiotics penicillin G, vancomycin, gentamicin, colistin, D-cycloserine and erythromycin against E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923. We demonstrated synergism between Lf-cin B and erythromycin against E. coli, and partial synergism between Lf-cin B and penicillin G, vancomycin and gentamicin against E. coli. Only penicillin G acted in partial synergism with Lf-cin B against S. aureus. Lf-cin B antagonized vancomycin and gentamicin against S. aureus in low concentration. We conclude that Lf-cin B may facilitate the uptake of antibiotics across the cell envelope.

Antibacterial effects of lactoferricin B.

The antimicrobial peptide, lactoferricin, can be generated upon gastric pepsin cleavage of lactoferrin. We have examined the inhibitory efficacy of lactoferricin of bovine origin (Lf-cin B) on Escherichia coli, Proteus mirabilis and Staphylococcus aureus with or without a cell wall. We found that spheroplasts and protoplasts had a lower MIC than their counterparts with a cell wall. We also compared the efficacies of Lf-cin B (17-31) made of all L-amino acids and all D-amino acids. The peptide made of all D-amino acids was more active than the corresponding L-enantiomer. Furthermore, we examined the influence of Lf-cin B on the motility of E. coli and the influence of temperature on the susceptibility of bacteria exposed to Lf-cin B. Bacteria exposed to sub-MIC of Lf-cin B lost their motility. Bacteria exposed to Lf-cin B at 20 degrees C were more sensitive to Lf-cin B than when exposed at 37 degrees C. These findings indicate that the cell envelope is a limiting step for Lf-cin B to exert its antibiotic effect. We cannot rule out a receptor-mediated first step for Lf-cin B (17-31).

Lactoferricin of bovine origin is more active than lactoferricins of human, murine and caprine origin.

The antimicrobial peptide lactoferricin is generated by gastric pepsin cleavage of lactoferrin. We have examined the antimicrobial activity of lactoferricins derived from lactoferrin of human, murine, caprine and bovine origin with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against E. coli ATCC 25922 and S. aureus ATCC 25923. We found that lactoferricin of bovine origin (Lf-cin B) was the most efficacious of the lactoferricins tested. By comparing the linear and cyclic Lf-cin B we found the cyclic peptide to be the most active. Lactoferricin B was moderately active against E. coli ATCC 25922 and S. aureus ATCC 25923, but had no activity against P. mirabilis or Y. enterocolitica. Lf-cin B showed good activity against C. albicans, C. tropicalis and C. neoformans.