HLP6

Lactoferricin includes an 11-amino-acid amphipathic alpha-helical region which is exhibited on the outer surface of the amino-terminal lobe of lactoferrin. Synthetic peptides homologous to this region exhibited potent antibacterial activity against a selected range of both gram-negative and gram-positive bacteria. An analog synthesized with methionine substituted for proline at position 26, which is predicted to disrupt the helical region, abolished antibacterial activity against Escherichia coli and considerably reduced antibacterial activity against Staphylococcus aureus and an Acinetobacter strain. The mode of action of human lactoferrin peptide (HLP) 2 against E. coli serotype O111 (NCTC 8007) was established by using flow cytometry, surface plasmon resonance, and transmission electron microscopy. Flow cytometry was used to monitor membrane potential, membrane integrity, and metabolic processes by using the fluorescent probes bis-1,3-(dibutylbarbituric acid)-trimethine oxonol, propidium iodide, and carbonyl cyanide m-chlorophenylhydrazone, respectively. HLP 2 was found to act at the cell membrane, causing complete loss of membrane potential after 10 min and of membrane integrity within 30 min, with irreversible damage to the cell as shown by rapid loss of viability. The number of particles, measured by light scatter on the flow cytometer, dropped significantly, showing that bacterial lysis resulted. The peptide was shown to bind to E. coli O111 lipopolysaccharide by using surface plasmon resonance. Transmission electron microscopy revealed bacterial distortion, with the outer membrane becoming detached from the inner cytoplasmic membrane. We conclude that HLP 2 causes membrane disruption of the outer membrane, resulting in lysis, and that structural considerations are important for antibacterial activity.

Human lactoferrin contains a 47 amino acid peptide, named lactoferricin H, which is thought to be responsible for its antimicrobial activity. Lactoferricin includes a loop region, which resides on the outer surface of the N-lobe of lactoferrin, adopting an alpha helix with a hydrophobic tail. Peptides have been synthesised corresponding to the highly charged alpha helix (HLP 2) and hydrophobic tail region (HLP 5). HLP 2 has potent antibacterial activity whereas HLP 5 had no activity. To investigate the relationship between structure and function of HLP 2, HLP 6 was synthesised with a proline replacing methionine. This substitution was predicted to disrupt the helical region of the peptide and the orientation of the positively charged residues. Antibacterial activity was significantly reduced when tested against Escherichia coli serotype 0111, NCTC 8007. The mode of action of HLP 2 against the bacterial membrane was investigated by flow cytometric analysis, using Escherichia coli, NCTC 8007. Membrane potential and integrity were monitored using the fluorescent probes, bis 1,3-(dibutylbarbituric acid) trimethine oxonol and propidium iodide respectively. HLP 2 caused complete loss of membrane potential and integrity, with irreversible damage to the cell as shown by rapid loss of viability. We conclude that HLP 2 causes membrane disruption and that helicity is an important factor for antibacterial activity.

Antimicrobial peptides bovine lactoferricin (LfcinB) and human lactoferricin (LfcinH) are produced from the respective lactoferrin, but are more active than their precursors. Despite sequence homology, the bovine peptide and its derivatives are more active than their human homologs. Such differences between not only the peptides and their precursor but also between the bovine and the human peptides could relate to structural differences. Upon sequence alignment of both peptides with their parental proteins, the structural differences observed between the bovine lactoferrin (BLf) and LfcinB were also found between the human lactoferrin (HLf) and the LfcinH. The helical structures in HLf are replaced by beta-strands separated by a strong turn in LfcinH suggesting an antiparallel beta-sheet structure similar to LfcinB. MIC assays with HLP-2 and BLP-2, 11-residue peptides derived from the active core of both Lfcins, against Escherichia coli, showed that the bovine derivative, BLP-2, is more active than its human homolog HLP-2. Both 3D models for HLP-2 and BLP-2 showed that the beta-strand is centred between the aromatic residues giving both side chains the same orientations. The displacement towards the N-terminus observed for the beta-strand in HLP-2, compared with its central location in BLP-2, could be less favourable to membrane interaction and therefore responsible for the decrease in activity. Such a model suggests for LfcinH a mechanism similar to the one observed for LfcinB, where the absence of long-range interaction, present in lactoferrin, destabilises the first alpha helix, as observed in solution and, upon interaction with the membrane, could result in the formation of a beta-strand, as observed in the presence of LPS. The location of the beta-strand in relation to the positive charges, seems to define the efficiency of the activity of the peptide and may explain the difference in activity obtained between HLP-2 and BLP-2.