Structure-microbicidal activity relationship of synthetic fragments derived from the antibacterial alpha-helix of human lactoferrin

Abstract

There is a need for new microbicidal agents with therapeutic potential due to antibiotic resistance in bacteria and fungi. In this study, the structure-microbicidal activity relationship of amino acid residues 14 to 31 (sequence 14-31) from the N-terminal end, corresponding to the antibacterial alpha-helix of human lactoferrin (LF), was investigated by downsizing, alanine scanning, and substitution of amino acids. Microbicidal analysis (99% killing) was performed by a microplate assay using Escherichia coli, Staphylococcus aureus, and Candida albicans as test organisms. Starting from the N-terminal end, downsizing of peptide sequence 14-31 showed that the peptide sequence 19-31 (KCFQWQRNMRKVR, HL9) was the optimal length for antimicrobial activity. Furthermore, HL9 bound to lipid A/lipopolysaccharide, as shown by neutralizing endotoxic activity in a Limulus assay. Alanine scanning of peptide sequence 20-31 showed that Cys20, Trp23, Arg28, Lys29, or Arg31 was important for expressing full killing activity, particularly against C. albicans. Substituting the neutral hydrophilic amino acids Gln24 and Asn26 for Lys and Ala (HLopt2), respectively, enhanced microbicidal activity significantly against all test organisms compared to the amino acids natural counterpart, also, in comparison with HL9, HLopt2 had more than 10-fold-stronger fungicidal activity. Furthermore, HLopt2 was less affected by metallic salts than HL9. The microbicidal activity of HLopt2 was slightly reduced only at pH 7.0, as tested in the pH range of 4.5 to 7.5. The results showed that the microbicidal activity of synthetic peptide sequences, based on the antimicrobial alpha-helix region of LF, can be significantly enhanced by optimizing the length and substitution of neutral amino acids at specific positions, thus suggesting a sequence lead with therapeutic potential.

FIG. 1.

Amino acid sequence of HLBD1. A synthesized lactoferricin-like cyclic peptide, HLBD1, was based on the first α/β region of the LF molecule and comprised amino acid residues 16-40. The full α/β region of the LF molecule comprises the α-helix with amino acid residues 14-31 and a following β-sheet with amino acid residues 35-40. The bracket denotes the cyclization by a disulfide bond.

FIG. 2.

Kinetics of the killing activity of HLBD1 and peptide CFAWKRMRKVR. The peptides were incubated with E. coli strain O6K5, S. aureus strain 1800, and C. albicans in BP for 20 h using 200 μg/ml (65.4 μM) of HLBD1 and 100 μg/ml (62.7 μM) of peptide CFAWKRMRKVR. Samples for viable count were collected directly after mixing and at 5, 10, 30, 60, and 120 min. All samples were run in duplicate.

FIG. 3.

Effects of various pH levels on the microbicidal activities of HLopt2, HL9, and HLBD1. All three peptides were analyzed for 99% killing (MMC₉₉) of E. coli (a), and HLopt2 and HL9 were analyzed for 99% killing of C. albicans (b). The microbicidal assay was run at various pH levels by adjusting the pH of BP. Twofold serial dilutions were made of each peptide starting at 100 μg/ml of HLopt2 and HL9 and at 200 μg/ml for HLBD1. All samples were run in triplicate.

FIG. 4.

Neutralization of the Limulus activities of lipid A (a) and LPS (b) by the peptide sequences containing amino acid residues 20-31 to 17-31. (a) The peptides (25 ng/ml) of amino acid residues 20-31 to 17-31 were incubated with diphosphoryl and monophosphoryl lipid A, and the remaining Limulus activity was analyzed (expressed as percentage of maximal LAL activity). The experiment was repeated three times, except for peptide sequences 17-31 and 20-31 (single run for diphosphoryl lipid A; repeated once for monophosphoryl lipid A). *, P > 0.05; **, P > 0.01 (Student's t test). In panel b, the remaining activity of LPS O111 and O55 after incubation with 5 μg/ml of peptide sequence 19-31 (HL9), polymyxin B, and BPI peptide is shown. The experiment was repeated once except for O111 LPS (single run).