Physicochemical properties that enhance discriminative antibacterial activity of short dermaseptin derivatives

Abstract

Antimicrobial peptides are widely believed to exert their effects by nonspecific mechanisms. We assessed the extent to which physicochemical properties can be exploited to promote discriminative activity by manipulating the N-terminal sequence of the 13-mer dermaseptin derivative K(4)-S4(1-13) (P). Inhibitory activity determined in culture media against 16 strains of bacteria showed that when its hydrophobicity and charge were changed, P became predominantly active against either gram-positive or gram-negative bacteria. Thus, conjugation of various aminoacyl-lysin moieties (e.g., aminohexyl-K-P) led to inactivity against gram-positive bacteria (MIC(50) > 50 microM) but potent activity against gram-negative bacteria (MIC(50), 6.2 microM). Conversely, conjugation of equivalent acyls to the substituted analog M(4)-S4(1-13) (e.g., hexyl-M(4)-P) led to inactivity against gram-negative bacteria (MIC(50) > 50 microM) but potent activity against gram-positive bacteria (MIC(50), 3.1 microM). Surface plasmon resonance experiments, used to investigate peptides' binding properties to lipopolysaccharide-containing idealized phospholipid membranes, suggest that although the acylated derivatives have increased lipophilic properties with parallel antibacterial behavior, hydrophobic derivatives are prevented from reaching the cytoplasmic membranes of gram-negative bacteria. Moreover, unlike modifications that enhanced the activity against gram-positive bacteria, which also enhanced hemolysis, we found that modifications that enhanced activity against gram-negative bacteria generally reduced hemolysis. Thus, compared with the clinically tested peptides MSI-78 and IB-367, the dermaseptin derivative aminohexyl-K-P performed similarly in terms of potency and bactericidal kinetics but was significantly more selective in terms of discrimination between bacteria and human erythrocytes. Overall, the data suggest that similar strategies maybe useful to derive potent and safe compounds from known antimicrobial peptides.

FIG. 1.

Growth-inhibitory activities of representative derivatives against a range of bacterial strains. Relative MIC is a ratio of the average MIC of a derivative to that of P (an identical MIC is assigned a value of zero). Standard deviations for averaged MICs were ≤0.7 μM. Specific values are specified when they were higher or lower than ±5. Bc, Bacillus cereus; Ef, Enterococcus faecalis; Ml, Micrococcus luteus; Sa, Staphylococcus aureus; Se, Staphylococcus epidermidis; Sp, Streptococcus pyogenes; Ec, Escherichia coli; Pa, Pseudomonas aeruginosa; St, Salmonella enterica serovar Typhimurium; Vc, Vibrio cholerae; Yk, Yersinia kristensenii.

FIG. 2.

Selective bactericidal activity in coculture. Cocultures of S. aureus and E. coli were exposed to one of the specified peptides (3 μM; 1 h; 37°C) or to buffer (control) and then plated for a CFU count. (Right) A representative set of the resulting plates (inset) and an enlargement of each plate. (Left) Plot of the CFU counts from the plates shown on the right. The asterisks indicate negative cultures. The error bars represent standard deviations of the mean determined from two independent experiments performed in duplicate.

FIG. 3.

Effects of acylation on peptide organization. (A) Peptide self-assembly in PBS as investigated by light-scattering measurements. The values represent the means from two independent experiments performed in duplicate. The error bars represent standard deviations of the mean. (B) Effects of acylation on peptide secondary structure in the presence of 2 mM POPC-POPG (3:1) liposomes. Shown are far-UV CD spectra of P (dotted line), C6-M₄-P (dashed line), NC6-K-P (solid line), and NC12-P (dashed and dotted line). Spectra were scanned for peptide samples of 100 μM in sodium phosphate buffer and expressed as mean residue molar ellipticity (Θ). The plots represent average values from three separate recordings.

FIG. 4.

Comparative bactericidal kinetics and hemolytic activities. (A) Bactericidal kinetics as determined against E. coli no. 2 at four multiples of the MIC of each peptide: NC6-K-P, solid line; MSI-78, dotted line; IB-367, dashed line; control, line with open circles. (B) Peptide dose-dependent hemoglobin release activity as measured after 1 h of incubation in PBS with washed human erythrocytes (10% hematocrit). The symbols are as in panel A. The asterisks represent negative cultures. The error bars represent standard deviations from the mean determined from two independent experiments performed in duplicate.