Effect of repetitive lysine-tryptophan motifs on the bactericidal activity of antimicrobial peptides

Abstract

Previous studies identified lysine- and tryptophan-rich sequences within various cationic antimicrobial peptides. In the present study, we synthesized a series of peptides composed of lysine (K)-tryptophan (W) repeats (KW)( n ) (where n equals 2, 3, 4 or 5) with amidation of the C-terminal to increase cationicity. We found that increases in chain length up to (KW)(4) enhanced the peptides' antibacterial activity; (KW)(5) exhibited somewhat less bactericidal activity than (KW)(4). Cytotoxicity, measured as lysis of human red blood cells, also increased with increasing chain length. With (KW)(5), reduced antibacterial activity and increased cytotoxicity correlated with greater hydrophobicity and self-aggregation in the aqueous environment. The peptides acted by inducing rapid collapse of the bacterial transmembrane potential and induction of membrane permeability. The mode of interaction of the peptides and the phosphate groups of lipopolysaccharide was dependent upon the peptides' ability to permeate the membrane. Longer peptides [(KW)(4) and (KW)(5)] but not shorter peptides [(KW)(2) and (KW)(3)] strongly bound and partially inserted into negatively charged, anionic lipid bilayers. These longer peptides also induced membrane permeabilization and aggregation of lipid vesicles. The peptides had a disordered structure in aqueous solution, and only (KW)(4) and (KW)(5) displayed a folded conformation on lipid membranes. Moreover, (KW)(4) destroyed and agglutinated bacterial cells, demonstrating its potential as an antimicrobial agent. Collectively, the results show (KW)(4) to be the most efficacious peptide in the (KW)( n ) series, exhibiting strong antibacterial activity with little cytotoxicity.

Fig. 1

Model structures of (KW)_n–NH₂, the linear antibacterial peptides used in this study (n = 2, 3, 4 and 5). The peptide is shown as cap-sticks, with carbon atoms in green, nitrogen atoms in blue, and oxygen and hydrogen atoms in red and white, respectively (color figure online)

Fig. 2

Structure and organization of (KW)₄ and (KW)₅ in aqueous solution. Aggregation states of the peptides in aqueous solution were determined based on Trp fluorescence and are shown as functions of the peptide concentration. The wavelength at the emission maximum was taken for plotting. (KW)₄ (filled circles), (KW)₅ (filled triangles): water (a) and PBS (pH 7.2) (b). Concentration-dependent CD spectroscopy was used to examine the conformations of the soluble and aggregation states of the peptides. Concentration-dependent CD spectra for (KW)₄ (c) and (KW)₅ (d) in PBS (pH 7.2): 25 μM (solid line), 50 μM (dashed line), 100 μM (dotted line) and 150 μM (dashed-dotted line)

Fig. 3

Confocal laser-scanning micrographs images of E. coli CCARM 1229 (a) and S. aureus CCARM 3090 (b) cells treated with TAMRA-labeled-(KW)₄. The cells were treated with 12.5 μM TAMRA-(KW)₄ for 10 min at 37° C in PBS (pH 7.2). From left to right: TAMRA, differential interference contrast (DIC), merged images

Fig. 4

Binding affinities of the peptides for LPS, as measured using dansyl PMB displacement assays and CD spectroscopy. P. aeruginosa LPS (9 μg) was incubated with dansyl PMB (4 μM) for 5 min, after which fluorescence was measured at an excitation wavelength of 340 nm and emission wavelength of 485 nm. Peptides were added at different concentrations, and dansyl PMB fluorescence was measured after 5 min (a). CD spectra for the peptides (50 μM) were measured in the presence of 0.1 % LPS (b): (KW)₂ (dashed-dotted line), (KW)₃ (dotted line), (KW)₄ (dashed line), and (KW)₅ (solid line)

Fig. 5

Membrane-disruption and bactericidal activity. Dose–response curves for peptide-induced membrane depolarization in intact E. coli CCARM 1229 (a) and S. aureus CCARM 3090 (b). Membrane depolarization was monitored as an increase in the fluorescence of DiSC₃-5 (excitation wavelength 620 nm, emission wavelength 670 nm) after addition of various concentrations of peptide: (KW)₂ (filled diamonds), (KW)₃ (filled squares), (KW)₄ (filled circles), and (KW)₅ (filled triangles). The increase in fluorescence obtained using 0.1 % Triton X-100 was taken as 100 %. Membrane permeabilization was monitored based on entry of SYTOX Green dye. Bacterial cells (2 × 10⁷ CFU/ml) in PBS were incubated with 1 μM SYTOX Green dye. Peptides were added at a concentration of 12.5 μM, after which uptake of SYTOX Green through the E. coli (c) or S. aureus (d) plasma membrane was measured based on the time course of fluorescence changes at an excitation wavelength of 485 nm and emission wavelength of 520 nm: (KW)₂ (filled diamonds), (KW)₃ (filled squares), (KW)₄ (filled circles), (KW)₅ (filled triangles), and Melittin (open circles). Kinetics of the bactericidal activity against E. coli CCARM 1229 (e) and S. aureus CCARM 3090 (f). Bacteria treated with the respective peptides were diluted at the appropriate times and then plated on LB agar. CFUs were then counted after 16 h of incubation at 37° C. Black (KW)₄, white (KW)₅, squares and triangles 1 and 2 times the MIC, respectively; cells (2 × 10⁵ CFU/ml) incubated in the absence of any peptide served as controls

Fig. 6

Peptide actions on lipid bilayers. Percent leakage of calcein from PE:PG (7:3, w/w) (a) and PC:CH (10:1, w/w) vesicles (b) at pH 7.4 was measured for 25 min after the addition of various doses of peptide. c LUV aggregation. Solutions containing various concentrations of peptide were added to a suspension of 400 μM PE:PG (7:3, w/w) LUVs, after which aggregation was monitored based on changes in the absorbance of the LUVs at 405 nm: (KW)₂ (filled diamonds), (KW)₃ (filled squares), (KW)₄ (filled circles), and (KW)₅ (filled triangles)

Fig. 7

CD spectra for (KW)₂ (dashed-dotted line), (KW)₃ (dotted line), (KW)₄ (dashed line), and (KW)₅ (solid line) in the presence of PE:PG (7:3, w/w) (a) and PC:CH (10:1, w/w) vesicles (b)

Fig. 8

Schematic representation of the proposed mechanism of action of (KW)_n peptides in bacterial cells. (1) The peptide molecules associate with cell membranes or lipid bilayers, (2) the membrane/bilayer is disrupted, releasing the internal contents of the cell/vesicles; and (3) although higher peptide-lipid ratios necessary for cells/vesicles aggregate