Figure 1.
Antibacterial and hemolytic activity of peptides.
(A) Antibacterial activity of PEPscreen peptides as assessed by radial diffusion assay (RDA) in the presence and absence of 0.15 M NaCl against E. coli ATCC 25922 and S. aureus ATCC 29213, as well as hemolysis. For determination of antibacterial activity, bacteria (4×106 cfu/ml) were inoculated in 0.1% TSB agarose gel and 4 mm wells punched. In each well 6 µl of peptide (at 100 µM) was loaded. The zones of clearance correspond to the inhibitory effect of each peptide after incubation at 37°C for 18–24 h (mean values are presented, n = 3). For hemolysis, the cells were incubated with the peptides at 60 µM, while 2% Triton X-100 served as positive control. The absorbance of hemoglobin release was measured at 540 nm and is expressed as % of Triton X-100 induced hemolysis. (B) Dose dependent activity of selected high purity peptides KNK10, KNK7, and W-modified variants of these against E. coli ATCC 25922 and S. aureus ATCC 29213 in RDA. Shown also are effects of the peptides on human erythrocytes in the hemolysis assay. Throughout, mean values are presented, n = 3. (In Figure 1B, the difference between tagged and non-tagged peptides is statistically significant in all cases (P<0.001, two way ANOVA).) WWW tripeptide alone yielded non-measurable bactericidal effects (0 mm clearance zone in RDA) under the conditions used in Figure 1A, as well as hemolysis (2.3±0.2%) comparable to that in the negative control (2.5±0.05%). Due to poor aqueous solubility, longer oligotryptophan peptides could not be investigated.
Figure 2.
Effect of peptides on eukaryotic cells.
Effects of peptides on HaCaT cells and erythrocytes in absence and presence of 20% human serum. The MTT-assay (upper panel) was used to measure viability of HaCaT keratinocytes in the presence of KNK10 peptides with variable W tagging. In the assay, MTT is modified into a dye, blue formazan, by enzymes associated to metabolic activity. The absorbance of the dye was measured at 550 nm. Cell permeabilizing effects of the indicated peptides (middle panel) were measured by the LDH-based TOX-7 kit. Hemolytic effects of the indicated peptides are also shown (lower panel). Cells were incubated with peptides at 60 µM, while 2% Triton X-100 served as positive control. The absorbance of hemoglobin release was measured at 540 nm and is expressed as % of Triton X-100 induced hemolysis (mean values are presented, n = 3). (For MTT and LDH, the difference between tagged and non-tagged peptides is statistically significant in the absence of serum (P<0.001, one way ANOVA), whereas the difference in the presence of serum is not statistically significant. For hemolysis, the difference between tagged and non-tagged peptides is not statistically significant.)
Figure 3.
Peptide interaction with bacteria and LPS.
(A) Permeabilizing effects of peptides on bacteria. E. coli was incubated with KNK7, KNK10, and the indicated W-modified variants (all at 30 µM) in buffer at physiological salt (0.15 M NaCl) for 2 h at 37°C, after which permeabilization was assessed using the impermeant probe FITC. The upper images in each row are Nomarski Differential Interference Contrast images, while the lower show FITC fluorescence of bacteria. (B) LPS and heparin binding abilities of the KNK7, KNK10 and the indicated W-modified variants peptides.
Figure 4.
Peptide-mediated permeabilization of liposomes.
(A) Effects of KNK10 and indicated W-modified peptides on liposomes in the presence and absence of 0.15 M NaCl. The membrane permeabilizing effect, and resulting release of carboxyfluorescein from liposomes, was recorded by fluorescence spectroscopy. Left panel shows DOPC (zwitterionic) liposomes, the center panel DOPE/ DOPG (75/25 mol/mol; anionic) liposomes, and the right panel (anionic) liposomes formed by E. coli lipids (mean values are presented, n = 3). (B) Leakage kinetics on addition of 1 µM KNK10 and KNK10-WWWWW to DOPE/DOPG (75/25 mol/mol; anionic) liposomes in 10 mM Tris buffer, pH 7.4, with 150 mM NaCl.
Figure 5.
(A) Protease sensitivity of peptides. KNK10 and KNK10-WWWWW were incubated with (+) or without (−) the S. aureus enzymes aureolysin (Aur), V8 proteinase (V8), or human leukocyte elastase (HLE), and analyzed by SDS-PAGE (16.5% Tris-Tricine gels). (B) Activities of peptides in an ex vivo skin infection model. Pig skin was inoculated with E. coli ATCC 25922 (upper panel) or S. aureus ATCC 29213 (lower panel). Peptides at 1 mM were added after an incubation time of 4 h. Bacteria were collected after 2 h and cfu determined (mean values are presented, n = 6). Note the logarithmic scale on the y-axis. (In Figure 5B, the difference between tagged and non-tagged peptide is statistically significant in all cases (P<0.002, one way ANOVA).)