Figure 1.
Screening, antimicrobial and hemolytic activities of Kn2-7 in vitro.
(A) Multiple alignments of Kn2-7 and its seven derivatives. (B) Predicted secondary structure of Kn2-7 and its seven derivatives. (C) Antibacterial activity screening of Kn2-7 and its seven derivative peptides. (D) Hemolytic activity of Kn2-7. The hemolytic activities of the Kn2-7 and BmKn2 peptides were estimated by monitoring the increase in the absorbance at 570 nm after incubating human red blood cells with different peptide concentrations at 37 °C for 1 h. The positive control was 0.1% Triton X-100, and 0.85% saline was used as a blank.
Table 1.
MICs of Kn2-7 against Gram-positive and Gram-negative strains.
Table 2.
MICs of Kn2-7 against clinical isolated antibiotic-resistant bacterial strains.
Figure 2.
Gowth inhibitory activities of Kn2-7.
(A) Growth curves of S. aureus AB94004 treated with Kn2-7, BmKn2 or antibiotics. (B) Growth curves of MRSA P1386 treated with Kn2-7, BmKn2 or antibiotics. (C) Growth curves of E. coli AB94012 treated with Kn2-7, BmKn2 or antibiotics.
Figure 3.
In vivo antibacterial activity of Kn2-7.
(A) Mice were observed after being treated with the peptide on days 1 and 4. (B) Cutaneous viable counts in treated mice. Eight mice per group were euthanized, and the viable counts of the surviving S. aureus bacteria were then determined. (C) Histological morphologies of the skin in treated mice. (a) Normal dorsal skin of mice. (b) Immediately after the skin was scratched. (c) Four days after the skin was scratched. (d) Four days after S. aureus infection in skin treated with Kn2-7. (e) Four days after S. aureus infection in skin treated with BmKn2. (f) Four days after S. aureus infection in skin treated with a placebo. (g) Four days after S. aureus infection in untreated skin. Numbered arrows indicate the following: 1, corneum; 2, epidermis; 3, dermis; and 4, muscular layer.
Figure 4.
Secondary structure and enzyme release assay of Kn2-7.
(A) and (B) Secondary structure analysis of the Kn2-7 and BmKn2 peptides. (A) Circular dichroism spectra of 0.1 mg/mL of Kn2-7 in water, 30% TFE/H2O or 70% TFE/H2O. (B) Circular dichroism spectra of 0.1 mg/mL of BmKn2 in water, 30% TFE/H2O or 70% TFE/H2O. (C) and (D) Enzyme release assay. (C) S. aureus AB94004 treated with Kn2-7 or BmKn2 was harvested, and the catalase activities of the supernatants were measured; 0.9% saline was used as the negative control, and ampicillin sodium was used as the antibiotic control. (D) E. coli AB94012 treated with Kn2-7 or BmKn2 was harvested, and the catalase activities in the supernatants were measured; 0.9% saline was used as the negative control, and kanamycin was used as the antibiotic control.
Figure 5.
Bactericidal assays of the Kn2-7 and BmKn2 peptides against S. aureus and E. coli AB94012.
Set point indicates untreated bacteria, and 0 minutes was defined as the time of the first sample collection, which was immediately after mixing bacteria and Kn2-7 or BmKn2. The other samples were collected at 5 min, 15 min, 30 min or 60 min. All of the counts were the average of three dishes, and the experiment was repeated at least three times. (A) Time-killing curve of Kn2-7 against S. aureus AB94004. (B) Time-killing curve of BmKn2 against S. aureus AB94004. (C) Time-killing curve of Kn2-7 against E. coli AB94012.
Figure 6.
Transmission electron microscopy of S. aureus and E. coli treated with Kn2-7.
(A) Transmission electron microscopy was performed with Kn2-7-treated S. aureus. (A1), (A2) and (A3) were negative controls. (A4), (A5) and (A6) show S. aureus treated with Kn2-7 for 2 min. (A7), (A8) and (A9) show S. aureus treated with BmKn2 for 2 min. (B) Transmission electron microscopy was performed with Kn2-7-treated E. coli. (B1), (B2) and (B3) were the negative controls. (B4), (B5) and (B6) show E. coli treated with Kn2-7 for 2 min. (B7), (B8) and (B9) show E. coli treated with BmKn2 for 2 min.
Figure 7.
Binding assay of Kn2-7 and LTA or LPS.
(A) Interaction of 10 µg/ml of Kn2-7 with 500 µg/ml of LPS or 500 µg/mL of LTA. Curve A3 is LPS, curve B3 is LTA and curve C3 is buffer. (B) Interaction of 10 µg/mL of BmKn2 with 500 µg/mL of LPS or 500 µg/mL of LTA. Curve D3 is LPS, curve E3 is LTA and curve F3 is the buffer. (C) MICs of Kn2-7 and BmKn2 treated with LTA or LPS against S. aureus and E. coli.