Secondary structure and enzyme release assay of Kn2-7.

<p>(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) <i>S. aureus</i> 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) <i>E. coli</i> 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.</p

Binding assay of Kn2-7 and LTA or LPS.

<p>(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 <i>S. aureus</i> and <i>E. coli.</i></p

MICs of Hp1404 against bacteria.

<p>MICs of Hp1404 against bacteria.</p

Toxicity of Hp1404 <i>in vitro</i> and <i>in vivo</i>.

<p>(A) Hemolytic activity of Hp1404 against human red blood cells. BmKn2 is a highly hemolytic antimicrobial peptide from the scorpion <i>Mesobuthus martensii Karsch</i>. (B) Cytotoxicities of Hp1404 against HFF, HEK293T and A375 cell lines. Cytotoxicity was measured by MTT assay. (C) Acute toxicity of Hp1404 to mice by intravenous injection.</p

Antibacterial activity of Hp1404 <i>in vivo</i>.

<p>(A) Therapeutic efficacy of Hp1404 on MRSA infected mice by intraperitoneal injection. (B) Therapeutic efficacy of Hp1404 on MRSA infected mice by intravenous injection.</p

Antibacterial mechanism of the peptide Hp1404.

<p>(A) Time-kill kinetics of Hp1404 against <i>S. aureus</i> AB94004.The assay was performed by determining the counts of surviving bacteria, 0 h represents bacteria before treated. (B) Confocal fluorescence microscopic images of <i>S. aureus</i> treated with FITC-Hp1404. Left: normal image; right: fluorescence image. (C) Transmission electron microscopy of <i>S. aureus</i> treated with Hp1404. a: negative control. b: treated with Hp1404 at the concentration of 1×MIC for 20 min. c-d: treated with Hp1404 at the concentration of 5×MIC for 20 min. (D) Fluorescence measurements. Negative control, 0.9% saline. MSI78 is an amphipathic α-helical peptide with an antibacterial mechanism of disrupting the membrane. The MIC of MSI78 against <i>S. aureus</i> AB94004 is 6.25 µg/mL.</p

MICs of Kn2-7 against Gram-positive and Gram-negative strains.

<p>MICs of Kn2-7 against Gram-positive and Gram-negative strains.</p

<i>In vivo</i> antibacterial activity of Kn2-7.

<p>(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 <i>S. aureus</i> 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 <i>S. aureus</i> infection in skin treated with Kn2-7. (e) Four days after <i>S. aureus</i> infection in skin treated with BmKn2. (f) Four days after <i>S. aureus</i> infection in skin treated with a placebo. (g) Four days after <i>S. aureus</i> infection in untreated skin. Numbered arrows indicate the following: 1, corneum; 2, epidermis; 3, dermis; and 4, muscular layer.</p

Gowth inhibitory activities of Kn2-7.

<p>(A) Growth curves of <i>S. aureus</i> 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 <i>E. coli</i> AB94012 treated with Kn2-7, BmKn2 or antibiotics.</p

Screening, antimicrobial and hemolytic activities of Kn2-7 <i>in vitro</i>.

<p> (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.</p