Figure 1.
Sequence analysis, multiple alignment, and secondary structure analysis of Hp1404 peptide.
(A) cDNA and deduced amino acid sequences of Hp1404.The deduced amino acid residues are shown below the corresponding nucleotide sequences. The signal peptide residues are underlined. The mature peptide residues are shaded in gray.The potential cleavage site for prohormone convertase is marked in rectangle. The potential polyadenylation signal aataaa is double underlined. (B) Multiple alignment of Hp1404 with other antimicrobial peptides. Sim%, the percentage of sequence identity relative to Hp1404. The different color represents the different homology level. StCT1 and StCT2 from the scorpion Scorpiops tibetanus, CT1-NDBP-5.17 from the scorpion Urodacus yaschenkoi, IsCT from the scorpion Opisthacanthus madagascariensis, OcyC2 from the scorpion Opisthacanthus cayaporum, pantinin 1 from the scorpion Pandinus imperator. (C) Helical wheel diagram of Hp1404 determined by the Heliquest method. The representation of Hp1404 as a helical wheel shows the hydrophilic face and hydrophobic face. (D) CD spectra of Hp1404 peptide (100 µg/mL) in water alone or with 30 or 70% aqueous TFE.
Table 1.
MICs of Hp1404 against bacteria.
Figure 2.
Antibacterial mechanism of the peptide Hp1404.
(A) Time-kill kinetics of Hp1404 against S. aureus AB94004.The assay was performed by determining the counts of surviving bacteria, 0 h represents bacteria before treated. (B) Confocal fluorescence microscopic images of S. aureus treated with FITC-Hp1404. Left: normal image; right: fluorescence image. (C) Transmission electron microscopy of S. aureus 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 S. aureus AB94004 is 6.25 µg/mL.
Figure 3.
Resistance development of S. aureus AB94004 treated by Hp1404 or kanamycin.
Figure 4.
Toxicity of Hp1404 in vitro and in vivo.
(A) Hemolytic activity of Hp1404 against human red blood cells. BmKn2 is a highly hemolytic antimicrobial peptide from the scorpion Mesobuthus martensii Karsch. (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.
Figure 5.
Antibacterial activity of Hp1404 in vivo.
(A) Therapeutic efficacy of Hp1404 on MRSA infected mice by intraperitoneal injection. (B) Therapeutic efficacy of Hp1404 on MRSA infected mice by intravenous injection.