Hemolytic activities of Hst5, P113, di-18Hc, Halocidin-P113 hybrid peptides and control peptide.

<p>1% of triton-X100 was used as the control for 100% hemolysis and 0.01% acetic acid was used as the peptide-free control. Percent hemolysis was calculated with the following equation: Hemolysis (%) = (A₅₄₀ of sample—A₅₄₀ of peptide-free control) / (A₅₄₀ of 100% control—A₅₄₀ of peptide-free control) × 100.</p

The percentage of secondary structure of Hst5, P113, hybrid peptides and di-18Hc.

<p>The percentage of secondary structure of Hst5, P113, hybrid peptides and di-18Hc.</p

Killing kinetics of Hst5, P113, di-18Hc, hybrid peptides against <i>Candida albicans</i>.

<p>Killing kinetics of peptides against <i>C</i>. <i>albicans</i> was measured by colony count assay. Candida cells were treated with a different concentration of peptides at a determined time. 10 mM NaPB was used as a control. Killing percent was calculated with the following equation: Killing % = (number of control cell—number of treated cell)/control cell × 100.</p

Effects of salt on MICs of Hst 5, P113, di-18Hc and hybrid peptides against Candida strains.

<p>Effects of salt on MICs of Hst 5, P113, di-18Hc and hybrid peptides against Candida strains.</p

Amino acid sequence of histatin 5, halocidin and hybrid peptides.

<p>Amino acid sequence of histatin 5, halocidin and hybrid peptides.</p

The effects of azide on killing activity of peptides.

<p>Candida cells were pre-incubated with 10 mM NaPB containing 5 mM NaN₃ at 30°C for 60 min then washed prior to addition of F-peptides. F-Hst 5, F-P113, F-HHP1 and F-di-PH2 showed (A) reduced levels of cytosolic uptake and (B) killing activity in azide (NaN₃, 5 mM) treated cells, whereas F-di-WP2 and F-di-18Hc showed equivalent amounts of cell wall binding to azide-treated cells and killing activity as untreated controls.</p

CD spectra of Hst5, P113, di-18Hc and hybrid peptides.

<p>This study was performed with one concentration (200 μg/ml) of each peptide in various buffers: 10 mM sodium phosphate buffer (Red line), 50% TFE in phosphate buffer (green line) and 20 mg of laminarin in phosphate buffer.</p

CC₅₀ values of Hst 5, P113, di-18Hc and hybrid peptides against L929 cells.

<p>CC₅₀ values of Hst 5, P113, di-18Hc and hybrid peptides against L929 cells.</p

MICs of Hst 5, P113, di-18Hc and hybrid peptides against Candida strains.

<p>MICs of Hst 5, P113, di-18Hc and hybrid peptides against Candida strains.</p

Hst 5 has lower toxicity and intracellular uptake in <i>C. glabrata</i> compared to <i>C. albicans</i>.

<p><b>A.</b> Candidacidal assays were performed on <i>C. albicans CAI4</i>, <i>C. glabrata Cg10</i>, <i>Cg30</i>, and <i>Cg32</i> strains. Killing activity of Hst 5 was significantly higher in <i>C. albicans CAI4</i> (O) than in the <i>C. glabrata</i> strains tested. <i>C. glabrata Cg10</i> (*) was significantly more sensitive to Hst 5 than either <i>Cg30</i> (▵) or <i>Cg32</i> (◊) (n = 4). <b>B.</b> Hst 5 binding (cell wall) and uptake (cytosol) assays were performed by incubating <i>CAI4</i>, <i>Cg10</i>, <i>Cg30</i>, and <i>Cg32</i> strains with biotin-labeled Hst 5 (B-Hst 5) at 37°C for 30 min (31.5 µM). <i>C. albicans CAI4</i> (1) showed the highest amount of cell wall (white bars) and cytosolic (black bars) Hst 5 compared to <i>C. glabrata Cg10</i> (2), <i>Cg30</i> (3), and <i>Cg32</i> (4) strains (n = 3). Among <i>C. glabrata</i> strains, <i>Cg10</i> showed higher amounts of cell wall bound and cytosolic Hst 5 than the other two strains. <b>C.</b> Translocation of Hst 5 was visualized using time-lapse confocal microscopy with FITC Hst 5 (31.5 µM) for 30 min, and the percentage of Hst 5 positive cells was quantified in at least three independent fields from three independent experiments. <i>C. albicans CAI4</i> had the largest number of Hst 5 containing cells (95%) compared with <i>C. glabrata Cg10</i> (20–25%) and <i>Cg30</i> and <i>Cg32</i> (5–10%).</p