Antifungal activities of human beta-defensins HBD-1 to HBD-3 and their C-terminal analogs Phd1 to Phd3

Abstract

The activities of defensins HBD-1, HBD-2, and HBD-3 and their C-terminal analogs Phd1, Phd2, and Phd3 against Candida albicans were investigated. Phd1 to Phd3 showed lower-level activities than HBD-1 to HBD-3, although metabolic inhibitors did not render Phd1 to Phd3 inactive. Their activities were also less salt sensitive than those of HBD-1 to HBD-3. Confocal microscope images indicated that the initial site of action was the fungal membrane.

FIG. 1.

Effect of metabolic inhibitors on the candidacidal activities of HBD-1 to HBD-3 and Phd1 to Phd3 against C. albicans. Cells pretreated with 50 μM CCCP or 5 mM sodium azide for 2 h at 30°C were further incubated with peptides at the MFC in PB for 2 h at 30°C. Suitably diluted aliquots were plated onto YEPD agar plates, which were incubated for 24 h. The colonies formed were counted, and the percent killing of C. albicans cells was determined. Shaded bars and dark bars represent peptide activities in the presence of 50 μM CCCP and 5 mM sodium azide, respectively. The values represent averages of results from three independent experiments done with duplicate samples, and variations were 3%.

FIG. 2.

Effect of salts on candidacidal activities of HBD-1 to HBD-3 and Phd1 to Phd3 against C. albicans. Cells were incubated with peptides at their MFC in PB containing different concentrations of salts for 2 h at 30°C. Suitably diluted aliquots were incubated for 24 h on YEPD agar plates. The colonies formed were counted, and the percent killing of C. albicans cells was determined. Panels: A, 25 mM NaCl and 100 mM NaCl; B, 0.5 mM CaCl₂ and 5 mM CaCl₂; and C, 0.5 mM MgCl₂ and 25 mM MgCl₂. Light bars and dark bars represent lower and higher concentrations of salts, respectively. The values represent averages of results from three independent experiments done with duplicate samples.

FIG. 3.

Confocal microscope images showing the localization of CF-Phd1, CF-Phd2, and CF-Phd3 incubated with C. albicans. Cells were treated with 50% MFC of peptide and 4 μg/ml of PI. Arrows in the panels show membranes in the fluorescence images (CF-peptide) and also in the corresponding bright-field (BF) images. Panels: A, control cells without peptide; B, CF-Phd1; C, CF-Phd2; and D, CF-Phd3. The bar represents 5 μm.

FIG. 4.

Membrane permeabilization with peptides was measured by the influx of Sytox green into C. albicans. Cells (10⁷ CFU per ml) were incubated with 1 μM Sytox green. Once the basal fluorescence reached a constant value, peptides at the corresponding concentrations were added and the increase in fluorescence was monitored at 30°C with excitation at 488 nm and emission at 540 nm. (A) HBD-1 (1), HBD-2 (2), and HBD-3 (3); (B) C-terminal analogs Phd1 (1), Phd2 (2), and Phd3 (3). Concentrations of peptides were 4 μM for HBD-1, HBD-2, Phd1, and Phd2 and 1 μM for HBD-3 and Phd3.