Human lactoferrin induces apoptosis-like cell death in Candida albicans: critical role of K+-channel-mediated K+ efflux

Abstract

Human lactoferrin (hLf) induced an apoptosis-like phenotype in Candida albicans cells, which includes phosphatidylserine externalization, nuclear chromatin condensation, DNA degradation, and increased reactive oxygen species (ROS) production. Intracellular ROS accumulation was seen to correlate with candidacidal activity in hLf-treated cells. Mitochondrial activity was involved as indicated by mitochondrial depolarization and increased hLf resistance of cells preincubated with sordarin or erythromycin, the latter of which inhibits protein synthesis in mitoribosomes. Interestingly, Cl(-)- and K(+)-channel blockers prevented the hLf antimicrobial activity, but only when cells were pretreated with the blocking agent (tetraethylammonium) prior to the hLf-induced K(+)-release period. These results indicate for the first time that K(+)-channel-mediated K(+) efflux is required for the progression of apoptosis-like process in yeast, suggesting that this essential apoptotic event of higher eukaryotes has been evolutionary conserved among species ranging from yeasts to humans.

FIG. 1.

Apoptotic markers induced by lactoferrin on C. albicans cells. (A) Representative micrographs showing cells stained with TUNEL (row 1), DAPI (row 2), and FITC-annexin V (row 3) to detect DNA strand breakage, fragmented nuclei, and phosphatidylserine externalization, respectively. Subpanels g and h show augmented micrographs displaying DAPI-stained cells with abnormal (g) and normal (h) round compact nuclei (control). Subpanel k shows PI staining. The cells were previously treated with lactoferrin (b, f, g, and j) or not treated (control) (d and h). Subpanels a, c, e, and i are phase-contrast micrographs. (B) Lactoferrin-treated cells exhibiting apoptotic markers. ROS+, TUNEL+, F.N.+, and PI+ refer to the percentage of cells (± the standard deviation) exhibiting DHR-123 fluorescence, a positive TUNEL reaction, fragmented nuclei, and PI staining, respectively. Bar, 5 μm.

FIG. 2.

ROS accumulation in lactoferrin-treated C. albicans cells. (A) Fluorescence and phase-contrast micrographs showing cells stained with DHR-123 previously treated with rhLf or H₂O₂ (positive control). Bar, 5 μm. (B) Flow cytometric analysis of C. albicans cells treated with rhLf or H₂O₂ (positive control) incubated with DHR-123. NAC, cells preincubated (10 min, 30°C) with the antioxidant agent NAC (5 mM) prior to rhLf exposure. (C and D) Percentage of viability from cell suspensions treated with rhLf (5 μM) for 3 h that were preincubated with NAC (C) or precultured (1 h, 30°C) with menadione (D). *, P < 0.05 versus untreated controls; **, P < 0.01 versus untreated controls; ***, P < 0.001 versus untreated controls.

FIG. 3.

Effect of lactoferrin on mitochondrial transmembrane potential. C. albicans cells (10⁶ cells/ml) resuspended in 5 mM PPB were treated with 5 μM lactoferrin (rhLf) or 25 μM histatin 5 (positive control). The mitochondrial transmembrane potential was then measured at different times using 10 μM Rh-123; Rh-123 fluorescence quenching was measured by flow cytometry with excitation at 480 nm and emission at 530 nm.

FIG. 4.

Effect of protein synthesis inhibitors on lactoferrin activity. C. albicans cells were precultured with (A) 1 μg of sordarin (SOR)/ml or (B) 10 mg of erythromycin (ERY)/ml for 30 min and 24 h, respectively. Cell suspensions (10⁵ cells/ml) in 5 mM PPB were then treated with 1.25, 2.5, or 5 μM rhLf for 3 h, and appropriate dilutions were plated onto SDB plates. After incubation at 30°C for 24 to 48 h, the colonies were counted to determine the number of viable cells on each plate. (C) Cytochrome spectra of cells treated with erythromycin (ERY) or not treated (control). The absorption maxima for cytochromes a+a₃, b, and c are 602, 564, and 550 nm, respectively. The data are expressed as means ± the standard deviation of three independent assays. *, P < 0.05 versus untreated controls; ***, P < 0.001 versus untreated controls.

FIG. 5.

Effect of ion channel blocking agents on lactoferrin activity. (A and B) Cytoprotective effect on C. albicans cells preincubated with TEA (A) and other K⁺ or Cl⁻ channel blocking agents (B) before treatment with 5 μM lactoferrin. (C) Time course of K⁺ efflux from lactoferrin-treated cells preincubated (10 min) with or without 10 mM TEA. Nystatin (100 μg/ml) was used as a positive control of K⁺ release. (D) Time-kill curve of lactoferrin-treated cells. The K⁺ channel blocking agent TEA (10 mM) was added before (−15 min) or after (+5 and +15 min) lactoferrin addition. The data are expressed as means ± the standard deviation of three independent assays. *, P < 0.05 versus untreated controls; ***, P < 0.001 versus untreated controls.