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Calcium Blocks Fungicidal Activity of Human Salivary Histatin 5 through Disruption of Binding with Candida albicans

¹ Department of Oral Biology and
² Restorative Dentistry, School of Dental Medicine, State University of New York at Buffalo, 310 Foster Hall, SUNY at Buffalo Main Street Campus, 3435 Main Street, Buffalo, NY 14214, USA;

View larger version (16K): [in a new window]   Figure 1. Extracellular divalent cation inhibition of Hst-5-induced killing of C. albicans. C. albicans cells were incubated with 31 µM Hst 5 in 10 mM sodium phosphate buffer, pH 7.4, containing from 1 to 25 mM MgCl2 (open bars) or CaCl2 (solid bars) for 1.5 hrs at 37°C. Cell survival is expressed as percent of control and is calculated as (CFU of cells in buffer with Hst 5 + CaCl2 or Hst 5 + MgCl2 / CFU of cells in phosphate buffer) x 100. Each data point represents the mean ± SD of at least three independent experiments.

View larger version (16K): [in a new window]   Figure 2. Extracellular ions modulate Hst-5-induced release of ATP from cells. C. albicans cells (106) were mixed with Hst 5 (31 µM) and incubated for 20 min to establish ATP efflux. The following salts were then added to the cells after 20 min (arrow): CaCl2 (5 mM, open squares; 10 mM, solid squares); KCl (20 mM, open triangles; 50 mM, solid triangles); MgCl2 (5 mM, open diamonds; 10 mM, solid diamonds). Control cells (closed circles) were treated with Hst 5 alone and maintained in 10 mM sodium phosphate buffer. Cells were then incubated for an additional 40 min, and extracellular ATP was measured by luminometry with the use of an ATP assay kit at 2, 5, 10, 25, and 40 min. Extracellular ATP concentrations are expressed as nmoles of ATP per 106 cells.

View larger version (30K): [in a new window]   Figure 3. Effects of extracellular ions on Hst 5 binding to C. albicans cells. C. albicans cells (106) were treated with FITC-Hst 5 (31 µM) for 1 hr (filled areas) and analyzed on a FACSCAN flow cytometer (Becton Dickinson). For pre-treatment experiments, cells were mixed with (A) KCl (50 mM), (B) MgCl2 (10 mM), or (C) CaCl2 (10 mM) for 20 min (thin black lines), after which FITC-Hst 5 (31 µM) was added and incubated for an additional 40 min. For post-treatment experiments, cells were first treated with FITC-Hst 5 (31 µM) for 20 min, after which KCl (50 mM), MgCl2 (10 mM), or CaCl2 (10 mM) was added for an additional 40 min (bold lines) (A, B, C, respectively).