Extracellular calcium and magnesium, but not iron, are needed for optimal growth of Blastomyces dermatitidis yeast form cells in vitro

Abstract

In the present study, we demonstrate that the yeast form of Blastomyces dermatitidis can proliferate for short periods of time in the absence of ferric iron but not in the absence of calcium or magnesium. The results of this study shed light on the resistance of B. dermatitidis to chelating agents, such as deferoxamine, and may explain how B. dermatitidis resists the iron-binding activity of serum transferrin.

FIG. 1.

Growth of B. dermatitidis yeast is inhibited by deferoxamine only at concentrations of 0.5 mM or greater. Yeast was inoculated into RPMI 1640 medium containing the indicated concentrations of deferoxamine (Def) and incubated for the indicated times at 37°C. Numbers of viable yeast cells were then determined using alamarBlue. Deferoxamine at 0.5 or 1 mM significantly inhibited yeast growth at 48 h (P < 0.05) but not at the 72-h time point, whereas deferoxamine at 10 mM significantly inhibited the proliferation of B. dermatitidis yeast at all time points (P < 0.05). Yeast incubated in untreated RPMI 1640 medium served as a control. Data presented are the means ± standard errors of the means (SEM) of results from three separate experiments. The asterisk indicates significant inhibition of growth compared to control cultures (P < 0.05).

FIG. 2.

Effects of chelation of RPMI 1640 medium with Chelex-100 on growth of B. dermatitidis yeast. (A) When B. dermatitidis yeast was grown in Chelex-100-treated RPMI 1640 medium ▴ or RPMI 1640 medium □ for up to 72 h, no difference in growth was observed. (B) When B. dermatitidis yeast was first grown in Chelex-100-treated RPMI 1640 medium for 48 h, washed, and then subcultured in fresh Chelex-100-treated RPMI 1640 medium, there was a substantial reduction in yeast growth compared with that of yeast incubated in untreated RPMI 1640 medium as a control (P, <0.05 at 24 to 72 h). Data presented are the means ± SEM of results from two separate experiments, although the error bars are obscured by the size of the symbols. The asterisk indicates significant inhibition of growth compared to control cultures (P < 0.05).

FIG. 3.

Addition of calcium and magnesium, alone or in combination, restores the ability of B. dermatitidis yeast to grow in Chelex-100-treated medium. (A) The addition of calcium [Ca(NO₃)₂; 0.1 mg/ml (□)] or magnesium (MgSO₄; 0.1 mg/ml ▪) to Chelex-100-treated RPMI 1640 medium (▴) enhanced the proliferation of B. dermatitidis yeast. (B) Calcium and magnesium in combination (both at 0.1 mg/ml ○) completely restored the proliferation of B. dermatitidis yeast compared to that of yeast incubated in Chelex-100-treated RPMI 1640 medium (▴). (C) Addition of ferric iron alone (50 mM [•]) neither restored the ability of B. dermatitidis to grow in Chelex-100-treated RPMI 1640 medium nor inhibited the ability of calcium and magnesium to completely restore yeast growth (+). In all three panels, yeast incubated in untreated RPMI 1640 medium (▵) served as a positive control for yeast growth. Data presented are the means ± SEM of results from three separate experiments.