Eukaryotic resistance to fluoride toxicity mediated by a widespread family of fluoride export proteins

Abstract

Fluorine is an abundant element and is toxic to organisms from bacteria to humans, but the mechanisms by which eukaryotes resist fluoride toxicity are unknown. The Escherichia coli gene crcB was recently shown to be regulated by a fluoride-responsive riboswitch, implicating it in fluoride response. There are >8,000 crcB homologs across all domains of life, indicating that it has an important role in biology. Here we demonstrate that eukaryotic homologs [renamed FEX (fluoride exporter)] function in fluoride export. FEX KOs in three eukaryotic model organisms, Neurospora crassa, Saccharomyces cerevisiae, and Candida albicans, are highly sensitized to fluoride (>200-fold) but not to other halides. Some of these KO strains are unable to grow in fluoride concentrations found in tap water. Using the radioactive isotope of fluoride, (18)F, we developed an assay to measure the intracellular fluoride concentration and show that the FEX deletion strains accumulate fluoride in excess of the external concentration, providing direct evidence of FEX function in fluoride efflux. In addition, they are more sensitive to lower pH in the presence of fluoride. These results demonstrate that eukaryotic FEX genes encode a previously unrecognized class of fluoride exporter necessary for survival in standard environmental conditions.

Keywords: dual membrane topology; environmental toxin; ion transport; toxicity resistance.

Fig. 1.

FEX proteins architecture and distribution. (A) Eubacterial, archaeal, and eukaryotic FEX domain arrangments. TM represents transmembrane domain. (B) Phylogenetic tree for eukaryotic FEX proteins.

Fig. 2.

Sensitivity of the N. crassa fex-1 KO strain to NaF by race tube assay. All strains are cultured on solid agar media with different concentrations of NaF. (Inset) Curve for the KO strain. The results are from three independent repeats. Gray bars represent SDs.

Fig. 3.

Fluoride sensitivity of the S. cerevisiae double KO strain. (A) Serial dilution assay of strains grown on YPD + NaF at the concentrations stated. All plates were grown at 30 °C and imaged after 3 d of growth. (B) Liquid growth assay of the double KO strain grown in YPD + NaF at the concentrations stated. Cultures were grown at 30 °C. (C) Representative IC₅₀ curves for wild-type S. cerevisiae and the double KO strain. AUC is area under the growth curve.

Fig. 4.

Sensitivity of the C. albicans FEX1 KO strain to NaF. Growth curves of (A) WT and (B) homozygous KO.

Fig. 5.

Intracellular fluoride concentrations in S. cerevisiae and C. albicans. Values graphed are the average of at least three measurements ± the SD. (A) Intracellular concentration of fluoride for S. cerevisiae strains after incubation with 50 μM NaF. (B) Intracellular fluoride concentration as a function of time for WT and double KO S. cerevisiae. Cells were incubated with 50 μM NaF. (C) Intracellular fluoride concentration as a function of external fluoride concentration for WT and double KO S. cerevisiae. All samples were incubated for 2 h before scintillation counting. (D) Intracellular fluoride concentration of C. albicans strains. Cells were incubated with 500 μM fluoride for 2 h.

Fig. 6.

Effects of NaF on the growth and survival of S. cerevisiae and C. albicans. Data were measured in triplicate but only one data set is shown as an example. Colonies formed by (A) WT S. cerevisiae, (B) S. cerevisiae double KO, (C) WT C. albicans, and (D) C. albicans homozygous KO cultures.