No abstract

Organisms are exposed to fluoride in the air, water, and soil. Yeast and other microbes utilize fluoride channels as a method to prevent intracellular fluoride accumulation and mediate fluoride toxicity. Consequently, deletion of fluoride exporter genes (FEX) in S. cerevisiae resulted in over 1000-fold increased fluoride sensitivity. We used this FEX knockout strain to identify genes, that when overexpressed, are able to partially relieve the toxicity of fluoride exposure. Overexpression of five genes, SSU1, YHB1, IPP1, PHO87, and PHO90, increase fluoride tolerance by 2- to 10-fold. Overexpression of these genes did not provide improved fluoride resistance in wild-type yeast, suggesting that the mechanism is specific to low fluoride toxicity in yeast. Ssu1p and Yhb1p both function in nitrosative stress response, which is induced upon fluoride exposure along with metal influx. Ipp1p, Pho87p, and Pho90p increase intracellular orthophosphate. Consistent with this observation, fluoride toxicity is also partially mitigated by the addition of high levels of phosphate to the growth media. Fluoride inhibits phosphate import upon stress induction and causes nutrient starvation and organelle disruption, as supported by gene induction monitored through RNA-Seq. The combination of observations suggests that transmembrane nutrient transporters are among the most sensitized proteins during fluoride-instigated stress.

Hydrofluoric acid elicits cell cycle arrest through a mechanism that has long been presumed to be linked with the high affinity of fluoride to metals. However, we have recently found that the acid stress from fluoride exposure is sufficient to elicit many of the hallmark phenotypes of fluoride toxicity. Here we report the systematic screening of genes involved in fluoride resistance and general acid resistance using a genome deletion library in Saccharomyces cerevisiae. We compare these to a variety of acids-2,4-dinitrophenol, FCCP, hydrochloric acid, and sulfuric acid-none of which has a high metal affinity. Pathways involved in endocytosis, vesicle trafficking, pH maintenance, and vacuolar function are of particular importance to fluoride tolerance. The majority of genes conferring resistance to fluoride stress also enhanced resistance to general acid toxicity. Genes whose expression regulate Golgi-mediated vesicle transport were specific to fluoride resistance, and may be linked with fluoride-metal interactions. These results support the notion that acidity is an important and underappreciated principle underlying the mechanisms of fluoride toxicity.

Fluoride is highly abundant in the environment. Many organisms have adapted specific defense mechanisms against high concentrations of fluoride, including the expression of proteins capable of removing fluoride from cells. However, these fluoride transporters have not been identified in all organisms, and even organisms that express fluoride transporters vary in tolerance capabilities across species, individuals, and even tissue types. This suggests that alternative factors influence fluoride tolerance. We screened for adaptation against fluoride toxicity through an unbiased mutagenesis assay conducted on Saccharomyces cerevisiae lacking the fluoride exporter FEX, the primary mechanism of fluoride resistance. Over 80 independent fluoride-hardened strains were generated, with anywhere from 100- to 1200-fold increased fluoride tolerance compared to the original strain. The whole genome of each mutant strain was sequenced and compared to the wild type. The fluoride-hardened strains utilized a combination of phenotypes that individually conferred fluoride tolerance. These included intracellular acidification, cellular dormancy, nutrient storage, and a communal behavior reminiscent of flocculation. Of particular importance to fluoride resistance was intracellular acidification, which served to reverse the accumulation of fluoride and lead to its excretion from the cell as HF without the activity of a fluoride-specific protein transporter. This transport mechanism was also observed in wild-type yeast through a manual mutation to lower their cytoplasmic pH. The results demonstrate that the yeast developed a protein-free adaptation for removing an intracellular toxicant.