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. 2022 Oct 25;35(11):2085–2096. doi: 10.1021/acs.chemrestox.2c00222

Cells Adapt to Resist Fluoride through Metabolic Deactivation and Intracellular Acidification

Nichole R Johnston †, Gary Cline ‡, Scott A Strobel †,§,*
PMCID: PMC9683101  PMID: 36282204

Abstract

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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.

1. Introduction

As the 13th most abundant element of the earth’s crust, fluoride can be found throughout the air, soil, and water. It is highest in concentration at areas with marine sedimentation, industrial waste, or volcanic activity.1 For the past 80 years, fluoride has been added to the water of many developed countries due to its benefits in oral health. Exposure to a small amount of fluoride reduces dental caries, inhibits bacterial plaque formation, and stimulates bone proliferation.2,3 While low fluoride exposure can improve overall bone health, there is no evidence of an organism requiring fluoride. Furthermore, high levels of fluoride cause widespread adverse effects.

Cellular sensitivity to fluoride depends on the external pH. Fluoride has the highest pKa of any halide at 3.2. Consequently, it is the only halide that remains protonated in mildly acidic environments. As extracellular environments become more acidic, a greater fraction of fluoride forms hydrofluoric acid (HF). Given that HF is uncharged, it can readily diffuse through the lipid bilayer and into the cytoplasm of a cell. The cytoplasm of cells is typically at neutral pH. In this state, the equilibrium shifts to the charged form F–, which cannot pass back through the lipid bilayer. The net effect of exposure of cells to fluoride in acidic environments is the accumulation of fluoride intracellularly, causing downstream toxicity.

Heightened fluoride exposure causes a range of toxic effects on organisms. In multicellular organisms, these effects include inflammation, skeletal defects, and tissue damage.4−6 In single cells, fluoride causes oxidative stress, DNA stress, nitrosative stress, and cell cycle arrest.7−10 One of the most widely reported phenotypes of fluoride exposure is metabolic inhibition, marked by a decreased rate of glycolysis and reduced concentration of ATP.11 Prolonged fluoride exposure also damages the nucleus, mitochondria, endoplasmic reticulum, cell membrane, and ribosomes.12−16 Recently, our lab reported that these phenotypes are largely caused by general acid stress from fluoride exposure followed by nutrient starvation.10,17

Despite the myriad of stress effects caused by high concentrations of fluoride, organisms readily tolerate fluoride at most biological concentrations. In 2012, Breaker et al. discovered a bacterial response pathway to fluoride, providing some of the first evidence for fluoride adaptation.18 A principle component of the bacterial response pathway is the expression of the fluoride channel Fluc. Fluc selectively removes unprotonated fluoride from cells. Fluc has over 8000 homologs across archaea, prokaryotes, and eukaryotes.19 Recently, we identified ion transporters in yeast that act as the predominant response pathway to fluoride. Yeast express two copies of a fluoride exporter (FEX), which efficiently removes fluoride from the cytoplasm.19 Deletion of both copies, or FEX double-knockout (DKO), results in over 1000-fold sensitivity to fluoride. Yeast lacking FEX undergo rapid intracellular fluoride accumulation, as well as growth and metabolic arrest at micromolar fluoride exposure.10 As this knockout model is hypersensitive to fluoride and lacks export capabilities, it serves as an optimal organism for testing adaptations.

Fluoride tolerance varies dramatically across organisms expressing fluoride channels. One example of this is in plants, wherein the flowering plant gladiolus is sensitive to 20 ppm (1 mM) fluoride, while the cotton plant appears healthy in excess of 4000 ppm (210 mM) fluoride.20 Furthermore, fluoride tolerance varies significantly across vertebrates, in which no fluoride transporter has been identified. Collectively, these observations suggest that organisms possess mechanisms outside of the Fluc family of protein transporters for combating fluoride.

Directed mutagenesis is a powerful tool for investigating adaptation. Several studies have previously identified bacteria with enhanced fluoride tolerance.21−25 These bacteria were found either through serendipity or manually exposed to fluoride and screened for adaptation. In general, organisms gained resistance by changing the expression levels of fluoride transporters or known fluoride targets.17 However, each of these studies focused on a single strain of fluoride-resistant bacteria. Given that the bacteria underwent random mutagenesis, there is a high probability of background mutations. The authors of each study then looked for changes in already-established fluoride targets, which prohibited new discoveries of potentially more important targets. In order to perform a more unbiased approach, multiple organisms must undergo mutagenesis. Then, common mutations and phenotypes can be identified without prejudice.

Here, we report the random selection of 81 strains of yeast with genetic mutations conferring fluoride tolerance that lack the fluoride transporter FEX. Rather than following a single mechanism, these yeast each displayed a combination of metabolic, growth, and pH alterations to enhance fluoride tolerance. These mechanisms demonstrate how cells can respond to fluoride exposure, even without a specific channel to remove the toxicant.

2. Experimental Procedures

For further experimental protocols and a more detailed description of methods, please see the Supporting Information Experimental Procedures section.

2.1. Strains and Reagents

A list of reagents is available in the SI Experimental Procedures. Yeast used in this study are the same as described previously; the wild type is BY4741, and the FEX DKO strain was previously generated using the hphMX4 and kanMX6 resistance cassettes (MATa his3Δ1 leu2Δ0 ura3Δ0 FEX1Δ::kanMX6 FEX2Δ::hphMX).19

2.2. Generating Fluoride-Hardened Mutants

Yeast mutants were selected for resistance to fluoride through four independent methods. Each method involved exposing an FEX double-knockout strain of S. cerevisiae to a mutagen (either UV or NaF) followed by screening the yeast for retained fluoride resistance.

2.2.1. UV Method

FEX double-knockout yeast cells were grown for 48 h on YPD-agar plates in a 30°C incubator. These plates were exposed directly to UV light using a UV lamp (UVP UVGL-58 handheld at 0.12 Amps, 254/365 nm UV) for 20 min. The cells were replica plated onto fresh YPD-agar plates and allowed to grow for 24 h at 30 °C. Individual colonies were tested for fluoride resistance by initial screening on 500 μM and 5 mM NaF-agar plates followed by liquid growth assays to attain the IC50 values of each colony.

2.2.2. Spontaneous Method

FEX double-knockout yeast cells were grown for 48 h on YPD-agar plates in a 30°C incubator. Yeast were first grown on 50 μM NaF-agar plates, and then individual colonies were directly plated onto 500 μM and 5 mM NaF-agar plates. Any yeast that grew on these plates were stored on YPD-agar, and their IC50 was obtained using a liquid growth assay.

2.2.3. NaF Method

FEX double-knockout yeast cells were grown for 48 h on YPD-agar plates in a 30°C incubator. Each yeast colony was incrementally added to increasing fluoride at the concentrations of 50, 125, 250, 500, 750, 1000, 2500, 5000, 7500, 10,000, 15,000, 25,000, 30,000, and 50,000 μM NaF. For colonies that did not grow at higher fluoride concentrations, yeast from that colony was repeatedly exposed to NaF until the colony adapted to grow on that NaF. Cells able to grow on 30 mM NaF plates were streaked onto YPD plates for storage, and their IC50 was assessed using liquid growth assays.

2.2.4. YPD Method

The YPD method is the same protocol as the NaF Method, except that in-between each fluoride plate of increasing concentration, yeast colonies are streaked onto YPD plates for 48 h growth to allow recovery.

2.3. Sequencing Analysis

Fluoride resistant yeast strains were whole-genome sequenced. Sequencing results were analyzed using a computer cluster system. The published S. cerevisiae genome from the Saccharomyces Genome Database (yeastgenome.org) was used as a reference index. DNA from FEX DKO yeast that had not been exposed to fluoride was also sent for sequencing as a control. DNA for each mutant was aligned to the reference genome, and the files were restructured using BWA and SAM Tools. Picard was used to mark duplicates. Freebayes was used for calling variants. The online Variant Effect Predictor (useast.ensembl.org/info/docs/tools/vep/index.html) was used to establish yeast annotations.

2.4. Intracellular pH

Intracellular pH was measured using the pH-sensitive dye 5(6)-carboxyfluorescein diacetate (CFDA). Unless otherwise noted, cells were grown to the log phase. Mutants differed in growth rate, so the amount of incubation time varied by mutant. Cells were washed three times in PBS (pH 7.0) and resuspended at O.D. 0.8 in 100 μL of PBS containing 50 μM 5(6)-CFDA. Cells were placed in a 37°C water bath for 8 min before being transferred to a 96-well plate. Fluorescence (492 nm excitation, 517 nm emission) was monitored using a plate reader. The fluorescence of each sample was then compared with a standard generated of yeast cells at O.D. 0.8 permeabilized in 70% ethanol for 45 min before being transferred to PBS at increasing pH ranging from 3.5 to 7.0. After 20 min, permeabilized cells were washed and placed in PBS (pH 7.0) with 50 μM 5(6)-CFDA and placed in a 37 °C water bath for 8 min. The pH of experimental cells was compared with the standard curve generated from the permeabilized cells. Each cell’s intracellular pH was measured in triplicate.

2.5. 13C Metabolomics

Extracellular and intracellular carbohydrates of FEX DKO yeast and yeast with 500- and 1200-fold increased fluoride resistance were monitored using sugar isolation and 13C NMR. Cells were grown overnight in 50 mL of YPD media. Yeast were then transferred into fresh tubes of 100 mL of YPD media containing 2% 1-13C glucose. The starting optical density of yeast was at 0.1 for 30 h growth, 1.5 for 9 h growth, and 5.0 for 2 h growth. Cells were grown for the indicated time at 30 °C with shaking. The optical density was measured for each sample, and cells were harvested by centrifugation. 600 microliters of supernatant was collected, and 120 μL of D2O was added before analyzing the extracellular contents by NMR. The yeast cell pellets were washed twice with water and then resuspended at O.D. 20 in 25 mL of water. 750 microliters of (2:0.8) methanol–water was added to cells, and yeast were frozen, thawed, and sonicated. 750 microliters of (2:1) chloroform–water was added to cells, and yeast were vortexed. Yeast were then spun for 5 min at 15,000 rcf on a benchtop centrifuge. The top fraction (containing metabolites and methanol) was transferred to glass tubes. Methanol was evaporated using nitrogen, and the remaining metabolites were resuspended in 550 μL of water and 110 μL of D2O. Solutions of metabolites were stored at −20 °C until analysis. 13C NMR spectra were acquired with an AVANCE 500-MHz spectrometer (Bruker Instruments, Inc., Billerica, MA). Analysis was conducted on true triplicates, harvested on separate days.

3. Results

3.1. Generation of Fluoride-Hardened S. Cerevisiae

Here, we report the assessment of adaptation against fluoride stress in yeast lacking fluoride exporters.

FEX DKO yeast were hardened to fluoride using four mutational approaches: the “UV Method”, the “YPD Method”, the “NaF Method”, and the “Spontaneous Method” (Supp. Figure 1). The UV Method is a classical mutagenesis assay wherein cells are exposed to UV light in order to induce random DNA damage in yeast. This method was the sole technique in which cells were not exposed to fluoride in order to gain resistance.

In the other three approaches, fluoride was used as the mutagen to drive adaptation. Cells were either immediately exposed to high levels of fluoride (the Spontaneous Method) or given incremental dosages of fluoride (NaF and YPD Methods). Under the YPD Method, yeast were also given a recovery step in normal YPD agar before exposure to the next higher dose of fluoride.

Each technique offers unique advantages. The UV and Spontaneous Methods were the fastest protocols for attaining mutants, but because of the high degree of cell stress involved, the vast majority of strains died. The NaF Method was adapted from the technique put forth by Liu et al., who generated two fluoride resistant mouse fibroblast cell lines by incrementally increasing their exposure to fluoride.26 The rate of cell recovery of the NaF Method is much higher than those of the UV and Spontaneous Methods. However, a technique like the NaF Method could result in cells inducing genes as part of a stress response pathway, thus gaining resistance without mutating the cells. To address this concern, we introduced the YPD Method, in which yeast were allowed to recover in between fluoride exposures. This recovery step allowed cells to return to basal conditions such that any resistance mechanism to fluoride must be retained either genetically or epigenetically for yeast to continue growing when re-exposed to fluoride.

Through this combination of mutagenesis assays, we generated 81 unique yeast strains. These isolates had between 100- and 1200-fold increased fluoride resistance compared with FEX DKO (Figure 1A). The majority of fluoride-hardened yeast strains gained between 100- and 400-fold increased resistance to fluoride compared with FEX DKO (Figure 1B). Four strains had over 700-fold resistance, with three of those having over 1000-fold increased resistance. These strains had almost as great of resistance to fluoride as the wild type despite lacking a fluoride exporter (1200-fold for the best mutant vs 1300-fold for the wild type).

Figure 1.

Figure 1

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General characteristics of yeast mutated to gain resistance against fluoride. (A) Liquid growth assay of control yeast (FEX DKO), mutants, or wild type (+ FEX1 and FEX2). Data is represented as the loss of total growth over 24 h, calculated as the area under the curve of growth, in increasing fluoride media. (B) Gain of resistance of yeast against fluoride, plotted as the fold increase in the IC50 of fluoride compared with FEX DKO yeast. Each dot on the graph represents a single strain.(C) Growth rate of yeast in YPD without fluoride. Growth is measured at optical density 600. (D) Morphology and clustering of FEX DKO and fluoride-hardened yeast. Cells taken at the same O.D. are suspended in PBS with no dye after growth into the log phase. 500× and 1200× denote the fold increase in resistance against fluoride compared with FEX DKO.

Each of these mutants adapted to harden against fluoride toxicity in the absence of the fluoride transporter, FEX. Given that FEX efficiently excretes intracellular fluoride, this suggests that other adaptive mutations approach but do not supplant the benefits of removing fluoride in preventing cellular toxicity.

3.2. Fluoride-Hardened Yeast Have Reduced Growth and Switch to Anaerobic Metabolisms

In general, as yeast gained resistance to fluoride, they also displayed an unusual clumping phenotype and reduced their overall growth rate, even in the absence of fluoride. Indeed, we found that the delay in slower growing yeast occurred during both the transition out of the lag phase and during the overall log phase growth (Figure 1C). The most fluoride-resistant mutant took three times longer to enter the lag phase than FEX DKO yeast. This is consistent with several previous studies indicating that cells with slower growth are more resistant to stressors through an unknown mechanism.27−30 Yeast of higher-fold resistance also tended to cluster with one another during imaging (Figure 1D). Some mutants also varied in size and shape, but there was no obvious correlation to resistance (data not shown).

We next examined if metabolism was reduced in these strains along with the reduced growth. Fluoride is well established to inhibit metabolic enzymes, so it stands to reason that mutant yeast may have reduced activity of some key fluoride targets.

We first examined intracellular ATP levels across the three phases of growth in fluoride-hardened and FEX DKO yeast while growing in normal media (Figure 2A). In general, yeast that gained resistance to fluoride had lower intracellular ATP through all growth phases. This trend had the greatest correlation with fold resistance to fluoride during the log phase, which is also the phase that is prolonged in fluoride-hardened yeast. We next quantified intracellular glucose and phosphate in yeast during the log phase and observed that mutant yeast in general had less intracellular nutrients than normal (Figure 2B). However, the drop in intracellular glucose and phosphate did not correlate with fold resistance as strongly as the drop in intracellular ATP. Further analysis of intracellular phosphorous levels using 31P NMR revealed that while fluoride resistance correlated proportionally with intracellular orthophosphate, it was inversely related to total intracellular phosphate and polyphosphate (Figure 2C and Supp. Figure 2). This was particularly the case for vacuolar polyphosphate, of which none could be detected in the NMR spectra of higher resistance mutants. This could either be because the cell is breaking down polyphosphate or because the vacuole has lost function. We assessed organelle function using dyes that incorporate into active membranes and found that the organellar membranes of mitochondria and vacuoles were significantly disrupted in higher resistance mutants (Figure 2D).

Figure 2.

Figure 2

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Quantification of intracellular nutrients. (A) Intracellular ATP at lag, log, and plateau phases of growth. (B) Intracellular glucose and phosphate at log growth. (C) Fold change in concentration of orthophosphate and vacuolar phosphate, compared to FEX DKO yeast and as determined by 31P NMR (see the Supporting Information for spectra). (D) Imaging of mitochondrial and vacuolar activity using Rhodamine 123 and FM4-64 dye, respectively, in FEX DKO yeast and a mutant with 1200×-increased fluoride resistance. Strains are (1, black) FEX DKO, (2 and 3, red) 100×-, (4, 5, orange) 300×-, (6, 7, green) 500×-, (8, blue) 1000×-, and (9, purple) 1200×-increased resistance to fluoride.

We next assessed the concentration of extracellular glucose in two resistant strains (mutants 500x and 1200x) in comparison to the FEX DKO control yeast (Supp. Figure 3). Extracellular glucose fluctuated in all three strains during the initial lag phase. Interestingly, FEX DKO yeast underwent major glucose uptake as the yeast transitioned into the plateau/stationary phase, while both fluoride-hardened yeast strains took in the most glucose as the yeast transitioned into the log phase. This is consistent with our previous observations that fluoride stress inhibits glucose and general nutrient uptake and induces starvation.10 Mutations that enhance glucose uptake would be predicted to confer significant resistance to fluoride stress.

We next assessed the full changes in carbohydrate metabolomics between FEX DKO yeast and 500x and 1200x fluoride-hardened mutants using 1-13C-labeled glucose. In this assay, yeast are fed 13C-labeled glucose and then grown under normal conditions. By following the 13C labeling of all carbon-based nutrients over time, we are able to directly compare the activities of metabolic pathways across yeast strains.

Overall, mutant yeast shifted their sugar from an active metabolic pathway and into storage. Fluoride-hardened mutants had up to 30-fold and 50-fold more intracellular trehalose than FEX DKO yeast after 30 h of growth (Figure 3B and Supp. Figure 4). Trehalose is the stored form of polyglucose, and its presence in yeast confers significant resistance to oxidative stressors.31,32 Similarly, fluoride-hardened yeast also had 40-fold increased intracellular concentrations of the other form of stored sugar, glycogen, after 30 h of growth. Given that fluoride induces nutrient starvation, large deposits of stored sugar would confer a significant survival advantage in yeast. Fluoride-hardened yeast also showed signs of increased anaerobic fermentation, with greater abundance of both ethanol and glycerol in the media (Figure 3C and Supp. Figure 5). Conversely, pyruvate, acetyl-CoA, and citrate, the starting substrates of the Krebs cycle, had reduced intracellular concentrations in fluoride-hardened yeast during early growth phases. This is consistent with the general observation that mutant yeast are more dormant than FEX DKO. However, the reduced initial metabolism enabled the conservation of nutrients such that by 30 h, the rates of aerobic metabolism were much higher than the FEX DKO control yeast. This suggests that prolonged metabolism with reduced initial growth confers a significant advantage against fluoride and subsequent nutrient starvation toxicity.

Figure 3.

Figure 3

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Global changes in metabolite abundancies with enhanced fluoride resistance. (A) Diagram depicting overall trend of relative metabolite concentrations in fluoride-hardened yeast compared with FEX DKO control yeast. (B) Fold change in concentration of extracellular or (C) intracellular substrates in 500-fold (green)- or 1200-fold (purple)-increased fluoride resistance yeast after 30 h growth. Fold change is compared to FEX DKO cells at 30 h growth and monitored through initial feeding of yeast with 1-13C glucose followed by analysis with 13C NMR. See the Supporting Information for the full comparisons at 2, 9, and 30 h growth.

Strikingly, fluoride-hardened yeast had large shifts in amino acid homeostasis. Neutral and basic amino acids, most notably alanine, glycine, arginine, lysine, and histidine, increased in concentration in the extracellular media. Conversely, the acidic amino acids aspartate and glutamate increased in intracellular concentration. The same pattern of differing localization based on pH was consistent in respiration products; the alkaline ethanol was largely excreted into the media, while the more acidic acetate accumulated intracellularly. Subsequently, the peaks of intracellular substrates in fluoride-hardened yeast taken by NMR were shifted in a manner consistent with enhanced protonation. Together, this data strongly suggests an overall trend of fluoride-hardened yeast globally increasing acidic components in their cytoplasm and alkaline components in the extracellular space compared to the control.

3.3. Intracellular Acidification Prevents Fluoride Accumulation

Collectively, the broad changes in phenotype of fluoride-hardened yeast suggest an overall altered ion gradient. As reported above, mutated strains grew slowly, and they shifted alkaline substrates to the media and acidic substrates to the cytoplasm. These two phenotypes are potentially linked; under stress, yeast halt growth through specialized transporters that acidify the cytoplasm.33 We also found that fluoride-hardened yeast did not incorporate dyes that were specific to active vacuolar and mitochondrial membranes (Figure 2D). This would occur if the membrane potentials were disrupted, as would occur during a pH shift. Third, the substrates measured either through 13C or 31P NMR had higher protonation in fluoride-hardened yeast than the wild type (data not shown). Together, this collection of observations led us to hypothesize that fluoride-hardened yeast had acidified their cytoplasm.

We quantified cytoplasmic pH using a pH-sensitive dye (Figure 4A). In general, mutants had a more acidic cytoplasm than the wild type. Five mutants were higher in pH than FEX DKO, while 58 of 81 had significantly lower intracellular pH. The intracellular pH of wild-type yeast fluctuates based on the growth phase and metabolism, but in general, it ranges from 6.5 to 7. Conversely, mutants with the highest resistance to fluoride averaged around pH 5.0. This is consistent with our observations of slowed growth and altered metabolism in fluoride-hardened yeast.

Figure 4.

Figure 4

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Intracellular pH and resulting fluoride excretion in resistant mutants. (A) Graph of increased fold resistance to fluoride (as determined by liquid growth assays) and intracellular pH during the log phase for each mutant. The blue dot represents the measured value for FEX DKO, and the dashed line is set at the average intracellular pH value for FEX DKO. (B) Fluoride-hardened mutants (black) and FEX DKO (blue), plotted by their intracellular pH versus concentration of intracellular fluoride after 24 h exposure to 50 μM NaF. (C) Intracellular fluoride concentration over time. The clustered columns represent 2, 4, 6, 12, 18, and 24 h of exposure to 50 μM NaF for each strain. (D) Reduction in growth over increasing fluoride for FEX DKO and mutant strains transformed with pRS416-FEX1, or (E) wild-type BY4741 strain (+ FEX1, FEX2), and the wild type with the gene VMA11 deleted. Vma11p is an essential component of V-ATPase, and non-functional V-ATPase eliminates the pH gradient between the cytoplasm and vacuole, resulting in intracellular acidification.

Fluoride toxicity is highly dependent on environmental pH. Hydrofluoric acid can readily permeate cells; therefore, organism sensitivity to fluoride correlates with the proportion of protonated fluoride in solution.34 In the case of fluoride-hardened yeast, the equilibrium is shifted such that the pH gradient drives the fluoride to diffuse out of the cell where it is less likely to be protonated and therefore reverses the directionality of fluoride accumulation in the cytoplasm. To test this hypothesis, we monitored the accumulation of intracellular fluoride over 24 h in 15 mutants at neutral extracellular pH (Figure 4B). One mutant strain tested had a higher intracellular pH than FEX DKO; this mutant had no significant difference in intracellular fluoride concentration compared to FEX DKO. Conversely, the acidic mutants all showed significantly reduced intracellular fluoride. There was approximately a threefold drop in intracellular fluoride in mutants at pHintra 6.0 and an additional twofold drop in intracellular fluoride in more acidic mutants. To determine whether the reduced intracellular fluoride accumulation was consistent over time, we monitored fluoride accumulation at 2, 4, 6, 12, 18, and 24 h for three mutants (Figure 4C). These mutants consistently showed lower intracellular fluoride over time compared to FEX DKO.

We next directly tested the hypothesis that intracellular pH can reverse fluoride accumulation independent of a fluoride transporter. V-ATPase is a major non-essential regulator of pH in yeast. Disruption of V-ATPase results in the immediate drop of intracellular pH to 5.9.17 We deleted the V-ATPase subunit VMA11 from normal FEX DKO yeast. Over 24 h, the VMA11 knockout had similarly reduced intracellular fluoride as the hardened mutants, supporting that intracellular acidification was directly responsible for fluoride excretion (Figure 4C).

FEX is the only known yeast protein to transport fluoride. We expressed FEX1 in fluoride-hardened mutants to determine how the combination of intracellular acidity and a fluoride transport would affect fluoride accumulation. Surprisingly, mutants expressing FEX1 were sensitized to fluoride compared with the wild type (Figure 4D). Yeast sensitization correlated with intracellular pH, whereby the most sensitized mutants were also the most acidic. However, rather than a linear decrease, only strains with more than 300-fold increased fluoride resistance demonstrated a three to seven-fold loss in fluoride resistance with FEX1 expression. As indicated by 31P NMR, this range of mutants have lost vacuolar function (Figure 2C). This is consistent with our lab’s previous finding that the vacuole is crucial for pH maintenance and fluoride resistance.17 We then monitored the sensitivity to fluoride in ΔVMA11 cells and found that abolition of V-ATPase function is sufficient to reduce fluoride resistance by five-fold (Figure 4E). Together, the indicated trends suggest that FEX activity is dependent on intracellular pH and vacuolar function, though the mechanism remains unknown.

3.4. Whole-Genome Sequencing of Fluoride-Hardened Yeast

Given the many differences between FEX DKO yeast and various fluoride-hardened yeast strains, we sought to identify the underlying genetic changes that resulted in fluoride resistance. To do this, we performed whole-genome sequencing on all 81 mutants as well as the FEX DKO strain from which the mutants were derived. The majority of genetic differences between FEX DKO and fluoride-hardened yeast were in the upstream or downstream sequence regions as opposed to the coding regions of genes. Alterations in these regions have a higher potential to affect the expression level of genes rather than the gene function. Across all strains, we identified 1725 unique open reading frames affected by mutations (approximately one-fourth of the known genome), with anywhere from 19 to 106 mutations per strain (Table 1). This result is not surprising given that the fluoride hardened yeast were generated using random mutagenesis. As such, it would be expected that there are a large number of total mutations across strains. By examining commonly mutated genes and pathways, we tried to separate potentially significant phenotypes from the noise of irrelevant, background mutations.

Table 1. Summary of Fold-Resistance Gained by Assay Typea.

  UV spontaneous NaF YPD total
mutants (#) 23 4 18 36 81
fold resistance (range) 100–1000 100–105 102–707 101–1200 100–1200
fold resistance (average) 233 102 283 300 274
mutations per strain (range) 19–64 20–41 19–50 26–106 19–106
mutations per strain (average) 31 23 34 38 35
ORFs affected per strain (range) 74–232 78–155 87–202 62–578 50–578
ORFs affected per strain (average) 115 125 128 138 129
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a

Fold-resistance was determined by the increase in IC50 to fluoride compared with FEX DKO (52.67 μM). Mutations per strain and ORFs affected were determined with bioinformatics, as outlined in Experimental Procedures.

Overall, the novel phenotypes identified in fluoride-hardened mutants were slowed growth, altered metabolism, clumping, and acidified cytoplasm. We therefore looked for mutations that could explain these phenotypes, as well as commonly observed mutations that might affect a phenotype for which we had not explicitly screened.

Many individual genes were mutated across fluoride-hardened yeast strains, most notably those involved in cell adherence and nutrient transport. FLO9, involved in flocculation, was the most frequently mutated gene across all strains, with a total of 444 mutations (Supp. Table 1). Every fluoride-hardened strain had at least one mutation to FLO9. The next most frequently mutated genes were FET4 (a plasma membrane iron transporter) with 335 mutations, ADH6 (an alcohol dehydrogenase linked to DNA stress), and DIA1 (involved in pseudo-hyphal growth) both with 283 mutations. Also among the most commonly mutated genes were IPP1 (inorganic pyrophosphatase) with 157 mutations and ERR3 (an enolase mimic) with 101 mutations. Ipp1p and enolase are well-established targets for fluoride inhibition in vitro. The majority of mutated genes, such as DAN4, FLO9, and DIA1, relate to the cell surface of yeast, its adherence, and ability to bud. GDH3, IPP1, ERR3, FET4, and DAN4 are each involved in metabolism and cell adaptation to nutrient starvation.

We also looked at the broad conservation of mutations to organelles and biological pathways. In the wild-type yeast genome, approximately 60% of corresponding proteins are cytoplasmic, 30% are in the nucleus, and 25% are located in the membrane (Yeast Genome Database). For the 81 fluoride-hardened mutants, the distribution of genes per organelle appears largely random (Figure 5A). The most non-random organelle mutated was the Golgi, which had threefold more mutations than would be expected by random mutagenesis. Apart from that, there was not a clear pattern for mutations specific to a particular organelle. This does not necessarily imply that organelle activities are not important for fluoride resistance; significant alterations to cellular processes can be made through a single mutation.

Figure 5.

Figure 5

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Changes in genotype of fluoride-hardened mutants. (A) Conserved components and (B) conserved cellular processes of yeast genome that are mutated in fluoride-hardened yeast. Gene ontology pie charts were generated using the SGD Gene Ontology Slim Mapper tool, and only values above 2% were included. (C) Gene expression of highly mutated alleles, measured through RT-qPCR during log phase growth of yeast. Each mutant yeast chosen had a mutation in all six genes listed. Cells are labeled as (1) FEX DKO, (2, 3) 100×-, (4) 300×-, (5) 500×-, and (6) 100×-increased resistance to fluoride. p values were determined using Welch’s t test on the ddCt values, which in turn were determined from three or more biological replicates. A p value of ≤0.05 is marked with an asterisk (*).

We also looked for the enrichment of mutations within genes conserved across cellular processes. There was a twofold decrease in abundancy to DNA repair and metabolism than would be expected randomly (Figure 5B and Supp. Figure 6). As fluoride is known to cause DNA damage and metabolic arrest, cells may face selection pressure to maintain DNA repair and metabolism.

Four strains of mutants had at least 700-fold increased fluoride sensitivity. Given their greatly enhanced resistance, they are the strains most likely to contain mutations to critical genes and pathways. Between these four strains, there were 603 total mutations (Supp. Table 2). These ultra-resistant strains had a threefold enrichment for ion and nutrient transport genes, as well as enrichment in genes pertaining to the cell surface (Supp. Figure 7). This mutation rate differed significantly from yeast with lower fluoride resistance. Of the genes assigned to transport, the majority were involved in either vesicle-mediated transport, mitochondrial nutrient regulation, or single nutrient uptake, including sugars, amino acids, ATP, and cation transporters. All four strains also had mutations affecting genes IPP1 (inorganic pyrophosphatase), DAN4 (cell wall mannoprotein important for cell adhesion and iron uptake), and FLO9 (flocculation). Third, each had expanded telomere regions. Telomeres are known to shorten during oxidative stress, and the lengthening of the telomeres could potentially confer resistance during fluoride-induced oxidative stress. The telomeric region on the left arm of chromosome VIII (TEL08L) was mutated in all four strains, suggesting that DNA near the left arm of this chromosome may be particularly important. TEL08L is located next to COS8 (an endosomal protein) and ARN2 (an iron transporter). Each of these genes and pathways is therefore likely candidates to participate in fluoride resistance.

We next tested whether the mutations to genes changed their expression levels in cells. Overall, the genome of the 81 fluoride-hardened yeast was largely altered in those affecting metabolism, cell surface, and nutrient uptake genes. We used RT-qPCR to monitor the expression of genes involved in nutrient acquisition (Figure 5C). We selected strains with mutations to the particular gene being monitored. Overall, hardened yeast had lower expression in genes involved in nutrient acquisition compared with FEX DKO yeast. The exceptions to this were IPP1 and PHO87. This is consistent with our lab’s previous finding that a higher copy number of IPP1 and PHO87 increases fluoride resistance.10 Nonetheless, overall fluoride-hardened mutants had an overall decline of nutrient uptake, which suggests that a decline in nutrient uptake helps confer improved fluoride resistance.

3.5. Flocculation Confers Significant Survival to Fluoride-Hardened Yeast

The whole-genome sequencing results strongly suggest that mutations in flocculation and cellular adherence conferred fluoride survival. Every resistant strain had at least one mutation to a flocculation gene. Flocculation is a process of cellular aggregation, consistent with the clumping we observe in fluoride-hardened mutants (Figure 1D). Flocculation is induced by the binding of flocculin lectin proteins of one cell to the mannose of another cell’s wall and is similar to bacterial biofilms.35 Many stimuli can activate flocculation, most notably nutrient starvation. During environmental stress, flocculating yeast divert nutrients to other yeast in their community to enhance overall survival. Previously, our lab found that fluoride activates the nutrient starvation response in yeast.10 We therefore hypothesized that the fluoride-hardened yeast in this study had induced flocculation as a defense against starvation.

FEX DKO yeast are derivatized from S. cerevisiae BY4741, which is a non-flocculating strain. The BY4741 genome contains all of the FLO genes necessary for flocculation, but these are transcriptionally silenced. Mutations in S. cerevisiae BY4741 FLO genes can activate flocculation.36−38 Given the aggregation phenotype observed in the fluoride-hardened mutants, we predicted that mutations to flocculating genes resulted in their active transcription. We monitored the transcription of FLO genes in vivo through RT-qPCR (Figure 6A). The filamentous growth genes FLO9, DIA1, and FLO1 were commonly mutated in upstream regions. FLO1 and FLO9 are lectins that function in cell adhesion, while DIA1 is involved in pseudo-hyphal growth. FLO1, FLO9, and DIA1 had increased expression in the overwhelming majority of fluoride-hardened strains compared with FEX DKO. FLO9 and DIA1 had the highest degree of correlation between increased expression and degree of fluoride resistance. However, the expression of FLO1, FLO9, and DIA1 was not a perfect correlation to fluoride resistance, suggesting that flocculation and cell adhesion offered some but not all of the enhanced survival.

Figure 6.

Figure 6

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Flocculation ability of fluoride-hardened mutants. (A) Change in expression of FLO1, FLO9, and DIA1 genes in strains grown to the saturated phase, as compared to expression in FEX DKO. Gene expression was quantified using RT-qPCR and using actin as a housekeeping gene. p values were determined using Welch’s t test on the ddCt values, which in turn were determined from three or more biological replicates. A p value of ≤0.05 is marked with an asterisk (*). (B) Flocculation rate in wild-type and FEX DKO cells. The percent of cells in solution at O.D. 5 is monitored for 2 h in either 2 mL of (black/gray) YPD, (blue) 2% mannose, which disrupts flocculation, or (red) 5 mM calcium chloride, which promotes flocculation. (C) Flocculation number of yeast growing in YPD, as determined by the percent of yeast no longer in solution after 1 min at rest. (D) Extracellular concentration of glucose and amino acids (measured by Bradford assay) of cells at a final O.D. of 5.0, grown in either YPD or (E) 50 μM NaF for 24 h. Samples in panels A, C, D, and E are colored as (0) media only (no cells), (1) FEX DKO, (2, 3) 100×-, (4, 5) 300×-, (6, 7) 500×-, (8) 750×-, (9) 1000×-, and (10) 1200×-increased resistance to fluoride.

Flocculation can be monitored by counting the number of cells that precipitate out of solution within 1 min. The standard nomenclature in the field is that high flocculating yeast have ≥75% of cells precipitate in 1 min, while moderate flocculating yeast precipitate ≥25% of total cells out of solution in 1 min. The flocculation number for FEX DKO at 1 min is less than 10%, which is classically considered non-flocculating (Figure 6B). However, the rate by which FEX DKO and wild-type yeast sediment out of solution was dependent on conditions known to enhance or disrupt flocculation. The addition of calcium promoted sedimentation, while mannose (which disrupts flocculation) decelerated cell clustering. Collectively, this suggests that BY4741 yeast have some basal flocculation abilities, which are enhanced in fluoride-hardened strains. In regular YPD media, fluoride-hardened mutants had a flocculation number of around 20–45% (Figure 6C). The majority of fluoride-resistant strains were able to flocculate efficiently in regular media, whereas wild-type yeast could not.

Flocculation is induced during nutrient starvation, and cells subsequently use flocculation to form nutrient-sharing communities. Nutrients are released from cells into the surrounding buffer so that they can be distributed throughout the clustering cells. Of particular prevalence are sugars and amino acids. Our metabolomics data suggest that sugars are not consumed as quickly in hardened strains and that neutral and basic amino acids are diverted to the extracellular components (Figure 3A). We confirmed this by quantifying extracellular amino acids and glucose through standard colorimetric kits (Figure 6D). For both amino acids and glucose, FEX DKO and mutants released nutrients into the buffer. More nutrients were present in the buffer of the mutant strains, consistent with nutrient sharing. We also tested the effect of fluoride on nutrient sharing (Figure 6E). FEX DKO and mutant cells were exposed to 50 μM fluoride for 24 h. The extracellular glucose concentration for FEX DKO cells decreased while remaining largely unchanged in mutant strains. The extracellular amino acid concentration decreased for both FEX DKO and mutant strains, although the mutants still retained higher extracellular concentrations. In all, the fluoride-hardened strains had a higher extracellular nutrient concentration, reminiscent of the mechanism of rescue from nutrient starvation by flocculation. This implicates that the ability for coagulation and nutrient sharing promotes survival in fluoride, and cells with the highest ability to coagulate through flocculation also have the highest degree of fluoride resistance.

4. Discussion

Here, we report the generation of 81 S. cerevisiae strains that are mutated to resist fluoride toxicity without a functional fluoride channel. Fluoride-resistant mutants had slower growth, enhanced nutrient storage, and increased cytoplasmic acidification. Together, these phenotypes served to conserve metabolites and shift the pH equilibrium such that intracellular fluoride is directed out of the cell without the need of a transport protein.

The most striking initial phenotype was that fluoride-resistant mutations resulted in slow growth phenotypes. These yeast were found to spend more time in the lag phase and have a slower overall growth rate during the log phase. Yeast are known to have enhanced resistance to stress during reduced growth through an unknown mechanism.27−30 The current hypothesis in the field is that cellular dormancy allows for a longer response time to toxicant exposure. Rapidly growing cells demand a constant supply of nutrients and would be particularly sensitized to the disruption of an essential pathway. Our lab previously found that fluoride triggers nutrient starvation and inhibits glucose uptake.10 A key survival mechanism for wild-type yeast is endocytosis, which can acquire glucose independent of specific glucose transporters.17 Reduced growth in fluoride-hardened cells reduces the need for nutrients, providing enhanced resistance to fluoride.

Fluoride-hardened yeast also dramatically increased their nutrient storage. Mutated yeast greatly increased stored sugar and amino acid deposits. Increased nutrient storage has been found to counteract stressors, most notably oxidative stress.31,39−41 It provides considerable resistance to fluoride-induced nutrient starvation. The net trend for fluoride-hardened yeast was a reduced rate in Krebs cycle activity until the stationary phase. This allowed for unused substrate to accumulate and for yeast to prolong and even enhance Krebs cycle activity in late stages of growth. It also allowed for the shifting of the ion gradient in cells by accumulating acidic substrates and excreting alkaline substrates. The most prominent example of this trend was an increase in amino acids in the extracellular media in a manner known to occur in starvation-induced flocculation.42,43

From whole-genome sequencing, we found that every fluoride-hardened strain had at least one mutation to genes involved in flocculation. Flocculation is the process in which single-celled yeast clump together and develop community-like behavior. Yeast share nutrients among their community along their cell surfaces, thus conferring survival during starvation.44 We have found a similar phenomenon in fluoride-hardened yeast. This appeared to be through the combination of several mutations to flocculation-related genes, most notably FLO9. FLO1 is considered the predominant gene in driving flocculation.35 It is unclear why FLO9 is the top mutated gene instead of FLO1. Prior research has found that FLO9 is responsive to changes in nutrient levels.36 Given fluoride’s ability to trigger nutrient starvation, this could lead to a more sensitive response pathway.

There are many possible mechanisms to explain why flocculation provides resistance to high fluoride exposure. Fluoride triggers predominantly intracellular toxicity. Clustering cells would have a reduced total surface area compared with suspended cells and therefore less available area for fluoride entry. Furthermore, as this effect is only seen in the weakly flocculating strains, sedimentation may help drive a more flocculant state. Cell adhesion and flocculation has been reported by several labs to result in enhanced survival to aging, oxidative stress, and nutrient starvation.35,45 As fluoride toxicity leads to metabolic arrest and oxidative stress, flocculation appears to be a significant contributor to resistance.

Another global trend for enhanced fluoride resistance was intracellular acidification. Fluoride is novel as a halide in that its acidic form, hydrofluoric acid (HF), has a positive pKa of 3.2. HF is able to pass through the neutral lipid bilayer independent of an ion channel. Organisms are therefore highly sensitized to intracellular fluoride accumulation in acidic environments. Fluoride-hardened yeast have seemingly reversed this principle, flipping the equilibrium such that fluoride is protonated and exported from the cell before it can confer significant toxicity. We were able to reproduce this phenotype by manually acidifying the cytoplasm of FEX DKO yeast through V-ATPase inhibition. This succeeded in reducing intracellular fluoride without the need of a fluoride protein exporter.

Not all fluoride-hardened cells achieved the acidic adaptation. Five fluoride-hardened mutants had higher intracellular pH compared to FEX DKO yeast. These strains still had a large degree of overlap in genomic mutations with other fluoride-hardened mutants. The most commonly mutated genes across the alkaline mutants included IPP1, CSS1, FLO proteins, tRNAs, telomeres, DAN4, and ERR3. These genes were also commonly mutated in acidic mutants. As such, these alkaline mutants appear to follow the same general mechanisms of resistance to fluoride as acidic mutants. However, the alkaline mutants only achieve 100- to 300-fold increased fluoride resistance, as opposed to up to 1200-fold resistance achieved through intracellular acidification. Given that the majority of higher-fold fluoride-resistant mutants show intracellular acidification, acidification appears to be an important, independent mechanism for resistance to fluoride.

In all, there was no gene that appeared solely responsible for influencing fluoride tolerance. Yeast gained significant resistance to fluoride through stockpiling and sharing nutrients, clustering together into communities, and exporting fluoride as HF. This is consistent with fluoride acting predominantly to induce nutrient starvation and suggests that organisms in the wild can vary in fluoride resistance by the pH gradient along their cellular membranes, their overall rate in metabolism, and their ability to store nutrients.

Acknowledgments

The authors of this study would like to thank the members of the Strobel lab for their discussions and proofreading of this manuscript, as well as David Hiller for his contribution. We also would like to thank Dr. Gerald I. Shulman for his valuable contribution to the metabolomics of this study.

Glossary

Abbreviations

FEX

fluoride exporter protein

DKO

double knockout

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.chemrestox.2c00222.

  • Supplementary methods, overview of mutational assays, 31P NMR spectra of yeast, correlation of extracellular glucose with cellular growth, fold change in concentration of intracellular and extracellular substrates, gene ontology map for yeast and yeast with at last 700-fold increased fluoride resistance, select comparative 13C NMR peaks, summary of the top 100 most frequently mutated genes across 81 strains of fluoride-hardened yeast, summary of top 30 conserved mutations across the four highest fluoride-resistant strains, and summary of top 50 conserved mutations across the five fluoride-hardened strains with alkaline intracellular pH (PDF)

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. CRediT: Nichole R Johnston conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, software, validation, visualization, writing-original draft; Gary Cline data curation, resources, supervision, writing-review & editing. Scott A. Strobel supervision, writing-review & editing.

This work was supported by funding provided by the N.I.H. Chemical Biology Interface training grant (to N.R.J.), by the National Science Foundation grant no. GR108639 (to S.A.S.), and by grants R01 DK116774 and P30 DK045735 (to Gerald I. Shulman and G.C.).

The authors declare no competing financial interest.

Supplementary Material

tx2c00222_si_001.pdf (1.5MB, pdf)

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