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Cells Adapt to Resist Fluoride through Metabolic Deactivation and Intracellular Acidification

Johnston, Nichole R., Cline, Gary, Strobel, Scott A.·2022·Chemical Research in Toxicology
DOI 10.1021/acs.chemrestox.2c00222PMC PMC9683101
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Figure 1 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.
Figure 1 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.CC BY-NC-ND · PubMed Central
Figure 2 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.
Figure 2 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.CC BY-NC-ND · PubMed Central
Figure 3 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.
Figure 3 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.CC BY-NC-ND · PubMed Central
Figure 4 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.
Figure 4 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.CC BY-NC-ND · PubMed Central
Figure 5 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 (*).
Figure 5 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 (*).CC BY-NC-ND · PubMed Central
Figure 6 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.
Figure 6 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.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Johnston, N. R., Cline, G., & Strobel, S. A.
Venue
Chemical Research in Toxicology
Publisher
American Chemical Society (ACS)
Published
2022-10-25
Volume
35
Issue
11
Pages
2085-2096
ISSN
  • 0893-228X
  • 1520-5010
DOI
10.1021/acs.chemrestox.2c00222
PMC
PMC9683101
Cited by
9
References
1
Language
en
Licence
creativecommons.org/licenses/by-nc-nd/4.0/ (vor, 365 d)
Funders
  • National Institute of Diabetes and Digestive and Kidney Diseases (P30 DK045735, R01 DK116774)
  • National Science Foundation (GR108639)

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