Fluoride export is required for the competitive fitness of pathogenic microorganisms in dental biofilm models

In the feast/famine biofilm experiment, total counts were slightly reduced relative to growth in chloride-exposed biofilms (P-values between 0.031 and 0.221 at each timepoint beyond day 2).

“over the course of the experiment, there was a small reduction in total counts relative to its growth in chloride-exposed biofilms (P-values between 0.031 and 0.221 at each timepoint beyond day 2).”

Genetic knockout of the CLCF transporter in S. mutans and exposure to low fluoride concentrations increased the relative proportion of oral commensal S. gordonii in dental biofilms.

“For biofilms generated using S. mutans with the genetic knockout of the CLCF transporter, exposure to low fluoride concentrations decreased S. mutans counts, synergistically reduced the populations of C. albicans, increased the relative proportion of oral commensal S. gordonii, and reduced properties associated with biofilm pathogenicity, including acid production and hydroxyapatite dissolution.”

For WT and rescue strains, CFUs increased up to 24 h, indicating continued cell division.

“Indeed, for the WT and rescue strains, CFUs actually increased up to 24 h, indicating continued cell division (Fig. S1).”

For three-species biofilms composed of ΔFEX C. albicans, WT S. mutans, and WT S. gordonii, WT S. mutans population decreased by a small but significant factor of 4.4 compared to the chloride control.

“For the three-species biofilms composed of ∆FEX C. albicans, WT S. mutans, and WT S. gordonii, in addition to the reduction of ∆FEX C. albicans described above, WT S. mutans population decreased by a small, but significant factor of 4.4 compared to the chloride control.”

WT S. mutans cells exhibited normal morphology after 0.3 or 1 mM NaF treatment for 4 h.

“WT cells exhibited normal morphology after both treatments.”

For planktonic ΔCLCF S. mutans treated with 0.3 mM NaF, the number of viable colonies decreased as a function of time between 4 and 24 h.

“For cells treated with 0.3 mM NaF, the number of viable ∆CLCF S. mutans colonies decreased as a function of time between 4 and 24 h.”

In all-WT biofilms, S. gordonii counts peaked on day 3 before being outcompeted by S. mutans and C. albicans.

“By contrast, S. gordonii counts peaked on day 3, before being outcompeted by the other two species.”

In three-species biofilms with a single fluoride-sensitive species, that species was significantly reduced in fluoride-containing media relative to chloride.

“Unsurprisingly, in biofilms with a single fluoride-sensitive species, that species was significantly reduced in fluoride-containing media relative to chloride.”

Exposure to 0.2 mM fluoride reduced ΔCLCF S. mutans and ΔFEX C. albicans colony counts by 830- and 50-fold, respectively, compared to biofilms grown without fluoride treatment.

“Exposure to 0.2 mM (3.8 ppm F−) fluoride reduced ∆CLCF S. mutans and ∆FEX C. albicans colony counts by 830- and 50-fold, respectively, compared to biofilms grown without fluoride treatment (Fig. 2A).”

Only when biofilms were grown with both ΔFEX C. albicans and ΔCLCF S. mutans was there a significant decrease in the S. gordonii population compared to the chloride control.

“Only when biofilms were grown with both ∆FEX C. albicans and ∆CLCF S. mutans was there a significant decrease in the S. gordonii population compared to the chloride control.”

Genetic knockout of the CLCF transporter in S. mutans and exposure to low fluoride concentrations reduced hydroxyapatite dissolution in dental biofilms.

“For biofilms generated using S. mutans with the genetic knockout of the CLCF transporter, exposure to low fluoride concentrations decreased S. mutans counts, synergistically reduced the populations of C. albicans, increased the relative proportion of oral commensal S. gordonii, and reduced properties associated with biofilm pathogenicity, including acid production and hydroxyapatite dissolution.”

For biofilms prepared with ΔCLCF S. mutans, populations of other species were impacted as well.

“However, for biofilms prepared with ΔCLCF S. mutans, populations of other species were impacted as well.”

Both biofilms with ΔCLCF S. mutans exhibited significantly lower dry-weight biomass compared to all-WT or chloride-treated controls.

“Both biofilms with ∆CLCF S. mutans exhibited significantly lower dry-weight biomass compared to all-WT or chloride-treated controls, in accord with S. mutans’ role as a keystone pathogen whose fitness impacts biofilm stability and assembly (Fig. 3C).”

Among fluoride-treated biofilms, only the samples with both ΔCLCF S. mutans and ΔFEX C. albicans showed significantly higher media pH compared to biofilms with the WT counterparts.

“However, among the fluoride-treated biofilms, only the samples with both ∆CLCF S. mutans and ∆FEX C. albicans showed significantly higher media pH compared to biofilms with the WT counterparts (Fig. 3B).”

Genetic knockout of the CLCF transporter in S. mutans and exposure to low fluoride concentrations reduced acid production in dental biofilms.

“For biofilms generated using S. mutans with the genetic knockout of the CLCF transporter, exposure to low fluoride concentrations decreased S. mutans counts, synergistically reduced the populations of C. albicans, increased the relative proportion of oral commensal S. gordonii, and reduced properties associated with biofilm pathogenicity, including acid production and hydroxyapatite dissolution.”

The reduction in the C. albicans population upon knockout of the S. mutans fluoride exporter is almost as great as the reduction in C. albicans population due to knockout of its fluoride exporter, 5 × 10^4 fold.

“Strikingly, this reduction in the C. albicans population upon knockout of the S. mutans fluoride exporter is almost as great as the reduction in C. albicans population due to knockout of its fluoride exporter, 5 × 104 fold.”

In three-species biofilms with ΔCLCF S. mutans or ΔFEX C. albicans, no significant change in the S. gordonii population was observed.

“In both cases, no significant change in the S. gordonii population was observed.”

After just 1-h exposure to 1 mM NaF, viable ΔCLCF S. mutans CFUs were reduced 6.8-fold relative to WT.

“After just 1-h exposure, viable ∆CLCF S. mutans CFUs were reduced 6.8-fold relative to WT (Fig. 2B).”

Genetic knockout of the CLCF transporter in S. mutans and exposure to low fluoride concentrations synergistically reduced the populations of C. albicans in dental biofilms.

“For biofilms generated using S. mutans with the genetic knockout of the CLCF transporter, exposure to low fluoride concentrations decreased S. mutans counts, synergistically reduced the populations of C. albicans, increased the relative proportion of oral commensal S. gordonii, and reduced properties associated with biofilm pathogenicity, including acid production and hydroxyapatite dissolution.”

S. mutans is highly sensitive to fluoride, with the knockout strain undergoing complete lysis when exposed to low fluoride for a moderate amount of time.

“Imaging studies indicate that S. mutans is highly sensitive to fluoride, with the knockout strain undergoing complete lysis when exposed to low fluoride for a moderate amount of time.”

Genetic knockout of the CLCF transporter in S. mutans and exposure to low fluoride concentrations decreased S. mutans counts in dental biofilms.

“For biofilms generated using S. mutans with the genetic knockout of the CLCF transporter, exposure to low fluoride concentrations decreased S. mutans counts, synergistically reduced the populations of C. albicans, increased the relative proportion of oral commensal S. gordonii, and reduced properties associated with biofilm pathogenicity, including acid production and hydroxyapatite dissolution.”

No survival of ΔCLCF S. mutans was detected at timepoints ≥4 h of 1 mM NaF treatment.

“No survival of ∆CLCF S. mutans was detected at timepoints ≥ 4 h of treatment.”

Sensitization of cariogenic species S. mutans and C. albicans to fluoride by genetic knockout of fluoride exporters alters the microbial composition and pathogenic properties of dental biofilms.

“This work shows that sensitization of cariogenic species S. mutans and C. albicans to fluoride by genetic knockout of fluoride exporters alters the microbial composition and pathogenic properties of dental biofilms.”

No viable ΔCLCF S. mutans colonies were observed from biofilms treated with 0.3 mM fluoride for 24 h.

“In striking contrast, we observed no viable ∆CLCF S. mutans colonies from biofilms treated with 0.3 mM fluoride for 24 h.”

Complementation with plasmid-encoded CLCF restored WT-like survival of ΔCLCF S. mutans after fluoride treatment.

“By contrast, when the gene encoding CLCF was re-introduced on a plasmid, this strain remained viable after fluoride treatment for the entire timecourse Fig. 2B.”

• Also stated in: § Fluoride is bactericidal for ∆CLCF S. mutans · plain

  “As for the 0.3 mM experiments, complementation with a plasmid-encoded CLCF enabled the WT-like survival of the ∆CLCF S. mutans strain.”

For all biofilms with ΔFEX C. albicans, ΔCLCF S. mutans, or both, fluoride treatment significantly increased the pH of the biofilm media relative to the NaCl control.

“For all biofilms with ∆FEX C. albicans, ∆CLCF S. mutans, or both, fluoride treatment significantly increased the pH of the biofilm media relative to the NaCl control.”

∆1290 S. mutans showed only a marginal sensitivity to 0.3 mM NaF.

“and showed only a marginal sensitivity to 0.3 mM NaF (Fig. 6A).”

Sequence alignments indicate that both 1289 and 1290 possess the characteristic fluoride-binding motifs observed in structures of the CLCF from Enterococcus casseliflavus.

“Sequence alignments indicate that both 1289 and 1290 possess the characteristic fluoride-binding motifs observed in structures of the CLCF from Enterococcus casseliflavus (26) (Fig. S5)”

• cites [26] Last NB, Stockbridge RB, Wilson AE, Shane T, Kolmakova-Partensky L, Koide A, Koide S, Miller C. 2018. A CLC-type F(-)/H(+) antiporter in ion-swapped conformations. Nat Struct Mol Biol 25: 601– 606. doi: 10.1038/s41594-018-0082-0 29941917 PMC6044475 sourceCheck pending

S. gordonii did not show the population peak and collapse observed in the all-WT or ΔFEX C. albicans experiments.

“but without the population peak and collapse observed in those two samples.”

The ∆1289 ∆1290 double knockout was not significantly less fit relative to the ∆1289 single knockout in fluoride (p = 0.29).

“Similarly, the ∆1289 ∆1290 double knockout was not significantly less fit relative to the ∆1289 single knockout in fluoride (p = 0.29; Fig. 6B).”

In biofilms with both ΔFEX C. albicans and ΔCLCF S. mutans, counts for both S. mutans and C. albicans were significantly reduced compared with biofilms with either individual knockout strain.

“counts for both species were significantly reduced in biofilms with both knockout strains, compared with biofilms with either individual knockout strain (Fig. 4B through E; Fig. S3).”

Biofilms with ∆CLCF S. mutans or ΔFEX C. albicans exhibited significantly lower hydroxyapatite dissolution at most timepoints.

“The biofilms with ∆CLCF S. mutans or ΔFEX C. albicans also exhibited significantly lower hydroxyapatite dissolution at most timepoints.”

Fluoride efflux from proteoliposomes containing 1290 was indistinguishable from uncatalyzed fluoride leak exhibited by protein-free liposomes.

“By contrast, fluoride efflux from proteoliposomes containing 1290 was indistinguishable from uncatalyzed fluoride leak exhibited by protein-free liposomes (Fig. 6F).”

Much smaller reductions in pH are seen during the overnight famine period.

“much smaller reductions in pH are seen during the overnight famine period”

We did not observe fluoride-induced changes in the transcription of ORF 1290.

“Moreover, we did not observe fluoride-induced changes in the transcription of ORF 1290.”

Biofilms grown with ∆CLCF S. mutans exhibited less demineralization.

“and less demineralization.”

∆CLCF S. mutans underwent massive lysis under fluoride stress.

“∆CLCF S. mutans unexpectedly underwent massive lysis under fluoride stress.”

The pH at the end of the feast period was consistently higher throughout the experiment for fluoride-treated biofilms in which either pathogenic microorganism was fluoride export deficient.

“The pH at the end of the feast period was consistently higher throughout the experiment for fluoride-treated biofilms in which either of the pathogenic microorganisms was fluoride export deficient (Fig. 5A; Table 2; Table S3).”

Media calcium content oscillates corresponding to the periods of feast and famine.

“we observed oscillations in the media calcium content corresponding to the periods of feast and famine”

1290 contributed little to resistance, either in culture or in the biofilm context.

“we found that 1290 contributed little to resistance, either in culture or in the biofilm context.”

Statistically significant differences in calcium release compared to chloride-treated biofilms emerged at all timepoints after fluoride addition, which occurred after the famine treatment on the second day (D2-AM).

“with statistically significant differences compared to the chloride-treated biofilms emerging at all timepoints after fluoride addition, which occurred after the famine treatment on the second day (D2-AM) (Fig. 5B).”

ORF 1289 showed an increase in transcripts over time when the biofilm was treated with 0.2 mM NaF, with a 17-fold increase between 1 and 24 h of fluoride exposure.

“ORF 1289 showed an increase in transcripts over time when the biofilm was treated with 0.2 mM NaF, with a 17-fold increase between 1 and 24 h of fluoride exposure.”

Biofilms grown with ∆CLCF S. mutans exhibited higher pH.

“higher pH”

Biofilms grown with ∆CLCF S. mutans exhibited a lower C. albicans population.

“Biofilms grown with ∆CLCF S. mutans exhibited a lower C. albicans population”

Both S. mutans and C. albicans are rendered sensitive to hundreds of micromolar F− upon deletion of their respective fluoride exporters.

“both S. mutans and C. albicans are rendered sensitive to hundreds of micromolar F− upon deletion of their respective fluoride exporters.”

∆CLCF S. mutans was eliminated from biofilms at a low fluoride concentration of 0.3 mM (5.7 ppm F-).

“Particularly striking was the elimination of ∆CLCF S. mutans from biofilms at a low fluoride concentration of 0.3 mM (5.7 ppm F-)”

1289 catalyzed robust fluoride efflux from liposomes, with a unitary transport rate of 830 ± 20 ions/second.

“1289 catalyzed robust fluoride efflux from liposomes, with a unitary transport rate of 830 ± 20 ions/second, in line with rates measured for other CLCF transporters (43).”

• cites [43] Brammer AE, Stockbridge RB, Miller C. 2014. F-/Cl- selectivity in CLCF-type F-/H+ antiporters. J Gen Physiol 144: 129– 136. doi: 10.1085/jgp.201411225 25070431 PMC4113899 sourceCheck pending

Growth of ∆1290 S. mutans was not affected at 0.1 mM NaF, similar to the WT strain.

“growth of ∆1290 S. mutans was not affected at 0.1 mM NaF, similar to the WT strain”

Fluoride-treated all-WT biofilms showed lower calcium release into the media compared to chloride-treated biofilms.

“even all-WT biofilms showed lower calcium release into the media compared to the chloride-treated biofilms.”

• AssumptionRests on an assumption: Fluoride helps prevent demineralization via a physicochemical effect.

Total biofilm biomass was decreased in biofilms with ∆CLCF S. mutans.

“The total biofilm biomass was also decreased”

The biomass of the mature biofilm was significantly reduced for fluoride-treated biofilms prepared with ∆CLCF S. mutans compared to chloride-treated biofilms.

“the biomass of the mature biofilm was significantly reduced for fluoride-treated biofilms prepared with ∆CLCF S. mutans, compared to the respective chloride-treated biofilms (Fig. 5C).”

A similar trend in S. gordonii growth was present in the biofilm sample with both ΔFEX C. albicans and ΔCLCF S. mutans.

“A similar trend in S. gordonii growth was also present in the biofilm sample with both ΔFEX C. albicans and ΔCLCF S. mutans.”

Fluoride sensitivity in S. mutans did not cause overgrowth of S. gordonii.

“as fluoride sensitivity in S. mutans did not cause overgrowth of S. gordonii”

For biofilms grown with ∆1290, there was a slight, but not statistically significant, decrease in S. mutans CFUs harvested from the biofilm (p = 0.108).

“For biofilms grown with ∆1290, there was a slight, but not statistically significant, decrease in S. mutans CFUs harvested from the biofilm (p = 0.108; Fig. 6B; Fig. S6).”

Fluoride-treated biofilms prepared with both fluoride-sensitive strains exhibited a significant reduction in acid production (increased pH) even after the famine periods.

“Fluoride-treated biofilms prepared with both fluoride-sensitive strains exhibited a significant reduction in acid production (increased pH) even after the famine periods”

Both 1289 and 1290 were incorporated into proteoliposomes at similar levels.

“Western blot analysis showed that both 1289 and 1290 were incorporated into proteoliposomes at similar levels (Fig. S8).”

1290 did not exhibit chloride transport activity.

“We further tested chloride efflux by 1290, but did not observe any transport activity for this halide either (Fig. S9).”

When fluoride concentration is increased to 0.3 mM, causing lysis and elimination of S. mutans, fluoride accumulation in the biofilm is eliminated.

“When the fluoride concentration is increased to the level that causes lysis and elimination of S. mutans, 0.3 mM, fluoride accumulation in the biofilm is likewise eliminated (Fig. S4).”

Biofilms grown with ∆CLCF S. mutans exhibited a greater proportion of S. gordonii.

“a greater proportion of S. gordonii”

These biofilms also accumulate the highest levels of fluoride.

“Interestingly, these biofilms also accumulate the highest levels of fluoride (Fig. 5D).”

Drastic reductions in pH occur during every feast period.

“Drastic reductions in pH occur during every feast period due to sucrose fermentation”

Temporal release of calcium was the lowest in fluoride-treated biofilms grown from the combination of mutant strains of both pathogens.

“Temporal release of calcium was the lowest in the fluoride-treated biofilms grown from the combination of the mutant strains of both pathogens”

An unexpected finding was the retention of fluoride in biofilms with ∆CLCF S. mutans.

“An unexpected finding was the retention of fluoride in biofilms with ∆CLCF S. mutans.”

∆1289 S. mutans exhibited comparable fluoride sensitivity to the ∆CLCF S. mutans strain in which both open reading frames are deleted.

“∆1289 S. mutans exhibited comparable fluoride sensitivity to the ∆CLCF S. mutans strain used for earlier experiments, in which both open reading frames are deleted.”

Biofilms prepared with fluoride-sensitive strains exhibited even lower levels of hydroxyapatite dissolution.

“the biofilms prepared with fluoride-sensitive strains exhibited even lower levels of hydroxyapatite dissolution.”

S. gordonii reached a similar population at day 5 as in the all-WT or ΔFEX C. albicans experiments.

“reaching a similar population at day 5 as in the all-WT or ΔFEX C. albicans experiments”

The lowest acid production was observed in fluoride-treated biofilms grown with mutant strains of both S. mutans and C. albicans.

“The lowest acid production was observed in the fluoride-treated biofilms grown with mutant strains of both S. mutans and C. albicans.”

Sequence alignments show that most of the fluoride-binding residues identified in the structure of the Enterococcus casseliflavus CLCF are conserved in 1290, except T320.

“Sequence alignments show that most of the fluoride-binding residues identified in the structure of the Enterococcus casseliflavus CLCF (26) are conserved in 1290, except T320 (E. casseliflavus numbering) (Fig. S5).”

• cites [26] Last NB, Stockbridge RB, Wilson AE, Shane T, Kolmakova-Partensky L, Koide A, Koide S, Miller C. 2018. A CLC-type F(-)/H(+) antiporter in ion-swapped conformations. Nat Struct Mol Biol 25: 601– 606. doi: 10.1038/s41594-018-0082-0 29941917 PMC6044475 sourceCheck pending

In 1290, there is methionine at position T320.

“In 1290, there is methionine at this position.”

Microbial fluoride sensitivity is amplified by low pH, as is characteristic of dysbiotic dental biofilms.

“First, microbial fluoride sensitivity is amplified by low pH, as is characteristic of dysbiotic dental biofilms (20).”

• cites [20] Ji C, Stockbridge RB, Miller C. 2014. Bacterial fluoride resistance, Fluc channels, and the weak acid accumulation effect. J Gen Physiol 144: 257– 261. doi: 10.1085/jgp.201411243 25156118 PMC4144673 sourceNot checked yet

Candida albicans extrudes intracellular fluoride using a FEX fluoride channel.

“Eukaryotes, including C. albicans, express a third type of fluoride exporter called FEX (Fluoride Exporter) (18).”

• cites [18] Li S, Smith KD, Davis JH, Gordon PB, Breaker RR, Strobel SA. 2013. Eukaryotic resistance to fluoride toxicity mediated by a widespread family of fluoride export proteins. Proc Natl Acad Sci USA 110: 19018– 19023. doi: 10.1073/pnas.1310439110 24173035 PMC3839697 sourceCheck pending

Most microbes possess fluoride export proteins to maintain cytoplasmic fluoride at low levels.

“Because most microbes possess fluoride export proteins to maintain cytoplasmic fluoride at low levels (16), the antimicrobial effect is clinically irrelevant at these concentrations.”

• cites [16] McIlwain BC, Ruprecht MT, Stockbridge RB. 2021. Membrane exporters of fluoride ion. Annu Rev Biochem 90: 559– 579. doi: 10.1146/annurev-biochem-071520-112507 33492991 PMC8217344 sourceNot checked yet

S. mutans possesses two genes that encode F-/H+ antiporters from the CLC family, termed CLCF.

“S. mutans possesses two genes that encode F-/H+ antiporters from the CLC (chloride channel) family of anion channels and transporters, termed CLCF (22).”

• cites [22] Stockbridge RB, Lim HH, Otten R, Williams C, Shane T, Weinberg Z, Miller C. 2012. Fluoride resistance and transport by riboswitch-controlled CLC antiporters. Proc Natl Acad Sci USA 109: 15289– 15294. doi: 10.1073/pnas.1210896109 22949689 PMC3458365 sourceCheck pending

Oral eubiota including S. gordonii, S. oralis, and S. sanguinis export F- via Fluc proteins.

“By contrast, oral eubiota including S. gordonii, S. oralis, and S. sanguinis all export F- via the action of Fluc (Fluoride channel) proteins (also known by their gene name, crcB), which harness the electrical component of the proton motive force to expel cytoplasmic fluoride (23).”

• cites [23] Stockbridge RB, Robertson JL, Kolmakova-Partensky L, Miller C. 2013. A family of fluoride-specific ion channels with dual-topology architecture. Elife 2: e01084. doi: 10.7554/eLife.01084 23991286 PMC3755343 sourceCheck pending

In the absence of a fluoride export mechanism, impermeable fluoride ions become trapped and accumulate intracellularly.

“In the absence of a fluoride export mechanism, these impermeable ions become trapped and accumulate intracellularly (20).”

• cites [20] Ji C, Stockbridge RB, Miller C. 2014. Bacterial fluoride resistance, Fluc channels, and the weak acid accumulation effect. J Gen Physiol 144: 257– 261. doi: 10.1085/jgp.201411243 25156118 PMC4144673 sourceNot checked yet

Pathogenic and health-associated oral species possess fluoride export proteins from different molecular families.

“Second, pathogenic and health-associated oral species possess fluoride export proteins from different molecular families (Fig. 1A).”

Streptococcus mutans extrudes intracellular fluoride using a CLCF F-/H+ antiporter.

“S. mutans possesses two genes that encode F-/H+ antiporters from the CLC (chloride channel) family of anion channels and transporters, termed CLCF (22).”

• cites [22] Stockbridge RB, Lim HH, Otten R, Williams C, Shane T, Weinberg Z, Miller C. 2012. Fluoride resistance and transport by riboswitch-controlled CLC antiporters. Proc Natl Acad Sci USA 109: 15289– 15294. doi: 10.1073/pnas.1210896109 22949689 PMC3458365 sourceCheck pending

Fluoride exhibits antimicrobial activity due to broad-spectrum inhibition of essential metabolic enzymes, including enolase in the glycolytic pathway, pyrophosphatase, and ATP-consuming enzymes.

“Fluoride also exhibits antimicrobial activity due to its broad-spectrum inhibition of essential metabolic enzymes, including enolase in the glycolytic pathway, pyrophosphatase, and ATP-consuming enzymes (10).”

• cites [10] Adamek E, Pawłowska-Góral K, Bober K. 2005. in vitro and in vivo effects of fluoride ions on enzyme activity. Ann Acad Med Stetin 51: 69– 85. 16519100Not checked yet

Streptococcus gordonii exports fluoride using a Fluc fluoride channel.

“oral commensal eubacteria, such as Streptococcus gordonii, export fluoride using a Fluc fluoride channel.”

• Also stated in: § INTRODUCTION · plain

  “oral eubiota including S. gordonii, S. oralis, and S. sanguinis all export F- via the action of Fluc (Fluoride channel) proteins (also known by their gene name, crcB), which harness the electrical component of the proton motive force to expel cytoplasmic fluoride (23).”

• cites [23] Stockbridge RB, Robertson JL, Kolmakova-Partensky L, Miller C. 2013. A family of fluoride-specific ion channels with dual-topology architecture. Elife 2: e01084. doi: 10.7554/eLife.01084 23991286 PMC3755343 sourceCheck pending

During the first hour after using over-the-counter fluoride treatments, fluoride concentrations in oral fluids drop to <1 mM.

“However, during the first hour after using these treatments, fluoride concentrations in the oral fluids drop to <1 mM (12–15).”

• cites [12] Vogel GL, Tenuta LMA, Schumacher GE, Chow LC. 2010. No calcium-fluoride-like deposits detected in plaque shortly after a sodium fluoride mouthrinse. Caries Res 44: 108– 115. doi: 10.1159/000287239 20185917 PMC2883841 sourceCheck pending
• cites [13] Cury JA, do Amaral RC, Tenuta LMA, Del Bel Cury AA, Tabchoury CPM. 2010. Low‐fluoride toothpaste and deciduous enamel demineralization under biofilm accumulation and sucrose exposure. Eur J Oral Sci 118: 370– 375. doi: 10.1111/j.1600-0722.2010.00745.x 20662910 sourceNot checked — no full text of the source
• cites [14] Zamataro CB, Tenuta LMA, Cury JA. 2008. Low-fluoride dentifrice and the effect of postbrushing rinsing on fluoride availability in saliva. Eur Arch Paediatr Dent 9: 90– 93. doi: 10.1007/BF03262616 18534177 sourceNot checked — no full text of the source
• cites [15] Vogel GL, Schumacher GE, Chow LC, Tenuta LMA. 2015. Oral fluoride levels 1 h after use of a sodium fluoride rinse: effect of sodium lauryl sulfate. Caries Res 49: 291– 296. doi: 10.1159/000381192 25924684 sourceNot checked — no full text of the source

Fluoride export by oral pathogens can be targeted by specific inhibitors to restore biofilm symbiosis in dental biofilms.

“Together, these findings suggest that fluoride export by oral pathogens can be targeted by specific inhibitors to restore biofilm symbiosis in dental biofilms and that S. mutans is especially susceptible to fluoride toxicity.”

• AssumptionRests on an assumption: Genetic knockout of fluoride exporters is a valid proxy for pharmacological inhibition of fluoride export in oral pathogens.
• AssumptionRests on an assumption: Specific inhibitors of fluoride exporters can be developed that inhibit pathogenic fluoride exporters without unacceptable effects on host or commensal fluoride exporters.

S. mutans is especially susceptible to fluoride toxicity.

“Together, these findings suggest that fluoride export by oral pathogens can be targeted by specific inhibitors to restore biofilm symbiosis in dental biofilms and that S. mutans is especially susceptible to fluoride toxicity.”

The decreased S. gordonii population in biofilms with both ΔFEX C. albicans and ΔCLCF S. mutans is attributed to an overall reduction in microbial growth due to diminished extracellular matrix production by S. mutans.

“As for the C. albicans result described above, we attribute the decreased S. gordonii population to an overall reduction in microbial growth due to diminished extracellular matrix production by S. mutans.”

If unable to export fluoride, S. mutans is killed and eliminated from biofilms by fluoride in the hundreds of micromolar range.

“Together, these experiments show that, if unable to export fluoride, S. mutans is killed and eliminated from biofilms by fluoride in the hundreds of micromolar range.”

Biofilms composed of fluoride-sensitive strains will be less pathogenic than WT biofilms.

“The experiments described above suggest that biofilms composed of fluoride-sensitive strains will be less pathogenic than WT biofilms.”

Development of drugs that inhibit fluoride exporters could potentiate the anticaries effect of fluoride in over-the-counter products like toothpaste and mouth rinses.

“These results suggest that the development of drugs that inhibit fluoride exporters could potentiate the anticaries effect of fluoride in over-the-counter products like toothpaste and mouth rinses.”

• AssumptionRests on an assumption: Genetic knockout of fluoride exporters is a valid proxy for pharmacological inhibition of fluoride export in oral pathogens.
• AssumptionRests on an assumption: Specific inhibitors of fluoride exporters can be developed that inhibit pathogenic fluoride exporters without unacceptable effects on host or commensal fluoride exporters.

The high fluoride accumulation in biofilms with ∆CLCF S. mutans is due to fluoride accumulation in the cytoplasm of species that lack a fluoride exporter.

“We propose that this is due to fluoride accumulation in the cytoplasm of the species that lack a fluoride exporter, as has been shown to occur in other fluoride export-deficient bacteria (17, 20).”

• cites [17] Baker JL, Sudarsan N, Weinberg Z, Roth A, Stockbridge RB, Breaker RR. 2012. Widespread genetic switches and toxicity resistance proteins for fluoride. Science 335: 233– 235. doi: 10.1126/science.1215063 22194412 PMC4140402 sourceCheck pending
• cites [20] Ji C, Stockbridge RB, Miller C. 2014. Bacterial fluoride resistance, Fluc channels, and the weak acid accumulation effect. J Gen Physiol 144: 257– 261. doi: 10.1085/jgp.201411243 25156118 PMC4144673 sourceNot checked yet

The drastic pH reductions during feast periods are due to sucrose fermentation.

“Drastic reductions in pH occur during every feast period due to sucrose fermentation”

For cells in a pH 5 environment with cytoplasmic pH 7, fluoride will accumulate intracellularly to a concentration 100-fold greater than external fluoride.

“For example, for cells in a pH 5 environment, with a cytoplasmic pH maintained at 7, fluoride will accumulate intracellularly to a concentration 100-fold greater than that of the external fluoride.”

The smaller pH reductions during famine are because the limiting glucose concentration minimizes fermentation.

“because the limiting glucose concentration minimizes fermentation.”

The S. mutans CLCF is essential to the stability of the cariogenic dental biofilm under conditions of fluoride exposure.

“The experiments thus far suggest that the S. mutans CLCF is essential to the stability of the cariogenic dental biofilm under conditions of fluoride exposure.”

Knockout of the S. mutans fluoride exporter influences the properties of the entire biofilm under fluoride stress.

“Knockout of the S. mutans fluoride exporter influences the properties of the entire biofilm under fluoride stress.”

In cells, intracellular fluoride accumulation quantitatively follows the pH gradient.

“In these cells, intracellular fluoride accumulation quantitatively follows the pH gradient (20).”

• cites [20] Ji C, Stockbridge RB, Miller C. 2014. Bacterial fluoride resistance, Fluc channels, and the weak acid accumulation effect. J Gen Physiol 144: 257– 261. doi: 10.1085/jgp.201411243 25156118 PMC4144673 sourceNot checked yet

ORF 1289 contributes more toward the fluoride resistance phenotype than ORF 1290.

“These data suggest that ORF 1289 contributes more toward the fluoride resistance phenotype.”

In clinically relevant conditions, fluoride sensitivity of the biofilm's constituent species has downstream effects on several markers of dental caries.

“These results show that, in clinically relevant conditions, fluoride sensitivity of the biofilm’s constituent species has downstream effects on several markers of dental caries.”

• AssumptionRests on an assumption: The three-species biofilm model and hydroxyapatite disc conditions are clinically relevant to dental caries.

At moderate fluoride concentrations, bacteria themselves could act as fluoride reservoirs, slowly releasing fluoride into the biofilm over several hours as the population lyses, and exposing tooth enamel to protective fluoride after treatment.

“This implies that at moderate fluoride concentrations, the bacteria themselves could act as fluoride reservoirs, slowly releasing fluoride into the biofilm over several hours as the population lyses, and exposing the tooth enamel to protective fluoride for some time after fluoride treatment.”

At higher fluoride concentrations, cell lysis releases fluoride from the biofilm where it can be washed away.

“at higher concentrations, we observe cell lysis, which would release fluoride from the biofilm where it can be washed away.”

Synergy between S. mutans and C. albicans growth occurs in these biofilms.

“synergy between S. mutans and C. albicans growth was observed”

S. mutans contributes to dysbiosis by producing acid that eliminates species that are not tolerant to low pH.

“and further by producing acid that eliminates species that are not tolerant to low pH (50).”

• cites [50] Bradshaw DJ, Marsh PD. 1998. Analysis of pH-driven disruption of oral microbial communities in vitro. Caries Res 32: 456– 462. doi: 10.1159/000016487 9745120 sourceNot checked — no full text of the source

The fluoride retention in biofilms with ∆CLCF S. mutans mirrors intracellular fluoride accumulation measured in other microbes that lack fluoride exporters.

“This effect mirrors the intracellular fluoride accumulation that has been measured in other microbes that lack fluoride exporters (17, 20, 52).”

• cites [17] Baker JL, Sudarsan N, Weinberg Z, Roth A, Stockbridge RB, Breaker RR. 2012. Widespread genetic switches and toxicity resistance proteins for fluoride. Science 335: 233– 235. doi: 10.1126/science.1215063 22194412 PMC4140402 sourceCheck pending
• cites [20] Ji C, Stockbridge RB, Miller C. 2014. Bacterial fluoride resistance, Fluc channels, and the weak acid accumulation effect. J Gen Physiol 144: 257– 261. doi: 10.1085/jgp.201411243 25156118 PMC4144673 sourceNot checked yet
• cites [52] Calero P, Gurdo N, Nikel PI. 2022. Role of the CrcB transporter of Pseudomonas putida in the multi-level stress response elicited by mineral fluoride. Environ Microbiol 24: 5082– 5104. doi: 10.1111/1462-2920.16110 35726888 PMC9796867 sourceNot checked yet

Although biofilms are protective against many antimicrobial stressors, fluoride is inhibitory to a similar extent in a biofilm context.

“Although biofilms are protective against many antimicrobial stressors, fluoride is inhibitory to a similar extent in a biofilm context.”

In the E. casseliflavus protein, the T320A mutation inhibited transport.

“In the E. casseliflavus protein, the T320A mutation inhibited transport (26)”

• cites [26] Last NB, Stockbridge RB, Wilson AE, Shane T, Kolmakova-Partensky L, Koide A, Koide S, Miller C. 2018. A CLC-type F(-)/H(+) antiporter in ion-swapped conformations. Nat Struct Mol Biol 25: 601– 606. doi: 10.1038/s41594-018-0082-0 29941917 PMC6044475 sourceCheck pending

Even if 1290 is expressed, the resulting protein does not contribute to fluoride efflux.

“our functional studies suggest that even if 1290 is expressed, the resulting protein does not contribute to fluoride efflux.”

We report substantial changes in dental biofilm communities based on a single factor, fluoride export.

“In summary, we report substantial changes in dental biofilm communities based on a single factor, fluoride export (Fig. 7).”

Fluoride efflux inhibitors that specifically target the CLCF of S. mutans or the FEX channel of C. albicans could serve to reduce or eliminate cariogenic species while preserving commensal microbes in the dental biofilm.

“Our results imply that fluoride efflux inhibitors that specifically target the CLCF of S. mutans or the FEX channel of C. albicans could serve to reduce or eliminate cariogenic species while preserving commensal microbes in the dental biofilm”

• AssumptionRests on an assumption: Commensal microbes in the dental biofilm mainly export fluoride via the action of the molecularly distinct Fluc channels.
• AssumptionRests on an assumption: Humans lack known fluoride exporters, including CLCF or FEX proteins.