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

Microorganisms resist fluoride toxicity using fluoride export proteins from one of several different molecular families. Cariogenic species Streptococcus mutans and Candida albicans extrude intracellular fluoride using a CLCF F-/H+ antiporter and FEX fluoride channel, respectively, whereas oral commensal eubacteria, such as Streptococcus gordonii, export fluoride using a Fluc fluoride channel. In this work, we examine how genetic knockout of fluoride export impacts pathogen fitness in single-species and three-species dental biofilm models. 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. Biofilms prepared with C. albicans with genetic knockout of the FEX channel also exhibited reduced fitness in the presence of fluoride but to a lesser degree. 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. Biochemical purification of the S. mutans CLCF transporter and functional reconstitution establishes that the functional protein is a dimer encoded by a single gene. 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. IMPORTANCE: Dental caries is a globally prevalent condition that occurs when pathogenic species, including Streptococcus mutans and Candida albicans, outcompete beneficial species, such as Streptococcus gordonii, in the dental biofilm. Fluoride is routinely used in oral hygiene to prevent dental caries. Fluoride also has antimicrobial properties, although most microbes possess fluoride exporters to resist its toxicity. 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. 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. This is a novel strategy to treat dental caries.

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

Microorganisms resist fluoride toxicity using fluoride export proteins from one of several different molecular families. Cariogenic species Streptococcus mutans and Candida albicans extrude intracellular fluoride using a CLCF F-/H+ antiporter and FEX fluoride channel, respectively, whereas commensal eubacteria, such as Streptococcus gordonii, export fluoride using a Fluc fluoride channel. In this work, we examine how genetic knockout of fluoride export impacts pathogen fitness in single-species and three-species dental biofilm models. For biofilms generated using S. mutans with 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 commensal S. gordonii, and reduced properties associated with biofilm pathogenicity, including acid production and hydroxyapatite dissolution. Biofilms prepared with C. albicans with genetic knockout of the FEX channel also exhibited reduced fitness in the presence of fluoride, but to a lesser degree. 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, and biochemical purification the S. mutans CLCF transporter and functional reconstitution establishes that the functional protein is a dimer encoded by a single gene. 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.

Crystal structures of bacterial small multidrug resistance transporter EmrE in complex with structurally diverse substrates.

Proteins from the bacterial small multidrug resistance (SMR) family are proton-coupled exporters of diverse antiseptics and antimicrobials, including polyaromatic cations and quaternary ammonium compounds. The transport mechanism of the Escherichia coli transporter, EmrE, has been studied extensively, but a lack of high-resolution structural information has impeded a structural description of its molecular mechanism. Here, we apply a novel approach, multipurpose crystallization chaperones, to solve several structures of EmrE, including a 2.9 Å structure at low pH without substrate. We report five additional structures in complex with structurally diverse transported substrates, including quaternary phosphonium, quaternary ammonium, and planar polyaromatic compounds. These structures show that binding site tryptophan and glutamate residues adopt different rotamers to conform to disparate structures without requiring major rearrangements of the backbone structure. Structural and functional comparison to Gdx-Clo, an SMR protein that transports a much narrower spectrum of substrates, suggests that in EmrE, a relatively sparse hydrogen bond network among binding site residues permits increased sidechain flexibility.

The fluoride permeation pathway and anion recognition in Fluc family fluoride channels.

Fluc family fluoride channels protect microbes against ambient environmental fluoride by undermining the cytoplasmic accumulation of this toxic halide. These proteins are structurally idiosyncratic, and thus the permeation pathway and mechanism have no analogy in other known ion channels. Although fluoride-binding sites were identified in previous structural studies, it was not evident how these ions access aqueous solution, and the molecular determinants of anion recognition and selectivity have not been elucidated. Using x-ray crystallography, planar bilayer electrophysiology, and liposome-based assays, we identified additional binding sites along the permeation pathway. We used this information to develop an oriented system for planar lipid bilayer electrophysiology and observed anion block at one of these sites, revealing insights into the mechanism of anion recognition. We propose a permeation mechanism involving alternating occupancy of anion-binding sites that are fully assembled only as the substrate approaches.