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

Abstract

AbstractMicroorganisms resist fluoride toxicity using fluoride export proteins from one of several different molecular families. Cariogenic speciesStreptococcus mutansandCandida albicansextrude intracellular fluoride using a CLCFF−/H+antiporter and FEX fluoride channel, respectively, whereas commensal eubacteria, such asStreptococcus 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 usingS. mutanswith genetic knockout of the CLCFtransporter, exposure to low fluoride concentrations decreasedS. mutanscounts, synergistically reduced the populations ofC. albicans, increased the relative proportion of commensalS. gordonii, and reduced properties associated with biofilm pathogenicity, including acid production and hydroxyapatite dissolution. Biofilms prepared withC. albicanswith genetic knockout of the FEX channel also exhibited reduced fitness in the presence of fluoride, but to a lesser degree. Imaging studies indicate thatS. mutansis 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 theS. mutansCLCFtransporter 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 thatS. mutansis especially susceptible to fluoride toxicity.ImportanceDental caries is a globally prevalent condition that occurs when pathogenic species, includingStreptococcus mutansandCandida albicans, outcompete beneficial species, such asStreptococcus 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 speciesStreptococcus mutansandCandida albicansto 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 toothpastes and mouth rinses. This is a novel strategy to treat dental caries.