The fluoride channel genes crcB1 and crcB2 contribute to high-level fluoride resistance and virulence in Staphylococcus aureus

Staphylococcus aureus ( S. aureus ) can colonize the oral cavity and cause infections ranging from localized disease to systemic illness. Fluoride is widely used in oral care for caries prevention and can inhibit many oral microorganisms. However, S. aureus exhibits high-level fluoride resistance, and the molecular basis of this phenotype remains incompletely understood. This study investigated the roles of two adjacent predicted fluoride channel genes, crcB1 and crcB2 , in fluoride resistance and assessed the consequences of crcB locus deletion in S. aureus . Reverse transcription PCR indicated that crcB1 and crcB2 are co-transcribed. Both encoded proteins were predicted to contain four transmembrane helices and conserved sequence motifs characteristic of Fluc-family fluoride channels. Deletion of either crcB1 or crcB2 decreased the sodium fluoride minimum inhibitory concentration (MIC) from approximately 128 mM in the wild-type strain to approximately 4 mM, and the double mutant displayed further reduced fluoride resistance. Complementation of the double mutant with either gene alone did not restore high-level fluoride resistance, whereas co-complementation with crcB1 and crcB2 restored this phenotype. Under the same sodium fluoride exposure conditions, intracellular fluoride levels were highest in the Δ crcB1 Δ crcB2 mutant. Co-complementation with crcB1 and crcB2 significantly reduced intracellular fluoride accumulation to levels comparable to those in the wild type, supporting a role for the crcB locus in intracellular fluoride homeostasis. In the absence of fluoride, the double mutant showed a modest increase in membrane order and reduced fluorescence from a membrane potential sensitive probe. Proteomic analysis identified altered abundance of proteins involved in lipid metabolism, teichoic acid biosynthesis, membrane-associated functions, and virulence-related processes, including reduced FakA abundance. The double mutant also showed reduced hemolytic activity and extracellular alpha-toxin (a-toxin) accumulation, while producing less staphyloxanthin. Co-complementation with crcB1 and crcB2 restored the tested virulence-associated phenotypes, whereas ectopic fakA expression partially restored staphyloxanthin production, hemolytic activity, and extracellular a-toxin accumulation. In mice, infection with the double mutant resulted in lower mortality, fewer kidney abscesses, and less extensive histopathological changes than infection with the wild-type strain. crcB1 and crcB2 jointly contribute to high-level fluoride resistance and intracellular fluoride homeostasis in S. aureus . Deletion of the crcB locus altered virulence-associated phenotypes. These findings support further evaluation of the crcB locus as a potential target for fluoride sensitization and antivirulence strategies, although the underlying mechanisms require further investigation.