Supplementary Material for: Biotin Limitation Attenuates Streptococcus mutans Cariogenicity by Disrupting Metabolic Flux and Virulence Pathways

Introduction: Our previous metagenomic analysis revealed higher frequencies of biotin-related genes (i.e., bioY, bioM, bccP) in caries-active children, indicating a potential role of biotin in caries pathogenesis. This study investigated a biotin limitation strategy against the primary cariogenic bacterium Streptococcus mutans (S. mutans), including its effects on cariogenic phenotypes, gene expression, and metabolomics.
Methods: S. mutans UA159 was cultured in biotin-free medium supplemented with different biotin concentrations. The cariogenic phenotypes of the strain, including growth kinetics, biofilm formation, exopolysaccharide (EPS) production, lactate synthesis, acid/oxidative tolerance, and membrane fluidity, were measured and compared across biotin concentrations. Biofilm architecture was visualized via confocal laser-scanning microscopy (CLSM) and scanning electron microscopy (SEM). Quantitative real-time polymerase chain reaction (RT-qPCR) was employed to analyze the expression of genes associated with virulence and biotin metabolism. Metabolomic analysis was performed to characterize metabolic perturbations induced by biotin limitation in S. mutans.
Results: Under biotin limitation, S. mutans exhibited significantly reduced cariogenic phenotypes, accompanied by cell elongation and reduced membrane fluidity. At the molecular level, biotin limitation suppressed the expression of key virulence-associated genes and induced a compensatory upregulation of genes involved in biotin uptake and biotin-dependent carboxylases. Metabolomic analysis under biotin-limited conditions in S. mutans revealed perturbed pathways in central carbon metabolism and nucleotide metabolism.
Conclusion: Biotin limitation significantly reduced the cariogenic potential of S. mutans by disrupting metabolic flux and virulence gene expression, highlighting biotin uptake and metabolism as potential targets for anti-caries therapies.