Regulation of NAD+ Homeostasis by SsNrtR in Streptococcus Sobrinus: A Critical Determinant of Its Cariogenic Potential

Nicotinamide adenine dinucleotide (NAD⁺) serves as a critical cofactor in redox reactions and metabolic transformations catalyzed by NAD-dependent enzymes and is essential for bacterial survival and virulence. The biosynthesis of NAD⁺ in the cariogenic pathogen <i>Streptococcus Sobrinus</i> (<i>S. sobrinus</i>), a pivotal participant in oral cavities of children and adolescents with a history of caries, has yet to be explored. Bioinformatics, genetics, and biochemical techniques were used to identify NAD⁺ biosynthesis pathways and corresponding regulator in <i>S. Sobrinus</i>. <i>S. sobrinus</i> lacks de novo NAD⁺ synthesis pathway but comprises NA and Nam salvage pathway I (PncA-PncB-NadD-NadE) and PnuC-NadR salvage pathway III. NiaY and PnuC were involved in the salvage pathways. N-terminal domain of <i>Ss</i>NrtR regulator was identified as DNA-binding domain binding to the <i>pnuC</i> and <i>pncB</i> probe, and addition of ADP-ribose reversed the binding of <i>Ss</i>NrtR to the target promoters to regulate NAD⁺ salvage pathways. C-terminal domain of <i>Ss</i>NrtR was non-catalytic, consistent with loss of Nudix motif conservation. Furthermore, the abrogation of <i>niaR</i> compromised multiple pathogenic traits, including cellular proliferation, acidogenesis, and the architecture/mechanical integrity of biofilms. Consequently, this mutant exhibited attenuated virulence in a rat caries model. Our findings conclusively demonstrate that <i>Ss</i>NrtR-mediated regulation of NAD⁺ homeostasis is a critical determinant of the cariogenic potential of <i>S. sobrinus</i>. This study identifies SsNrtR as a previously uncharacterized NAD⁺-responsive regulator that integrates metabolic homeostasis with the control of virulence in Streptococcus sobrinus. These findings elucidate a novel metabolic-virulence regulatory axis in this species and position SsNrtR as a promising target for the development of anti-caries interventions.