Abstract
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 Streptococcus Sobrinus (S. sobrinus), 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 S. Sobrinus. S. sobrinus 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 SsNrtR regulator was identified as DNA-binding domain binding to the pnuC and pncB probe, and addition of ADP-ribose reversed the binding of SsNrtR to the target promoters to regulate NAD(+) salvage pathways. C-terminal domain of SsNrtR was non-catalytic, consistent with loss of Nudix motif conservation. Furthermore, the abrogation of niaR 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 SsNrtR-mediated regulation of NAD(+) homeostasis is a critical determinant of the cariogenic potential of S. sobrinus. 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.