Construction of a genome-wide pooled CRISPRi library as a resource for exploring the acid tolerance mechanism in Streptococcus mutans

Streptococcus mutans is recognized as the primary etiological agent of dental caries, one of the most prevalent infectious diseases globally. Its remarkable acid tolerance enables survival and proliferation in the low-pH biofilm microenvironment, establishing S. mutans as the dominant species in dental plaque and a key contributor to cariogenesis. While numerous studies have identified genes associated with acid tolerance mechanisms, the full genomic complement of essential acid tolerance genes remains poorly characterized due to the absence of genome-scale investigative approaches. To address this knowledge gap, we constructed a genome-wide pooled CRISPR interference (CRISPRi) library targeting 95% of predicted S. mutans genes and employed next-generation sequencing to systematically identify acid tolerance determinants. Our screen revealed 95 acid tolerance-associated genes, a subset of which were functionally validated through gene knockout studies. Functional enrichment analysis demonstrated significant associations with metabolic pathways (including cofactor biosynthesis and amino/nucleotide sugar metabolism), tRNA modification, and transcriptional regulation. Protein-protein interaction (PPI) network analysis identified critical interactors (comYC, SMU_1979c, deoC, acpP, nadD, and SMU_1988c) and two functionally cohesive modules. These findings provide novel mechanistic insights into the acid adaptation strategies of S. mutans and highlight potential therapeutic targets for caries prevention.