Decoding the interactome for cyclic-di-AMP-producing enzyme diadenylate cyclase

In many gram-positive bacteria, the diadenylate cyclase DacA synthesizes the essential second messenger c-di-AMP. While regulators like GlmM and CdaR are known, the broader DacA interactome and its functional relevance remain poorly defined. Using Streptococcus mutans as a model, we identified novel protein-protein interactions using co-immunoprecipitation and mass spectrometry approaches. We identified 22 candidate interactors under non-crosslinked conditions and 18 under crosslinked conditions, with six shared candidates. Using AlphaFold-guided modeling and experimental split-luciferase complementation, we validated SMU_723—a putative calcium transporter—as a primary binding partner. Targeted mutagenesis identified threonine 147, glutamine 148, and threonine 149 as the key interacting interface; mutations in these residues impaired binding, confirming their structural importance. Disruption of the DacA–SMU_723 interaction resulted in a significant reduction in intracellular c-di-AMP levels, supporting a role for SMU_723 in modulating DacA enzymatic activity. Deletion of SMU_723 recapitulated several phenotypes observed in the ∆ dacA strain, including an extended lag phase, altered cell morphology, reduced acid production and acid tolerance, impaired sorbitol metabolism, decreased colonization in the Drosophila model, and delayed growth following calcium stimulation. However, ∆SMU_723 phenotypes were generally less severe than those of ∆ dacA , indicating partial functional overlap rather than a complete phenocopy. Together, this study identifies a functional link between SMU_723 and DacA that modulates c-di-AMP levels and calcium response and is vital for the survival of S. mutans . IMPORTANCE Mapping the DacA interactome reveals how environmental and intracellular cues tune c-di-AMP signaling to control stress adaptation, ion balance, and virulence traits in Streptococcus mutans . By identifying new DacA-associated proteins and validating SMU_723 as a previously unrecognized interactor with genetic and phenotypic linkage to DacA, this study provides new insights into the mechanistic framework for c-di-AMP regulation in S. mutans . The connection between DacA and a putative calcium transporter highlights a plausible axis that couples second-messenger signaling to calcium homeostasis, with implications for biofilm physiology and pathogenesis. These insights open new avenues to therapeutically modulate c-di-AMP pathways.