c-di-AMP–DasR signaling axis mediates mycobacterial acid resistance

Pathogenic mycobacteria encounter acidic environments during host invasion, necessitating sophisticated acid resistance mechanisms. Here, we identify the GntR family regulator DasR as a conserved cyclic di-AMP (c-di-AMP) receptor in <i>Mycobacterium tuberculosis</i> that orchestrates acid adaptation through a multilayer network. Biochemical analyses demonstrated that DasR binds c-di-AMP with 20-fold higher affinity under acidic conditions than under neutral conditions, as evidenced by a Kd shift from 226 μM to 11.4 μM. This pH-sensitive binding aligns with acidified host niches during infection. ChIP-seq revealed that DasR directly targets nucleotide second-messenger metabolism genes, dynamically balancing intracellular pools of (p)ppGpp, cyclic AMP (cAMP), and c-di-AMP via positive feedback regulation. Concurrently, DasR upregulated the expression of the molecular chaperone HtpG, which stabilizes the DasR complex under acid stress. Functionally, c-di-AMP enhances DasR-DNA binding capacity at low pH, whereas HtpG-mediated thermostability amplifies signal output. This integrated axis coupling pH sensing, transcriptional reprogramming of stress metabolites, and chaperone reinforcement confers robust acid resistance. These findings establish the c-di-AMP-DasR pathway as an evolutionarily optimized strategy for mycobacterial persistence in hostile environments and suggest that this axis could be targeted to disrupt <i>M. tuberculosis</i> resilience.IMPORTANCEThe findings identified a regulatory axis central to mycobacterial acid adaptation, and DasR was found to be a conserved c-di-AMP receptor in <i>Mycobacterium tuberculosis</i>. A key feature is its highly pH-sensitive binding to c-di-AMP, which exhibits a 20-fold increase in affinity under acidic conditions, indicating that environmental cues are linked to the transcriptional response. DasR directly targets genes governing (p)ppGpp, cyclic AMP (cAMP), c-di-AMP metabolism, and acid adaptation, creating a feedback loop that dynamically balances stress signaling pathways. The concurrent upregulation of the chaperone HtpG stabilizes the DasR complex, increasing signal output under stress. This integrated system, which combines allosteric enhancement of DNA binding with chaperone-mediated stabilization, constitutes an evolutionarily refined strategy for acid resistance. The c-di-AMP-DasR pathway is therefore a promising target that could enable researchers to address the persistence of <i>M. tuberculosis</i>.