Summary: Cyclic di‐AMP (c‐di‐AMP) is an emerging second messenger in bacteria. It has been shown to play important roles in bacterial fitness and virulence. However, transduction of c‐di‐AMP signaling in bacteria and the role of c‐di‐AMP in biofilm formation are not well understood. The level of c‐di‐AMP is modulated by activity of di‐adenylyl cyclase that produces c‐di‐AMP and phosphodiesterase (PDE) that degrades c‐di‐AMP. In this study, we determined that increased c‐di‐AMP levels by deletion of the pdeA gene coding for a PDE promoted biofilm formation in Streptococcus mutans. Deletion of pdeA upregulated expression of gtfB, the gene coding for a major glucan producing enzyme. Inactivation of gtfB blocked the increased biofilm by the pdeA mutant. Two c‐di‐ AMP binding proteins including CabPA (SMU_1562) and CabPB (SMU_1708) were identified. Interestingly, only CabPA deficiency inhibited both the increased biofilm formation and the upregulated expression of GtfB observed in the pdeA mutant. In addition, CabPA but not CabPB interacted with VicR, a known transcriptional factor that regulates expression of gtfB, suggesting that a signaling link between CabPA and GtfB through VicR. Increased biofilm by the pdeA deficiency also enhanced bacterial colonization of Drosophila in vivo. Taken together, our studies reveal a new role of c‐di‐AMP in mediating biofilm formation through a CabPA/VicR/GtfB signaling network in S. mutans.

Cyclic di-AMP (c-di-AMP) and cyclic di-GMP (c-di-GMP) are signaling molecules that play important roles in bacterial biology and pathogenesis. However, these nucleotides have not been explored inStreptococcus pneumoniae, an important bacterial pathogen. In this study, we characterized the c-di-AMP-associated genes ofS. pneumoniae. The results showed that SPD_1392 (DacA) is a diadenylate cyclase that converts ATP to c-di-AMP. Both SPD_2032 (Pde1) and SPD_1153 (Pde2), which belong to the DHH subfamily 1 proteins, displayed c-di-AMP phosphodiesterase activity. Pde1 cleaved c-di-AMP into phosphoadenylyl adenosine (pApA), whereas Pde2 directly hydrolyzed c-di-AMP into AMP. Additionally, Pde2, but not Pde1, degraded pApA into AMP. Our results also demonstrated that both Pde1 and Pde2 played roles in bacterial growth, resistance to UV treatment, and virulence in a mouse pneumonia model. These results indicate that c-di-AMP homeostasis is essential for pneumococcal biology and disease.

Cyclic di-AMP (c-di-AMP) has been shown to play important roles as a second messenger in bacterial physiology and infections. However, understanding of how the signal is transduced is still limited. Previously, we have characterized a diadenylate cyclase and two c-di-AMP phosphodiesterases in Streptococcus pneumoniae , a Gram-positive pathogen. In this study, we identified a c -di- A MP b inding p rotein (CabP) in S. pneumoniae using c-di-AMP affinity chromatography. We demonstrated that CabP specifically bound c-di-AMP and that this interaction could not be interrupted by competition with other nucleotides, including ATP, cAMP, AMP, phosphoadenylyl adenosine (pApA), and cyclic di-GMP (c-di-GMP). By using a bacterial two-hybrid system and genetic mutagenesis, we showed that CabP directly interacted with a potassium transporter (SPD_0076) and that both proteins were required for pneumococcal growth in media with low concentrations of potassium. Interestingly, the interaction between CabP and SPD_0076 and the efficiency of potassium uptake were impaired by elevated c-di-AMP in pneumococci. These results establish a direct c-di-AMP-mediated signaling pathway that regulates pneumococcal potassium uptake.

Little is known about cyclic AMP (cAMP) function in Mycobacterium tuberculosis , despite its ability to encode 15 adenylate cyclases and 10 cNMP-binding proteins. M. tuberculosis Rv3676, which we have designated CRP _Mt , is predicted to be a cAMP-dependent transcription factor. In this study, we characterized CRP _Mt 's interactions with DNA and cAMP, using experimental and computational approaches. We used Gibbs sampling to define a CRP _Mt DNA motif that resembles the cAMP receptor protein (CRP) binding motif model for Escherichia coli . CRP _Mt binding sites were identified in a total of 73 promoter regions regulating 114 genes in the M. tuberculosis genome, which are being explored as a regulon. Specific CRP _Mt binding caused DNA bending, and the substitution of highly conserved nucleotides in the binding site resulted in a complete loss of binding to CRP _Mt . cAMP enhanced CRP _Mt 's ability to bind DNA and caused allosteric alterations in CRP _Mt conformation. These results provide the first direct evidence for cAMP binding to a transcription factor in M. tuberculosis , suggesting a role for cAMP signal transduction in M. tuberculosis and implicating CRP _Mt as a cAMP-responsive global regulator.

Summary: Tuberculosis (TB) remains a major cause of morbidity and mortality worldwide. The pathogenesis by the causative agent, Mycobacterium tuberculosis, is still not fully understood. We have previously reported that M. tuberculosis Rv3586 (disA) encodes a diadenylate cyclase, which converts ATP to cyclic di‐AMP (c‐di‐AMP). In this study, we demonstrated that a protein encoded by Rv2837c (cnpB) possesses c‐di‐AMP phosphodiesterase activity and cleaves c‐di‐AMP exclusively to AMP. Our results showed that in M. tuberculosis, deletion of disA abolished bacterial c‐di‐AMP production, whereas deletion of cnpB significantly enhanced the bacterial c‐di‐AMP accumulation and secretion. The c‐di‐AMP levels in both mutants could be corrected by expressing the respective gene. We also found that macrophages infected with ΔcnpB secreted much higher levels of IFN‐β than those infected with the wild type (WT) or the complemented mutant. Interestingly, mice infected with M. tuberculosis ΔcnpB displayed significantly reduced inflammation, less bacterial burden in the lungs and spleens, and extended survival compared with those infected with the WT or the complemented mutant. These results indicate that deletion of cnpB results in attenuated virulence, which is correlated with elevated c‐di‐AMP levels.

Cyclic diguanosine monophosphate (c-di-GMP) and cyclic diadenosine monophosphate (c-di-AMP) are recently identified signaling molecules. c-di-GMP has been shown to play important roles in bacterial pathogenesis, whereas information about c-di-AMP remains very limited. Mycobacterium tuberculosis Rv3586 (DacA), which is an ortholog of Bacillus subtilis DisA, is a putative diadenylate cyclase. In this study, we determined the enzymatic activity of DacA in vitro using high-performance liquid chromatography (HPLC), mass spectrometry (MS) and thin layer chromatography (TLC). Our results showed that DacA was mainly a diadenylate cyclase, which resembles DisA. In addition, DacA also exhibited residual ATPase and ADPase in vitro. Among the potential substrates tested, DacA was able to utilize both ATP and ADP, but not AMP, pApA, c-di-AMP or GTP. By using gel filtration and analytical ultracentrifugation, we further demonstrated that DacA existed as an octamer, with the N-terminal domain contributing to tetramerization and the C-terminal domain providing additional dimerization. Both the N-terminal and the C-terminal domains were essential for the DacA's enzymatically active conformation. The diadenylate cyclase activity of DacA was dependent on divalent metal ions such as Mg²⁺, Mn²⁺ or Co²⁺. DacA was more active at a basic pH rather than at an acidic pH. The conserved RHR motif in DacA was essential for interacting with ATP, and mutation of this motif to AAA completely abolished DacA's diadenylate cyclase activity. These results provide the molecular basis for designating DacA as a diadenylate cyclase. Our future studies will explore the biological function of this enzyme in M. tuberculosis.