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

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Diadenylate cyclase (DacA) synthesizes the crucial second messenger cyclic di-AMP (c-di-AMP) in many Gram-positive bacteria.
• The DacA interactome and its functional significance are not fully understood, particularly in Streptococcus mutans.

Purpose of the Study:

• To comprehensively map the DacA interactome in S. mutans.
• To elucidate the functional role of DacA interactors in regulating c-di-AMP signaling and bacterial physiology.
• To investigate the link between c-di-AMP homeostasis, calcium ion balance, and virulence.

Main Methods:

• Co-immunoprecipitation and mass spectrometry to identify DacA interactors.
• AlphaFold-guided modeling and split-luciferase complementation for interaction validation.
• Site-directed mutagenesis to pinpoint the interaction interface.
• Genetic deletion and phenotypic analysis of DacA and SMU_723 mutants.

Main Results:

• Identified 22 (non-crosslinked) and 18 (crosslinked) candidate DacA interactors, with six shared.
• Validated SMU_723, a putative calcium transporter, as a primary binding partner.
• Mutations in Thr147, Gln148, and Thr149 of SMU_723 disrupted DacA binding.
• Disruption of the DacA-SMU_723 interaction reduced intracellular c-di-AMP levels.
• SMU_723 deletion recapitulated some, but not all, phenotypes of DacA deletion, including altered growth, acid production, and virulence.

Conclusions:

• SMU_723 is a novel, functionally relevant interactor of DacA in S. mutans.
• The DacA-SMU_723 interaction modulates c-di-AMP levels and is critical for calcium response and bacterial survival.
• This interaction provides a mechanistic link between c-di-AMP signaling and calcium homeostasis, impacting virulence.
• Targeting this pathway offers potential therapeutic strategies against S. mutans infections.