Regulation of Second Messenger Synthesis in Mycobacterium tuberculosis

• Leif Boddie, Medical Diagnostics, University of Delaware

• Vijay Parashar, Medical and Molecular Sciences, University of Delaware

The Mycobacterium tuberculosis DNA integrity scanning protein A (MtbDisA) acts as a diadenylate cyclase (DAC) that synthesizes the vital bacterial second messenger, cyclic di-adenosine monophosphate (c-di-AMP). The interaction of the host’s immune system and c-di-AMP is key; when released from the bacterium into the macrophage cytosol, it activates STING (Stimulator of Interferon Genes) to trigger a robust immune cascade. However, MtbDisA’s DAC activity is strongly inhibited by ATP concentrations exceeding 1mM, which prevents a robust immune response. This study sought to determine if a T104M mutation, proposed through computational modeling, could alleviate this inhibition and sustain c-di-AMP production. While experimental testing was limited to the T104M residue due to time constraints, we also conducted a parallel computational analysis using multiple sequence alignment, the crystal structure of MtbDisA, and structural assessments via PDBePISA to identify additional residues that may be critical for ATP binding, structural stability, and catalytic function. This will lead to a large-scale mutagenesis that will be continued in the coming months. Ultimately, the combined experimental and computational insights from this project aim to inform genetic modifications of the M. bovis BCG TB vaccine to enhance the c-di-AMP-mediated immune response against M. tuberculosis.