Exploring cyclic di-nucleotide signaling across the tree of life (Supplement)

This research program explores cyclic di-nucleotide signaling with a focus on elucidating mechanisms in Vibrio cholerae and translating those insights across biological systems. Using V. cholerae as a model, the laboratory has characterized the c-di-GMP signal transduction pathway in detail, demonstrating that two transcription factors central to biofilm formation and motility directly bind c-di-GMP. VpsR is activated by c-di-GMP binding to promote biofilm gene expression, while FlrA is inhibited, producing an opposing regulatory network in which c-di-GMP serves both co- and anti-activator roles. The lab developed the first in vitro transcription system showing c-di-GMP binding to VpsR drives open complex formation by RNA polymerase. Systematic analyses revealed high-specificity signaling: c-di-GMP synthesized by different diguanylate cyclases can differentially impact downstream phenotypes. Environmental cues such as bile and bicarbonate were identified as inverse regulators of c-di-GMP levels, suggesting these signals provide spatial information to V. cholerae within the human host. At the RNA level, the c-di-GMP-binding riboswitch Vc2 was shown to regulate motility by inhibiting a downstream transcription factor, TfoY, and to stabilize a novel cis-encoded sRNA by preventing 3' degradation, revealing a new mechanism of gene regulation.

Beyond classical roles in biofilm formation and motility, this work has expanded the repertoire of c-di-GMP-regulated phenotypes in V. cholerae to include Type II secretion, DNA repair, hydrogen peroxide stress responses, and even regulation of cell curvature. These phenotypes are coordinated by the VpsR/VpsT regulatory network, suggesting that many c-di-GMP-dependent traits contribute to fitness within biofilm communities.

A major discovery from this research is the identification of the first cyclic GMP-AMP (cGAMP) signaling pathway in bacteria. The enzyme DncV, encoded on a genomic island specific to the current pandemic El Tor lineage of V. cholerae, synthesizes cGAMP. The laboratory discovered that cGAMP activates a nearby putative phospholipase, named CapV, to remodel the bacterial membrane. The genomic colocation of dncV and capV implies a mobile functional cassette transferred by horizontal gene transfer, with implications for the evolution of pandemic V. cholerae strains. This work identified the first bacterial protein receptor for cGAMP.

Translational efforts leverage cyclic di-nucleotide biology to modulate the eukaryotic STING pathway. The team engineered non-replicating adenoviral vectors expressing V. cholerae diguanylate cyclases to produce c-di-GMP in vivo, demonstrating potent vaccine adjuvant activity and efficacy in multiple cancer animal models, where vectors delayed or resolved tumors. In parallel, the laboratory conducted high-throughput screens to identify small molecules that inhibit biofilm formation, discovering benzimidazole classes and the first chemical inhibitors of diguanylate cyclases. Collaborative work also showed that protonophores such as triclosan and oxyclozanide synergize with tobramycin to enhance killing of Pseudomonas aeruginosa biofilms by dissipating the proton motive force and inhibiting efflux. Together, these basic and translational studies map cyclic di-nucleotide signaling from molecular mechanisms to potential clinical applications in infection control, vaccine development and cancer immunotherapy.