3', 5'-cyclic diguanylic acid (c-di-GMP) is an intracellular bacterial second messenger that mediates the transition between a sessile, biofilm forming lifestyle to a motile, virulent lifestyle. Diguanylate cyclase (DGC) enzymes synthesize c-di-GMP, whereas c-di-GMP specific phosphodiesterase (PDE) enzymes hydrolyze the second messenger. Although numerous bacterial behaviors are regulated by c-di-GMP, the regulatory inputs of this system remain mostly undefined. Here, I examine how the marine bacterium and human pathogen Vibrio cholerae utilizes c-di-GMP signaling to interpret and respond to environmental cues. The central hypothesis that underpins my research is that V. cholerae senses environmental signals with DGCs and PDEs to modulate c-di-GMP concentrations in different environments. As c-di-GMP is a widely conserved second messenger utilized by many different bacteria, the mechanisms by which V. cholerae utilizes c-di-GMP in different environments can be applied to other bacterial systems to further comprehend how they behave in and adapt to various surroundings. To examine the influence of environmental factors on c-di-GMP synthesis and hydrolysis, I have developed a novel method named The Ex vivo Lysate c-di-GMP Assay (TELCA) that systematically measures total DGC and PDE cellular activity. I have shown that V. cholerae grown in different environments exhibits distinct intracellular levels of c-di-GMP, and using TELCA have determined that these differences correspond to changes in both c-di-GMP synthesis and hydrolysis. These findings highlight that modulation of both total DGC and PDE activity alters the intracellular concentration c-di-GMP.While searching for specific environmental cues that regulate c-di-GMP, I have found that bile acids, a prevalent constituent of the human proximal small intestine, increase intracellular c-di-GMP in V. cholerae. This bile-mediated increase in c-di-GMP is quenched by bicarbonate, the intestinal pH buffer secreted by intestinal epithelial cells. These findings lead me to propose that V. cholerae senses distinct microenvironments within the small intestine, using bile and bicarbonate as chemical cues, and responds by modulating intracellular c-di-GMP.In addition to its function as a bacterial second messenger, c-di-GMP has potent immunostimulatory properties in eukaryotes; these properties make c-di-GMP a prime candidate for use as a vaccine adjuvant. Here, I present a novel method of delivering c-di-GMP into eukaryotic cells using adenovirus. I have demonstrated that c-di-GMP can be synthesized in vivo by delivering DGC DNA into the cell, and that this c-di-GMP increases the secretion of numerous cytokines and chemokines. This novel adenovirus c-di-GMP delivery system offers a more efficient and cost-effective method to administer c-di-GMP as an adjuvant to stimulate innate immunity.
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