New Insights into cyclic GMP-AMP Signaling in Innate Immunity

Abstract

Second messengers are small molecules that orchestrate cellular changes in the face of altered environmental conditions. Signal transduction is mediated through the action of protein receptors whose function is modulated by second messenger binding, resulting in altered transcriptional, translational, and post-translational processes. Over the past decade, cyclic dinucleotides (CDNs) have emerged as ubiquitous second messengers in all three domains of life, where they regulate a myriad of physiological processes. Among these roles, cyclic dinucleotide signaling systems display evolutionarily conserved roles in the control of pathogen infection in both prokaryotes and eukaryotes. In response to pathogen infection, cGAS/DncV-like nucleotidyltransferases synthesize diverse cyclic oligonucleotide products which activate effector proteins to terminate the infection. Of these effectors, the receptor STING has emerged as a conserved effector protein in both prokaryotic and eukaryotic immune systems. In prokaryotes, STING domains are found fused to NAD+ hydrolyzing TIR domains as well as putative transmembrane domains (TM). Upon engaging CDNs, STING-TIR depletes cellular NAD+ stores resulting in loss of viability of the infected cell and termination of bacteriophage infection. In eukaryotes, STING domains contain an N-terminal transmembrane domain required for ER localization and a loosely structured C-terminal tail required for downstream signaling. Upon engaging CDNs, metazoan STING homologs trigger autophagy and downstream innate immune signaling programs to sterilize the cytosol of the infected host cell and to restrict further pathogen replication and spread. In addition to controlling pathogen infection, STING signaling systems also play key roles in restricting tumor development as well as in the pathogenesis of acquired and hereditary autoinflammatory diseases. For these reasons, there is immense interest in designing novel therapeutics that target the cGAS-STING pathway for use as immune adjuvants and treatments for various inflammatory disease processes. In this work, we leverage the latest advances in CRISPR-interference technology to identify the first metazoan transporter of cyclic dinucleotides. Next, we develop the first FRET-based biosensor for the cyclic dinucleotide 2’3’-cGAMP enabling the real time, in vitro and in vivo detection of cGAMP signaling. Finally, we develop a high throughput quantification assay for the bacterial cyclic dinucleotide 3’3’-c-di-AMP and leverage this technology to quantify the bacterial immune signaling molecule 3’3’-cGAMP. In total, the biological process uncovered in this work have important implications for cancer immunotherapy, vaccine development, host responses to pathogen infection, and the pathogenesis of autoimmune diseases. Moreover, the technologies outlined in this work will enable previously intractable questions in the fields of prokaryotic and eukaryotic innate immune signaling and will likely facilitate fundamental discoveries in CDN biology for years to come.