The innate immune system detects infection via germ-line encoded receptors that recognize specific microbial ligands. We have been particularly interested in characterizing the innate immune response to unique nucleic acids produced by bacteria called cyclic dinucleotides. These nucleotides, which include c-di-GMP and c-di- AMP, are best known as key regulators of bacterial physiology. However, since they are not produced by mammalian cells, cyclic dinucleotides are ideal targets for innate immune recognition. Indeed, we found that the presence of cyclic dinucleotides in the host cell cytosol induces an innate immune response characterized by the production of important cytokines called type I interferons (IFNs). We recently found that the host protein STING is a direct innate immune sensor of cyclic dinucleotides. STING binds to cyclic dinucleotides in the cytosol and is essential for subsequent induction of a host type I interferon response. Interestingly, however, it has also been shown that STING is essential for induction of the host interferon response to cytosolic DNA. Since STING is required for the IFN response to both DNA and cyclic dinucleotides, the field now faces a significant problem, namely, there is no way to determine in vivo whether interferon induction by any given bacterial pathogen is due to cytosolic recognition of DNA or cyclic dinucleotides. This is a critical issue since virtually all bacterial pathogens ar capable of producing both stimulatory ligands. To address this problem we identified a specific point mutation in STING (R231A) that selectively abolishes the IFN response to cyclic dinucleotides without affecting the cytosolic response to DNA. This mutant provides a unique tool for distinguishing the responses to DNA versus cyclic dinucleotides. In this application we propose to leverage our findings to pursue two specific aims: (1) Generate """"""""knock-in"""""""" mice in which the gene encoding wild-type STING is replaced by a gene encoding a STING R231A mutant (2) Validate and test STING R231A knock-in mice using various inducers and infection models. We plan to distribute the STING R231A knock-in mice without restriction to investigators that request them. Our hope is that these mice will represent a unique and powerful reagent for the field and allow investigators to determine for any pathogen of interest whether induction of the host IFN response is via DNA or cyclic dinucleotides.
Infectious diseases remain a major cause of global mortality and morbidity. The design of novel immunotherapeutics, adjuvants and vaccines is predicated on a better understanding of how the innate immune system detects pathogens and initiates protective responses. Our proposal aims to fulfill this goal by dissection of the molecular mechanisms by which intracellular bacterial pathogens are sensed by the immune system.
|Kranzusch, Philip J; Lee, Amy S Y; Wilson, Stephen C et al. (2014) Structure-guided reprogramming of human cGAS dinucleotide linkage specificity. Cell 158:1011-1021|
|Tenthorey, Jeannette L; Kofoed, Eric M; Daugherty, Matthew D et al. (2014) Molecular basis for specific recognition of bacterial ligands by NAIP/NLRC4 inflammasomes. Mol Cell 54:17-29|
|Barker, Jeffrey R; Koestler, Benjamin J; Carpenter, Victoria K et al. (2013) STING-dependent recognition of cyclic di-AMP mediates type I interferon responses during Chlamydia trachomatis infection. MBio 4:e00018-13|
|Conlon, Joseph; Burdette, Dara L; Sharma, Shruti et al. (2013) Mouse, but not human STING, binds and signals in response to the vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid. J Immunol 190:5216-25|
|Diner, Elie J; Burdette, Dara L; Wilson, Stephen C et al. (2013) The innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING. Cell Rep 3:1355-61|