PROJECT SUMMARY/ABSTRACT Mammals defend against infection by detecting foreign and/or danger signals to mount both cell-autonomous as well as paracrine immune responses. Among the microbial signatures sensed by mammalian hosts are a new class of signaling intermediates termed cyclic di-nucleotides (CDNs). CDNs are recognized by the host protein, STING (STimulator of INterferon Genes), to elicit type I IFN production. In this application we will characterize a new CDN recognition system that mobilizes protective immunity independently of STING or type I IFN release. This new circuit enlists four members of an immune GTPase family – the 65-73kDa Guanylate Binding Proteins (GBPs) – to activate distinct host defense programs in both humans and mice. Preliminary evidence implicates GBP1 as the proximal CDN sensor which recruits GBP2, GBP3 and GBP5 as adaptors that link the antimicrobial machinery further downstream. In Aim 1, we will test the importance of GBP1 to act as a broad-spectrum CDN sensor capable of binding c-diGMP, c-diAMP and cGAMPs under physiological conditions to elicit either inflammasome assembly, pyroptosis or autophagy in a STING-independent manner. This will be examined using a powerful host-pathogen genetic strategy where mice and cells lacking GBP1 or expressing GBP1 CDN-binding mutants will be infected with important bacterial pathogens (M. tuberculosis, S. typhimurium and L. monocytogenes) engineered to secrete a specific CDN moiety to define this relationship in vitro and in vivo. In Aim 2, we expand this CDN sensor circuit to include GBP2, GBP3 and GBP5 as heterotypic partners that direct core components of the inflammasome, pyroptotic or autophagy cascade to restrict bacterial replication. This aim will enlist newly-created Gbp2-/-, Gbp3-/- and Gbp5-/- mice as well as mixed sensor-adaptor chimeras as a novel binary approach to dissect how the GBP relay mobilizes different types of CDN-dependent immunity to bacterial pathogens. Collectively, our proposal examines a new set of host proteins that define a novel CDN sensor-adaptor circuit with important implications for developing host- directed therapies as an adjunct to standard antibiotic treatment.