PROJECT SUMMARY/ABSTRACT Inflammasomes alert the mammalian immune system to the presence of infection and tissue damage. These cytosolic protein complexes detect danger signals or microbial products released by a wide variety of intracellular pathogens. In the case of bacterial pathogens, a number of prokaryotic signatures are recognized including the major cell-wall constituent of most Gram-negative species, lipopolysaccharide (LPS). Detection of LPS inside host cells activates a “non-canonical“ inflammasome pathway where caspase-11 (Caspases 4 and 5 in humans) act as upstream sensors to stimulate inflammasome complex assembly and processing of the pore-forming protein, Gasdermin D (Gsdmd), further downstream. Gsdmd pores release protective cytokines and contribute to a lytic form of cell death termed pyroptosis that may help eliminated infected host cells. How these sequential events are co-ordinated and the host factors involved remains a major question in the field of innate immunity and host defense. Here, we focus on members of a new 65-73kDa immune GTPase family termed Guanylate- Binding Proteins (GBPs) that control distinct steps in the non-canonical pathway. Preliminary results suggest Gbp2 may target cytosolic bacteria to help liberate LPS for caspase-11 detection whereas Gbp3 acts further downstream to regulate Gsdmd trafficking to the plasma membrane. GBPs thus offer a unique opportunity to understand how this sequential hierarchy unfolds. In Aim 1, we will test the respective contributions of Gbp2 and Gbp3 to immunity against Gram-negative Salmonella typhimurium (Stm) infection via the non-canonical inflammasome in vitro and in vivo. CRISPR-Cas9 deleted human and mouse cells as well as newly-created Gbp2-/-, Gbp3-/- and GbpDchr.3H1 mice will be infected with Stm variants designed to interfere with GBP recruitment or responsiveness to LPS. Thereafter, we will dissect the molecular mechanisms enlisted by these GBPs to confer their intracellular functions as part of Aim 2. Here gene-deficient macrophages complemented with GBP mutants with distinct biochemical lesions will reveal how GBPs direct the inflammasome core machinery to LPS- positive bacteria or control downstream events such as Gsdmd trafficking and assembly on the plasma membrane. Cell-free studies will also attempt to reconstitute the GBP “coatomer” on the bacterial outer membrane that serves as a platform for inflammasome assembly. Collectively, our proposal examines a new set of host factors that act at different stages within the non-canonical signaling cascade as part of a unique functional hierarchy, helping choreograph these events with major implications for the treatment of sepsis and Gram-negative bacterial infections.