PROJECT SUMMARY The constant arms race between viruses and their hosts lead to the evolution of antiviral defense. Recent studies have revealed that bacteria developed diverse anti-phage defense systems that can be denoted as the “prokaryotic immune systems”. The well-characterized prokaryotic immune systems include restriction- modification (R-M) systems, CRISPR-Cas systems, abortive infection systems, and prokaryotic Argonautes. Mechanistic characterization of these systems has revolutionized biomedical research. For example, R-M systems provide restriction enzymes as invaluable tools for molecular cloning and genetic engineering while CRISPR-Cas has revolutionized the genome-editing field. In this proposal, we will focus on characterizing a novel bacteria immune system with unknown mechanisms. In this system, we have two components: A and B. A is predicted to have an ATPase domain, while B is predicted to be a nuclease. Our preliminary study showed that A and B can form a stable and large complex for anti-phage defense. This finding is reminiscent of inflammasomes, which are large immune complexes that are assembled via the ATPase domain in response to pathogen infection in higher organisms. We hypothesized that A and B also form large oligomers to combat phage infection. We will use cutting-edge cryogenic electron microscopy single-particle analysis techniques to define the structure and assembly mechanisms of the A/B complex and develop biochemical and cellular assays to characterize the functions of the A/B complex. Our studies will provide a mechanistic understanding of the assembly and functions of this novel inflammasome-like system in bacteria, which will shed light on the evolution of inflammasome signaling as well as provide a basis to develop novel tools for biomedical research and disease diagnosis and treatment.