Abstract Innate immunity is an ancient defense response that evolved with the earliest metazoan creatures to protect against microbial infection. These responses rely on the immediate recognition of microbes by germline-encoded receptors, and drive the production of numerous chemical, biological, and cellular responses to defend against infection. In the face of constant microbial assault, innate immunity is essential for the survival of nearly all multicellular organisms. On the other hand, over-exuberant or inappropriate innate immune responses are the underlying cause of morbidity and mortality associated with many infectious, autoimmune, and autoinflammatory diseases. Thus, a thorough mechanistic understanding of innate immunity has many potential applications in the development of the next generation of therapeutics. This proposal exploits the fruit fly Drosophila melanogaster as a model for the study of innate immunity. Flies offer many advantages for the study of innate immunity, including experimental tractability and a model system without the complexity of adaptive immunity. The Drosophila immune response is also an excellent model for vector insect species, and discoveries made in flies are being translated into new approaches to control vector-borne diseases. Furthermore, many aspects of the Drosophila innate immune responses are highly conserved with mammals, and this conservation has lead to paradigm shifting discoveries of Toll receptors and NF-κB transcription factors. In the current cycle of this project, we have uncovered another conserved aspect of innate immunity, implicating the SLC46 family of solute carriers in the delivery of peptidoglycan fragments (muropeptides) to cytosolic innate immune receptors, in flies and mammals. In Drosophila, microbial infections are recognized by two distinct NF-κB signaling pathways, the Toll and immune deficiency (Imd) pathways. The Imd pathway is triggered by DAP-type peptidoglycan from the cell wall of certain bacteria, which binds and activates cell surface or the cytosolic receptors PGRP-LC or PGRP- LE, respectively, and activates a NF-κB signal transduction system with significant similarity to the TNFR and TLR/TRIF-dependent pathways in mammals. The long-term objective of this project is to understand in molecular detail the mechanisms used by the Imd pathway to trigger effective immune responses, and to translate key discoveries, made in this invertebrate model system, to mammals. In this proposal, we build on past discoveries and move in important new directions, with two specific aims exploiting the Drosophila model, and the third aim translating our recent discoveries to mammals. Aim1 investigates the expanded SLC46 family of muropeptide transporters in flies, while Aim 2 is focused on the molecular and biochemical mechanisms controlling signal transduction in the Drosophila Imd pathway. Aim 3 utilizes Slc46a2-/- mice to elucidate the function of this transporter in the NOD1 path...