PROJECT SUMMARY The proposed research plan focuses on determining the importance of sphingolipids in host-microbiome interactions and specifically whether sphingolipids function as one of the few examples of reciprocal interkingdom communication molecules. Sphingolipids are a dynamic class of signaling and structural molecules that are produced in all eukaryotes and a select few bacterial species that are prominent beneficial symbionts. The unique ability of select beneficial microbes and their hosts to produce and utilize sphingolipids suggests that sphingolipids could serve as important host-microbe communication molecules. We hypothesize that the transfer of sphingolipids between host and microbe influences phenotypes important for symbiosis. Host-supplied sphingolipids, whether taken in through the diet or produced by de novo synthesis, could serve to promote a beneficial microbial community in the gut, while contributions of bacterial sphingolipids could stimulate beneficial sphingolipid-dependent pathways in the host. To address these possibilities, we propose to characterize the function of sphingolipids in host-microbe interactions. We have developed mass spectrometry- based methods to measure both bacterial and host sphingolipids and certain associated metabolites. Moreover, we use precursors to sphingolipid synthesis that are modified with a terminal alkyne to metabolically label sphingolipids and trace their transfer between microbe and host. Application of these methods in diet-controlled and gnotobiotic mouse models of host-microbe metabolite exchange, allows us to define mechanisms of sphingolipid-dependent crosstalk. Over the next five years, the goals of the proposed research are to (Project 1) define how bacterially-derived sphingolipids are processed and utilized by the host, (Project 2) define the effect of bacterial sphingolipid synthesis on microbiome composition, and (Project 3) define how host-controlled contributions to the intestinal sphingolipid environment influence microbiota community structure and metabolism. The long-term objective of the project is to characterize host-microbe lipid exchange to an extent where we could manipulate this system for the benefit of host health.