Project Summary Cellular metabolism instructs immune cell functions in both physiological and disease conditions. Activation of immune cells in response to invading pathogens induces a robust metabolic reprogramming, which has been indicated to play a critical role in orchestrating immune cell functions. The possibility of modulating immune cell function through metabolic regulation represents a new strategy to strengthen host defense mechanism. However, the role of individual metabolic pathways and enzymes during pathogen infection is not well understood, which represents a critical knowledge gap. Recently, we have identified the O-GlcNAc transferase (OGT), an important glucose metabolism enzyme, as a critical regulator of innate immune signaling during virus infection and septic inflammation. OGT mediates a ubiquitous post-translational modification (PTM) of cytosolic and nuclear proteins, known as O-GlcNAcylation. OGT-mediated O-GlcNAc signaling is well known as an essential regulator of many fundamental aspects of cell physiology and dysregulated O-GlcNAc signaling occurs in numerous human diseases. Despite a broad pathophysiological significance of OGT, the function of OGT in host defense mechanism against pathogen infection is just emerging and still underappreciated. Our preliminary results indicate that OGT directly restricts intracellular replication of both virus and bacteria, in addition to its previously established impacts on regulating innate immune signaling. By studying cell and mouse models with either OGT gene-deletion (Ogt-KO) or enzyme-inactive OGT (OgtK908A), we observed both enzyme-dependent and -independent functions of OGT in modulating innate immune signaling and limiting intracellular pathogen replication. We hypothesize that OGT serves as a central component of host defense mechanism by sensing pathogen infection and integrating innate immune signaling and cellular lipid metabolism via both enzyme activity-dependent and -independent manner. The long-term objective of our work is to determine the principles, functions and mechanisms of OGT and O-GlcNAc signaling in health and diseases. In this proposal, we aim to use our newly developed OGT enzyme-inactive mouse model and OGT gene-deletion model to understand the molecular mechanism by which OGT mediates host defense responses against invading virus and bacteria. We will advance this goal through three directions: 1) how does OGT sense virus infection and integrate innate immune response and cellular fatty acid synthesis pathway? 2) what is the functional importance of OGT-mediated protein O- GlcNAcylation in the detection of intracellular bacteria? and 3) how does the ubiquitination of OGT affect host defense? This study will significantly advance the field of immunometabolism by elucidating the function and mechanism of an important metabolic enzyme sensing and antagonizing pathogen infection. Successful development of OGT enzyme-inactive mouse model will also benefit...