Project Summary Bacterial endophthalmitis is a vision-threatening complication commonly occurring post penetrating eye injuries and ocular surgeries. Despite aggressive antibiotics and surgical interventions, endophthalmitis often results in partial or complete vision loss. The long-term goal of our research has been to study the pathobiology of endophthalmitis and identify potential therapeutic targets for treatment. In our recent study (PMID: 34095879), using transcriptomics and untargeted metabolomics, we identified several key pathways related to energy metabolism being perturbed during Staphylococcus aureus (SA) endophthalmitis. Among these pathways, we found that bacterial infection rapidly depletes the nicotinamide adenine dinucleotide (NAD+) pool in the mouse retina. NAD+ is not only crucial for oxidation-reduction reactions in the mitochondria, but metabolites of the NAD+ pathway also serve as substrates for various enzymes (e.g., PARPs, sirtuins, and CD38) to maintain cellular homeostasis. Thus, dysregulation in NAD+ metabolism has emerged as a contributing factor in the pathogenesis of several diseases. However, its role has not been investigated in ocular infections. Here, we propose that NAD+ depletion causes bioenergetics collapse, leading to the activation of receptor-interacting protein kinase-3 (RIPK3) mediated retinal cell death. In support, our preliminary data show disruption of the NAD+ synthesis via salvage pathway, increased activity of CD38 NADase, and the activation of RIPK3/MLKL signaling in SA-infected retina and cultured cells. Using a combination of mouse genetic tools, gene therapy, and pharmacological interventions, we will determine mechanisms of NAD+ depletion and restoration of salvage pathway (Aim 1), elucidate the crosstalk between CD38 NADase activity and RIPK3 in regulating retinal cell death (Aim 2), and test the hypothesis whether supplementation of NAD+ precursors can be used as an adjunct therapy to treat bacterial endophthalmitis (Aim 3). Collectively, the mechanistic insights on NAD+ dysregulation and NAD+ supplementation treatment strategies developed in this proposal could have a major impact in the field, not only with regards to ocular infections but other systemic infectious diseases as well.