Mitochondrial stress shapes host responses to bacterial infection Project Summary The mitochondrial network is a central hub for metabolism and sensing cellular stress, critical to shaping the immune response to infection. Cardiolipin (CL), an anionic phospholipid found in bacteria and in the inner mitochondrial membrane (IMM), anchors multi-protein respiratory chain complexes to the membrane and may also be deployed to nucleate immune supramolecular organizing centers such as the inflammasome. In conditions of stress, such as infection, cardiolipin is thought to translocate to the outer mitochondrial membrane (OMM) or the extracellular space. The regulatory steps that control CL translocation, remodeling and CL-dependent immune responses during bacterial infection are poorly understood. Mutations in the human CL remodeling enzyme, Tafazzin, result in an X-linked multi- system disorder known as Barth Syndrome, commonly associated with recurrent bacterial infections. The long-term goal of this proposal is to elucidate the mechanisms by which CL localization, modification and signaling enable mitochondrial control of the innate immune response to bacterial pathogens. Here we propose to test the hypothesis that CL translocation and modification regulates the switch between homeostatic and stress-responsive functions to drive innate immune responses to methicillin-resistant Staphylococcus aureus (MRSA) in vitro and in vivo. Specifically, we will perturb macrophage CL at four different steps: global biosynthesis, OMM-localization, oxidation and remodeling and define the signaling and effector mechanisms that are CL-dependent in the context of MRSA infection. Under these four experimental conditions, we will quantitatively track CL membrane localization and intracellular trafficking in macrophages using super resolution microscopy coupled with biochemical approaches. Finally, we will study the innate immune response to MRSA skin and soft tissue infection in mice genetically deficient in OMM-localized CL or enzymatically remodeled CL. We will also test the contribution of oxidized CL to in vivo antibacterial responses by treating MRSA-infected mice with a CL-targeted antioxidant. These studies will yield mechanistic insight into CL localization and remodeling during innate immune responses and potentially identify therapeutic targets for productively modulating inflammation. Additionally, this work will help contextualize CL-dependent innate immune signaling within the framework of a clinically relevant pathogen in vivo.