Project Summary / Abstract Objectives: This application seeks to train a dual degree DVM/PhD student, enhancing the student's skills in research and clinical veterinary medicine. The project proposed in this application will prepare this student for a career as an independent clinician-scientist in translational medical fields. Specifically, the research proposal in this application will address critical knowledge gaps in the understanding of Mycobacterium tuberculosis (Mtb) mediated shifts in host immune cell metabolism. Knowledge gained through this proposal will have translational applications to other mycobacterial diseases of animals and humans. Specific Aims: 1. Determine if mycobactin activates HIF-1α in a hypoxia-independent manner during early Mtb infection. 2. Determine if blocking glycolysis and mitochondrial fatty acid oxidation alters Mtb survival, macrophage viability, and Mtb-macrophage metabolomic profiles during early infection. Research Design and Methods: 1. Using mycobactin knock-out strains of Mtb, HIF-1α morpholino oligomers, and custom Agilent Extracellular Flux Analyzer protocols, guinea pig bone marrow derived macrophages (GpBMDMs), will be assessed for the effect of mycobactin on HIF-1α activation and changes in cell metabolism. WB, qRT-PCR, and Agilent protocols will be employed. Guinea pigs infected with knock-out and control Mtb strains will be sampled for bronchoalveolar lavage and tissues (lung, spleen, lymph node) post- mortem to complement in vitro results and to be analyzed in the same manner. 2. Targeted metabolomics analysis of carbohydrates, fatty acid, and amino acid profiles for Mtb infected and uninfected GpBMDMs will be compared. Two key metabolic pathways, glycolysis and mitochondrial fatty acid oxidation, will be inhibited and the effect of altered metabolism on infection outcome will be characterized by pairing metabolomic analyses with macrophage viability (Trypan Blue exclusion assay) and Mtb survival (CFU) data. Relatedness to Public Health: Mtb is the leading cause of death by an infectious disease worldwide and remains a prominent global health concern. More effective alternative approaches are needed to combat tuberculosis in endemic areas. Host-directed therapies (HDTs), which aim to modulate the host immune and metabolic response rather than target the pathogen, are a promising new area of study. This proposal will contribute to the knowledge of mechanisms by which Mtb infection modulates host immune cell metabolism. Understanding these mechanisms can aid in determining targets for HDTs and provide knowledge for the development of novel therapeutic strategies.