The aims of MITOvTOXO are to: 1) define the mechanism by which mitochondria enhance fatty acid oxidation to defend the cell against fatty acid siphoning by microbes; 2) determine whether mitochondrial fatty acid oxidation can be exploited to restrict microbial growth in vivo; and 3) determine whether mitochondria play a broader role in cellular defence by sequestering other essential metabolites from microbes. In addition, we seek to ask whether the mechanisms we identify also regulate cellular metabolic homeostasis independently of infection. In this reporting period we have made significant progress in aim 1, 2, and 3. Regarding aim 1, we have: a) defined a potential mechanism by which mitochondria enhance mitochondrial FAO—through the tethering of lipid droplets by MFN1 and MFN2 (unpublished data); b) shown that MFN1 and MFN2 restrict parasite use of host fatty acids for phospholipids important for membrane biogenesis (unpublished data); and c) discovered that mitochondria shed their outer membrane in response to parasite-induced and infection-independent import stress (Li et al, Science, 2022). Regarding progress in aim 2: our application has been accepted by the LANUV and we have dedicated significant efforts to establishing our in vivo infection protocols. Regarding aim 3, our identification of a role for mitochondria in regulating cholesterol homeostasis drove our efforts to understand the molecular mechanism by which they do so. In work in preparation, we have pinpointed the mechanism by which loss of MFN2 drives cholesterol synthesis and identified key metabolites derived/utilized by mitochondria that are required for the expression of the key enzymes in cholesterol synthesis.