Periodic Reporting for period 2 - MITOvTOXO (Understanding how mitochondria compete with Toxoplasma for nutrients to defend the host cell)
Période du rapport: 2021-12-01 au 2023-05-31
We understand little of the role of mitochondria, the energy producing organelles in our cells, in the cellular response to microbes. This translates into a lack of understanding of how human metabolism influences the progression of infectious disease.
The methodology we will develop and goals we will address for the study of mitochondria versus Toxoplasma nutrient competition will be applicable to the study of any microbe-organelle interaction in any cell type. The successful completion of the proposed research has the potential to not only advance our understanding of basic mitochondrial biology and the metabolic networks that determine host resistance or susceptibility to infection, but to also provide novel tools for the study of metabolism under these conditions and likely reveal novel strategies for the development of antimicrobial therapies.
The methodology we will develop and goals we will address for the study of mitochondria versus Toxoplasma nutrient competition will be applicable to the study of any microbe-organelle interaction in any cell type. The successful completion of the proposed research has the potential to not only advance our understanding of basic mitochondrial biology and the metabolic networks that determine host resistance or susceptibility to infection, but to also provide novel tools for the study of metabolism under these conditions and likely reveal novel strategies for the development of antimicrobial therapies.
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mechanism by which mitochondria enhance mitochondrial FAO—through the tethering of lipid droplets by MFN1 and MFN2 (unpublished data); b) shown that MFN1 and MFN2 restrict parasite use of host fatty acids for phospholipids important for membrane biogenesis (unpublished data); and c)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.
We are well on our way to accomplishing Aim 1. As mentioned previously, we have identified a potential mechanism by which MFN1/2 mediate fatty acid uptake during infection. Futhermore, we have optimized and implemented our mitochondrial isolation protocol and defined changes in the mitochodnria that occur during infection (Li et al., Science 2022). While we have generated the parasite strains needed for the experiments discussed in Aim 2, here we have made slower progress as our application for mouse work was delayed (the process is normally slow and the Corona situation exacerbated this) despite great support from our administration. However, we did received note that our protocol was finally approve so we foresee tackling experiments detailed in aim 2 starting next year. Aim 3 is also well underway, we have developed a method for the simultaneous purification of host mitochondria and the parasite vacuole membrane (Milestone 1.3) as well as have continued our efforts dissecting the contribution of mitochondria to cholesterol synthesis. Furthermore, we have identified another set of metabolites that mitochondria compete with Toxoplasma for, as well as the host mechanism that activates defensive mitochondrial metabolism.