Our lab has a long-standing interest in the biology of the mitochondrial and apicoplast organelles of apicompexan parasites.
The mitochondrion of apicomplexans has a range of canonical functions, including in the production of cellular energy (ATP), and the biosynthesis of important iron-containing protein co-factors such as haem and iron-sulfur clusters. There are, however, notable differences that set apicomplexan mitochondria apart from those in other eukaryotes, including some of the enzymes used to generate energy and the presence of additional metabolic pathways not found in typical mitochondria.
The apicoplast is a chloroplast-derived organelle that is no longer photosynthetic. The apicoplast has, however, retained numerous biosynthetic pathways that are essential for parasite survival. These 'plant-like' pathways are ideal drug targets, since equivalent pathways are absent from the host organisms that these parasites infect.
Our lab is particularly interested in the functions, biogenesis and metabolism of the apicoplast and mitochondrion.
- Organellar functions. We are particularly interested in novel functions of the mitochondria and apicoplasts of apicomplexans. Using proteomic approaches, we have identified 100's of organelle-localised proteins that have no predicted function. We aim to identify the functions of essential organellar proteins.
- Integration of organellar metabolism with cellular metabolism. Apicoplasts and mitochondria are bound by multiple membranes that segregate the metabolism of these organelles from the metabolism of the rest of the parasite. These organelles must harbour solute transporters that transport the substrates of organellar metabolism into the organelle, and solute transporters that transport products of organellar metabolism out of the organelle. Very little is known about the identity and function of these organellar transporters. We aim to identify and characterise organellar transport proteins, thereby understanding how the metabolism of these organelles is linked to that of the rest of the parasite.
- Organellar division. Apicoplasts and mitochondria can only form from pre-existing apicoplasts and mitochondria. This means that these organelles must divide and segregate into daughter cells upon cell division. We aim to identify key proteins that function in division and maintenance of these organelles.
- Protein targeting. The vast majority of proteins required in these organelles are encoded on nuclear genes, and must be post-translationally targeted across the multiple membranes surrounding these organelles. We are interested in identifying how nuclear-encoded proteins are targeted to apicoplast and mitochondrion.
Our approach in addressing these questions is:
- Identify candidate proteins that may have a role in organellar metabolism, solute transport, division or protein trafficking;
- Generate genetic mutants in candidate proteins, using the various inducible systems available in Toxoplasma research;
- Develop microscopic, biochemical and other assays to measure the effect of losing a particular protein on organellar functions. We do this through a range of techniques, including fluorescence and time-lapse microscopy, pulse-chase protein labelling analyses to measure protein targeting, heterologous expression systems to characterise transporters, and blue-native gel electrophoresis and co-immunoprecipiation experiments to measure protein-protein interactions;
- Determine whether the protein-of-interest is required for parasite survival. We have several ways to measure parasite growth, such as by introducing a super-bright fluorescent protein into our mutant parasites and measuring growth over time as a function of fluorescence intensity in a population of parasites;
- Where appropriate, to use the mutants we have generated to learn more about the function of the protein-of-interest. For example, we can attempt to complement the mutants we generate with a second copy of the protein-of-interest that is missing domains or residues that we hypothesise may be important for its function.
Recent papers from this project that summarise the sorts of questions we are interested in:
- van Dooren GG, Yeoh LM, Striepen B, McFadden GI (2016) The import of proteins into the mitochondrion of Toxoplasma gondii J Biol Chem. in press
- van Dooren GG, Striepen B (2013) The algal past and parasite present of the apicoplast. Annu Rev Microbiol 67: 271-289
- Glaser S*, van Dooren GG*, Agrawal S, Brooks CF, McFadden GI, Striepen B, Higgins MK (2012) Tic22 is an essential chaperone required for protein import into the apicoplast. J Biol Chem 287(47): 39505-39512. *these authors contributed equally
- Brooks CF*, Johnsen H*, van Dooren GG*, Muthalagi M, Lin SS, Bohne W, Fischer K and Striepen B (2010) The Toxoplasma apicoplast phosphate translocator links cytosolic and apicoplast metabolism and is essential for parasite survival. Cell Host Microbe 7(1): 62-73. *these authors contributed equally
- van Dooren GG, Reiff SB, Tomova C, Meissner M, Humbel BM, Striepen B (2009) A novel dynamin-related protein has been recruited for apicoplast fission in Toxoplasma gondii. Curr Biol 19(4): 267-276. Cover Image
- van Dooren GG, Tomova C, Agrawal S, Humbel BM, Striepen B (2008) Toxoplasma gondii Tic20 is essential for apicoplast protein import. Proc Natl Acad Sci U S A 105(36): 13574-13579.