Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes – a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle.

Summary Toxoplasma gondii proliferates within host cell vacuoles where the parasite relies on host carbon and nutrients for replication. To assess how T. gondii utilizes these resources, we mapped the carbon metabolism pathways in intracellular and egressed parasite stages. We determined that intracellular T. gondii stages actively catabolize host glucose via a canonical, oxidative tricarboxylic acid (TCA) cycle, a mitochondrial pathway in which organic molecules are broken down to generate energy. These stages also catabolize glutamine via the TCA cycle and an unanticipated γ-aminobutyric acid (GABA) shunt, which generates GABA and additional molecules that enter the TCA cycle. Chemically inhibiting the TCA cycle completely prevents intracellular parasite replication. Parasites lacking the GABA shunt exhibit attenuated growth and are unable to sustain motility under nutrient-limited conditions, suggesting that GABA functions as a short-term energy reserve. Thus, T. gondii tachyzoites have metabolic flexibility that likely allows the parasite to infect diverse cell types.

Apicomplexan parasites undergo cell division using an evolutionarily conserved mechanism first described in the positioning and assembly of flagella in algae.

Toxoplasma gondii is an aerobic protozoan parasite that possesses mitochondrial antioxidant enzymes to safely dispose of oxygen radicals generated by cellular respiration and metabolism. As with most Apicomplexans, it also harbors a chloroplast-like organelle, the apicoplast, which hosts various biosynthetic pathways and requires antioxidant protection. Most apicoplast-resident proteins are encoded in the nuclear genome and are targeted to the organelle via a bipartite N-terminal targeting sequence. We show here that two antioxidant enzymes—a superoxide dismutase (TgSOD2) and a thioredoxin-dependent peroxidase (TgTPX1/2)—and an aconitase are dually targeted to both the apicoplast and the mitochondrion of T. gondii. In the case of TgSOD2, our results indicate that a single gene product is bimodally targeted due to an inconspicuous variation within the putative signal peptide of the organellar protein, which significantly alters its subcellular localization. Dual organellar targeting of proteins might occur frequently in Apicomplexans to serve important biological functions such as antioxidant protection and carbon metabolism.

The mitochondrial electron transport chain (mETC) and F1Fo-ATP synthase are of central importance for energy and metabolism in eukaryotic cells. The Apicomplexa, important pathogens of humans causing diseases such as toxoplasmosis and malaria, depend on their mETC in every known stage of their complicated life cycles. Here, using a complexome profiling proteomic approach, we have characterised the Toxoplasma mETC complexes and F1Fo-ATP synthase. We identified and assigned 60 proteins to complexes II, IV and F1Fo-ATP synthase of Toxoplasma, of which 16 have not been identified previously. Notably, our complexome profile elucidates the composition of the Toxoplasma complex III, the target of clinically used drugs such as atovaquone. We identified two new homologous subunits and two new parasite-specific subunits, one of which is broadly conserved in myzozoans. We demonstrate all four proteins are essential for complex III stability and parasite growth, and show their depletion leads to decreased mitochondrial potential, supporting their assignment as complex III subunits. Our study highlights the divergent subunit composition of the apicomplexan mETC and F1Fo-ATP synthase complexes and sets the stage for future structural and drug discovery studies.