Toxoplasma gondii is one of the most successful human parasites, infecting nearly one-third of the total world population. T. gondii tachyzoites residing within parasitophorous vacuoles (PVs) can acquire fatty acids both via salvage from host cells and via de novo synthesis pathways for membrane biogenesis. However, although fatty acid fluxes are known to exist in this parasite, how fatty acids flow through Toxoplasma lipid metabolic organelles, especially mitochondria, remains unknown. In this study, we demonstrated that Toxoplasma expresses an active ankyrin repeat containing protein TgACBP2 to coordinate cardiolipin metabolism. Specifically, HMA acquisition resulting from heterologous functional expression of MAF1 rescued growth and lipid metabolism defects in ACBP2-deficient type II parasites, manifesting the complementary role of host mitochondria in parasite cardiolipin metabolism. This work highlights the importance of TgACBP2 in parasite cardiolipin metabolism and provides evidence for metabolic association of host mitochondria with T. gondii.

Toxoplasma gondii relies on apicoplast‐localised FASII pathway and endoplasmic reticulum‐associated fatty acid elongation pathway for the synthesis of fatty acids, which flow through lipid metabolism mainly in the form of long‐chain acyl‐CoA (LCACoAs) esters. Functions of Toxoplasma acyl‐CoA transporters in lipid metabolism remain unclear. Here, we investigated the roles of acyl‐CoA‐binding protein (TgACBP1) and a sterol carrier protein‐2 (TgSCP2) as cytosolic acyl‐CoA transporters in lipid metabolism. The fluormetric binding assay and yeast complementation confirmed the acyl‐CoA binding activities of TgACBP1 and TgSCP2, respectively. Disruption of either TgACBP1 or TgSCP2 caused no obviously phenotypic changes, whereas double disruption resulted in defects in intracellular growth and virulence to mice. Gas chromatography coupled with mass spectrometry (GC–MS) results showed that TgACBP1 or TgSCP2 disruption alone led to decreased abundance of C18:1, whereas double disruption resulted in reduced abundance of C18:1, C22:1, and C24:1. 13C labelling assay combined with GC–MS showed that double disruption of TgACBP1 and TgSCP2 led to reduced synthesis rates of C18:0, C22:1, and C24:1. Furthermore, high performance liquid chromatography coupled with high resolution mass spectrometry (HPLC‐HRMS) was used for lipidomic analysis of parasites and indicated that loss of TgACBP1 and TgSCP2 caused serious defects in production of glycerides and phospholipids. Collectively, TgACBP1 and TgSCP2 play synergistic roles in lipid metabolism in T. gondii.

Few information of the function of stearoyl-coenzyme A (CoA) desaturase (SCD) in apicomplaxan parasite has been obtained. In this study, we retrieved a putative fatty acyl-CoA desaturase (TGGT1_238950) by a protein alignment with Plasmodium falciparum SCD in ToxoDB. A typical Δ9-desaturase domain was revealed in this protein. The putative desaturase was tagged with HA endogenously in Toxoplasma gondii, and the endoplasmic reticulum localization of the putative desaturase was revealed, which was consistent with the fatty acid desaturases in other organisms. Therefore, the TGGT1_238950 was designated T. gondii SCD. Based on CRISPR/Cas9 gene editing technology, SCD conditional knockout mutants in the T. gondii TATi strain were obtained. The growth in vitro and pathogenicity in mice of the mutants suggested that SCD might be dispensable for tachyzoite growth and proliferation. The SCD-overexpressing line was constructed to further explore SCD function. The portion of palmitoleic acid and oleic acid were increased in SCD-overexpressing parasites, compared with the RH parental strain, indicating that T. gondii indeed is competent for unsaturated fatty acid synthesis. The SCD-overexpressing tachyzoites propagated slower than the parental strain, with a decreased invasion capability and weaker pathogenicity in mice. The TgIF2α phosphorylation and the expression changes of several genes demonstrated that ER stress was triggered in the SCD-overexpressing parasites, which were more apt toward autophagy and apoptosis. The function of unsaturated fatty acid synthesis of TgSCD was consistent with our hypothesis. On the other hand, SCD might also be involved in tachyzoite autophagy and apoptosis.