Functional Studies Of Acyl-CoA Binding Proteins In Toxoplasma Gondii

Toxoplasma gondii is an important parasitic protozoan.Lipid metabolism plays key roles in the growth,development and proliferation of T.gndii.In most cells,long-chain acyl-CoA esters are channeled towards different metabolic pathways via acyl-CoA binding protein and sterol carrier protein.Sequence alignment in database showed that T.gondii owns two acyl-CoA binding proteins,named as TgACBP1 and TgACBP2 respectively.In addition,T.gondii expresses a sterol carrier protein 2,named as TgSCP2.However,there still haven’t any reports about ACBP and SCP2 in T.gondii so far.Firstly,we showed that Toxoplasma expresses ACBP1 and ACBP2.Endogenous HA-tagged parasites showed that TgACBP1 is distributed throughout the cytoplasm,while there exists great difference of localization of ACBP2 in intracellular and extracellular parasites.TgACBP2 is localized to the mitochondria outer membrane and parasite periphery in intracellular and extracellular parasites respectively;ACBP2 is dephosphorylated and phosphorylated in intracellular and extracellular parasites respectively.High[K]and low[Ca2+]induced the dephosphorylation of ACBP2 in extracellular parasites.High[K+]induced translocation of TgACBP2 in extracellular parasites.Furthermore,recombinant TgACBP1 and TgACBP2 could bind to the fluorescent substrate NBD-C16:0-CoA,while yeast complementation assay confirmed that TgACBPl and TgACBP2 expression rescued the multi-lobed vacuole phenotype defects of ACBP-deficient yeast.These results demonstrated that TgACBPl and TgACBP2 are both able to bind to acyl-CoA esters in either vitro or yeasts.Based on TATi parasites,we constructed conditional knockout strain for TgACBP1,and found that under normal condition,loss of TgACBP1 did not affect phenotype of parasites,but led to enhanced incorporation of fatty acids from host cells.Loss of TgACBPl resulted in reduced ATP levels.In ddition,TgACBP1 knockdown led to increased TAG abundance,which was associated with increased numbers of lipid bodies.Under glucose-free condition,TgACBP1 disruption caused defects in replication and invasion.RT-PCR indicated that disruption of TgACBPl elevated the transcriptional level of TgSCP2.Loss of SCP2 caused no obvious phenotypic changes of parasites,while double knockout of TgACBP1 and TgSCP2 impaired intracellular growth,replication and pathogenicity to mice.Gas chromatgraphy coupled with mass spectrometrometry and isotope labeling technologies were used to detect 11 kinds of fatty acids in T.gondii.The results indicated that disruption of TgACBPl has no effects on fatty acid overall abundance and fatty acid synthesis.Loss of TgACBP1 or TgSCP2 resulted in reduced abundance of C18:1 but did not affect fatty acid synthesis.Double knockout of TgACBP1 and TgSCP2 reduced the synthesis rates of C18:0,C22:1 and C24:1.Also,the double disruption caused lessened abundance ofC18:1,C22:1 and C24:1,while other 8 kinds of fatty acids were not affected.Lipdomic analysis identified many different kinds of glyceride and phospholipid molecular species and showed that the double disruption caused reduced abundance of total and individual TAG,DAG,CDP-DAG,MAG,PC,PE,PI and Cl,while the double disruptionresulted in increased abundance of PS.These results show that ACBP1 and SCP are synergistically involved in the long chain fatty acid,glyceride and phospholipid metabolism.Based on RHAKu80 parasites,we disrupted ACBP2 and found that disruption of TgACBP2 in RHAKu80 parasites caused no obvious phenotype defects,while under high[K+]loss of TgACBP2 blockedreplication and pathogenicity of parasites to mice;Based on Pru parasites,we disrupted TgACBP2 and found that disruption of TgACBP2 blocked intracellular growth,replication and pathogenicity to mice.We complemented PruAACBP2 with ACBP2 mutants and found that ANK2 mutant rather than ACBD mutant could rescue the growth defect of Pru△ACBP2.By determining the mitochondrial changes in different parasites,we found that disruption of TgACBP2 did not impair the reactive oxygen species and inner membrane potential of RHAKu80 parasites,while under high[K+]the disruption of TgACBP2 in RHAKu80 parasites enhanced reactive oxygen species,reduced the inner membrane potential,casued Cytochrome C release,increased the apoptosis rate.However,loss of TgACBP2 in Pru parasites enhanced reactive oxygen species and reduced the inner membrane potential.Then we detected the mitochondrial specific phospholipid cardiolipin and identified totally 25 kinds of cardiolipins.We found that disruption of TgACBP2 reduced the abundance of 10 cardiolipins in Pru parasites,manifesting that TgACBP2 plays roles in cardiolipin metabolism.Expression of mitochondrial association factor 1 MAF1RHb1 in ACBP2-deficient Pru parasites recruited host mitochondria and thus rescued the defects of phenotype,apoptosis and mitochondrial inner membrane potential of ACBP2-deficient parasites.Lipidomic analysis showed that MAFIRHbl expression did not affect the abundance of fatty acids,PC,PE,PI,PS and MLCL,while MAF1RHb1 restored the abundance of PA,PG and CL in PruAACBP2.All the these results show that TgACBP2 plays a key role in the cardiolipin metabolism in Pru parasites.TgACBP1,TgACBP2 and TgSCP2 can all bind fatty acyl-CoA.TgACBP1 and TgSCP2 are synergistically involved in regulating long chain fatty acid,glyceride and phospholipidmetabolism.TgACBP2 participates in cardiolipin metabolism and maintaining mitochondrial functions.MAF1RHb1-mediated host mitochondrial association is closely related with the role of ACBP2 in cardiolipin metabolism.