Biological Function Of De Novo Synthesis Of Saturated Fatty Acids And Phosphatidylcholine In Toxoplasma Gondii

Toxoplasma gondii is an apicomplexan protozoan and can infect almost all warmblooded animals.To adapt to different parasitic environments,Toxoplasma has evolved a complicated and flexible metabolic network.Finding key nodes and catalytic enzymes in metabolic networks can provide theoretical support for antiparasitic drugs and vaccines design.Lipids are important metabolites that participate in life activities as components of cell membranes,energy storage forms and signaling molecules.As an intracellular parasitic protozoon,Toxoplasma gondii can not only salvage exogenous lipids and their precursors,but also de novo synthesize lipid,but the roles of different lipid synthesis or uptake pathways in different growth and development stage of Toxoplasma gondii remain unaddressed completely.Based on the key catalytic enzymes of fatty acid and phospholipid synthesis pathways,we used reverse genetics,metabolomics,cell biology and other technologies to explore the biological significance of de novo synthesis of saturated fatty acids and phosphatidylcholine in Toxoplasma gondii in this study.The main research content include:(1)The biological role of saturated fatty acids de novo synthesis pathwayExisting studies have shown that apicoplast is a non-photosynthetic organelle shared by most apicomplexans.The fatty acid type 2 synthesis pathway(FAS2)in the apicoplast of Toxoplasma gondii utilizes acetyl-CoA to catalyze the de novo synthesis of saturated fatty acids.The pyruvate dehydrogenase(PDH)of Toxoplasma gondii located in the apicoplast and may convert pyruvate to acetyl CoA to participate in the FAS2 pathway.Therefore,this study took PDH as an entry point to explore the physiological significance of FAS2 pathway to the growth and development of tachyzoites.PDH is a complex composed of multiple subunits.After knocking out different subunits of PDH in Toxoplasma,it showed that the depletion of PDH resulted in growth inhibition of the mutant,accompanied by the loss of apicoplast.When mice were used to infected with PDHdeficient strain,the virulence level of the mutant strain was basically equivalent to that of the wild-type strain.Using[13C6]-glucose as a marker,intracellularly and extracellularly parasite was labeled respectively.By detecting the 13C labeling level of sugar and lipid metabolites,it was found that the deletion of PDH leads to a reduced 13C labelled level of the FAS2 pathway main products,myristic acid and palmitic acid.The growth defects of PDH-deficient strains were significantly restored in the presence of myristic acid or palmitic acid.The above studies show that Toxoplasma can not only synthesize fatty acids by itself,but also obtain fatty acids from exogenous sources to meet its growth needs.Toxoplasma gondii fatty acid metabolism is flexible,and de novo saturated fatty acid synthesis pathway is not necessary for the tachyzoite stage.(2)Biological role of de novo phosphatidylcholine synthesis pathwayThe sources of fatty acids in Toxoplasma gondii are diverse.Fatty acids seldomly exist freely in parasite.Most of fatty acids,whether de novo synthesized or exogenous salvaged,are normally used to synthesize complex lipids such as phospholipids.Existing studies have shown that Toxoplasma gondii has catalytic enzymes and metabolic pathways that involved in de novo phospholipid synthesis,but whether Toxoplasma gondii phospholipid anabolism is flexible,and the biological significance of phospholipid synthesis still needs to be further explored.Tachyzoites are known to mainly contain 8 phospholipids including phosphatidylcholine(PtdCho),phosphatidylethanolamine(PtdEtn),phosphatidylthreonine(PtdThr),phosphatidylinositol(PtdIns),phosphatidylserine(PtdSer),phosphatidylglycerol(PtdGro),sphingomyelin(SM)and phosphoethanolamine ceramide.Among them,the content of PtdCho is the largest,accounting for about 80%of the total phospholipid.To locate the pathway and determine the biological significance of PtdCho de novo synthesis pathway in Toxoplasma gondii.We found two choline/ethanolamine phosphotransferases(TgCEPT1 and TgCEPT2).By insertional tagging of the endogenous genes,we showed that TgCEPT1 located in the endoplasmic reticulum and TgCEPT2 located in Golgi apparatus.After knocking them out respectively,it showed that deletion of TgCEPT1(Δceptl)resulted in severe growth inhibition,while deletion of TgCEPT2(Δcept2)barely have effect on the growth of tachyzoites.Lipidomic study showed that the deletion of TgCEPT1 leads to the inhibition of PtdCho synthesis,the content of more than 90%of PtdCho species decreased,and the total amount of PtdCho was about half of the wild type;while deletion of TgCEPT2 affected the synthesis of C28 and C30 PtdCho,and the total PtdCho content remained unchanged.In addition,TgCEPTl and TgCEPT2 are also partly involved in the synthesis of PtdEtn.Deletion of TgCEPT2 affects the synthesis of C36,C38,and C40 PtdEtn,and the total PtdEtn content decreases by about 10%,while it is not statistic significant;deletion of TgCEPT1 affects the synthesis of C34 PtdEtn.It implied that both TgCEPT1 and TgCEPT1 could act as bifunctional enzymes for the synthesis of PtdCho and PtdEtn in tachyzoite.When TgCEPTl and TgCEPT2(iCEPTIΔcept2)were double depleted,the growth of the mutant strain was arrested,manifested as decreased invasion,replication,egress and daughter cell forming ability,accompanied by abnormal morphology,and decreased mitochondrial membrane potential.This further convinced that TgCEPT1 and TgCEPT2 complementally contribute to the synthesis of PtdCho and de novo PtdCho synthesis is indispensable in Toxoplasma gondii.In conclusion,the fatty acid metabolism of Toxoplasma gondii is flexible.The parasite can flexibly utilize de novo synthesized fatty acids or exogenously salvaged to meet the needs growth and development.However,using fatty acid for PtdCho de novo synthesis is very important for the growth of Toxoplasma tachyzoites.As a key node in lipid metabolism,the de novo synthesis pathway of PtdCho has the potential to become an antitoxoplasmosis drug target.