Biological Function Of The Apicoplast Pyruvate Carrier In Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular parasite belonging to the Apicomplexa,with a host range that includes nearly all warm-blooded animals.According to epidemiological data,over 30%of the world’s population is infected with Toxoplasma.Toxoplasmosis can be life-threatening in immunocompromised individuals or in those with congenital infections.In livestock,toxoplasmosis has been found to be a significant cause of abortion and stillbirth.With the exception of Cryptosporidium,most apicomplexan parasites possess a unique organelle called the apicoplast.The apicoplast is essential for the parasite’s metabolism,including the biosynthesis of isoprenoids that require pyruvate as a substrate.Pyruvate is a key metabolic molecule in the apicoplast,however,the origin of pyruvate within the apicoplast remains unclear.The results of this laboratory have shown that pyruvate kinase 2（PYK2）catalyzed by phosphoenolpyruvate in the apicoplast is not the main source of pyruvate in the apicoplast,suggesting that apicoplast may have a pathway to enter pyruvate.In this study,using Toxoplasma as a model organism,through bioinformatics analysis,two potential apicoplast pyruvate carrier proteins were identified,named APC1 and APC2（Apicoplast Pyruvate Carrier）.Results confirmed that APC are responsible for pyruvate transport into the apicoplast using gene editing,targeted metabolomics,heterologous expression,and super-resolution microscopy,and their transport and biological functions were studied.Specific research contents include:（1）Screening and identification of apicoplast putative pyruvate transporters in ToxoplasmaAfter analyzing the characteristics of the discovered transporters such as MPC,BASS2,and MCT,this study successfully screened seven potential apicoplast transporters by setting the CRISPR phenotype score（≤-3）,species specificity（not homologous to Cryptosporidium）,and transmembrane characteristics（TM≥3）using the strategy analysis function in Toxo DB.After analyzing the characteristics of the identified transporters such as MPC,BASS2 and MCT,Toxo DB’s strategy analysis function was used to and setting criteria such as CRISPR phenotype score（≤-3）,species specificity（non-homology with Cryptosporidium）,and transmembrane features（TM≥3）,we successfully screened seven putative apicoplast transporters.Combining sequence analysis with in situ labeling,we identified two potential carriers of pyruvate,APC1 and APC2.This study focuses on APC2 and conducts an in-depth study of its functions.To validate the role of the APC2 protein in Toxoplasma growth,an APC2-m AID strain was constructed using a conditional degradation system.The results showed that conditional degradation of APC2 severely affected Toxoplasma growth,inhibiting intracellular growth and preventing the formation of plaques.Targeted metabolic flux analysis using ¹³C-labeled glucose revealed that APC2 degradation disrupted glycolysis and the tricarboxylic acid cycle,causing a decrease in ¹³C-labeled glucose entering the intermediate metabolites of glycolysis（such as 6-phosphogluconate and fructose-6-phosphate）and the tricarboxylic acid cycle（such as citric acid,cis-aconitic acid,succinic acid,etc.）.In brief,these studies show that APC2 is necessary to maintain the normal growth of the parasite,that is,APC2 is the key protein of the apicoplast in Toxoplasma.（2）APC2 is crucial for maintaining the function and integrity of the apicoplastPyruvate is the major substrate for the important metabolic pathways MEP and FASII in the apicoplast,and its stable production or source is crucial for maintaining the function of the apicoplast.Targeted lipid metabolism flow using ¹³C glucose labeling and targeted MEP mass spectrometry were performed on APC2-m AID strains,and the results showed that the ¹³C labeling content of the major products of the FAS II pathway,myristic acid（C14:0）and palmitic acid（C16:0）,both significantly decreased after conditional degradation of APC2.The content of the major products of the MEP pathway,IPP/DMAPP and MEP,also decreased significantly,to only 5%-17%of normal levels,indicating the crucial role of APC2 in maintaining normal metabolism in the apicoplast.Subsequently,the integrity of the apicoplast was tested using apicoplast counting and q PCR,and the results showed that significant defects occurred at the protein and gene level after conditional degradation of APC2.These results demonstrate that APC2 is crucial for maintaining the function and integrity of the apicoplast.（3）The APC complex has the ability to transport pyruvateUsing homologous recombination gene deletion technology,a strain of Escherichia coli lacking pyruvate transport protein was constructed.APC1 and APC2 were then expressed in E.coli,and the pyruvate transport capacity of strains expressing APC1 alone,APC2 alone,and both APC1 and APC2 were evaluated using dot blot assay and radiolabeled[2-¹⁴C]-pyruvate absorption experiments.The results showed that APC1 or APC2 alone did not have the ability to transport pyruvate,and only when APC1 and APC2were expressed simultaneously,stable pyruvate transport capacity was observed.Ion dependency experiments indicated that its transport ability was influenced by H⁺and Na⁺.To explore the inherent reasons why APC1 and APC2 have pyruvate transport capacity only when co-expressed,this study investigated the transmembrane topology and interaction of APC using STED,Split-GFP,and Bi FC technologies.The results showed that APC1 is located in the apicoplast membrane,with its N-terminus located on the outermost membrane and its C-terminus between the outermost and second outermost membranes.APC2 is also located in the apicoplast membrane,with both its N-terminus and C-terminus located on the outermost membrane.Bi FC technology confirmed an interaction between APC1 and APC2 in the apicoplast membrane.These results indicate that APC function as a complex in the apicoplast membrane to transport pyruvate.In summary,this study identified potential apicoplast pyruvate transporters and characterized it as a complex for transporting pyruvate into the apicoplast.The biological function of APC2 was demonstrated to be important for parasite growth and apicoplast function.This study provides important insights into understanding apicoplast metabolism,and the unique properties of APC also provide new targets for designing anti-parasitic drugs.