Roles Of Key Enzymes Involved In Glucose Storage And Catabolism In The Growth And Development Of Toxoplasma Gondii

Toxoplasma gondii is a ubiquitous pathogen infecting almost all warm-blooded animals and causes Toxoplasmosis.Toxoplasmosis is widespread throughout the world,causing serious issues to human society and animal husbandry.The complex life cycle and the variety of hosts of T.gondii make it difficult to prevent Toxoplasmosis.Therefore,the treatment and prevention of toxoplasmosis have become a globally significant public health concern.Glucose is a key carbon sources to utilize in T.gondii.Under abundant nutrition,glucose can be degraded by glycolysis and the TCA cycle to provide energy and substrates for cell growth.Meanwhile,glucose can be stored in amylopectin.The parasite’s ability to interconvert between acute and chronic infection(tachyzoites and bradyzoites)is one of the reasons for T.gondii pathogenesis and transmission.The obvious difference in the structure of life cycle stages is abundant amylopectin granules in encysted bradyzoites,oocysts and sporozoites,whereas these granules are barely detectable in tachyzoites.This phenomenon reminds us that glucose storage(synthesis of amylopectin)and catabolism(glycolysis and citric acid cycle)may be of substantial significance for the parasite’s growth,development and stage conversion.In order to further clarify the physiological significance of starch synthesis and complete glucose catabolism,starch synthase,SS),mitochondrial pyruvate carrier(MPC)and citrate synthase(CS),the key rate-limiting enzyme of the TCA cycle,were selected as research subjects in this study.And their biological significance in parasite growth and development was investigated using genetics,molecular biology,bioinformatics,metabolomics and biochemistry.The main research contents and results include:1.The biological function of SS in starch synthesis and the growth and development of T.gondii.In this study,the enzymatic pathways of amylopectin synthesis and catabolism of Toxoplasma gondii were predicted by bioinformatics methods.By constructing the Δss strain,we found that lack of SS could not produce amylopectin granules and cause growth defects in tachyzoite and bradyzoite in vitro and abnormal cyst morphology in vivo.However,starch synthase is not necessary for parasite growth and clinical infection.In addition,the activation experiment in vitro preliminarily verified that amylopectin played a certain role in bradyzoite activation.According to results,there was a significant difference in bradyzoite activation efficiency between the ME49 and SS mutant strain.Moreover,there was a significant difference in the spillage time between the two strains as time went on.ME49 had fully spillage at 48 hours,while ss strains only reached 50%spillage at 60 hours.These results also suggested that amylopectin accumulation might also be of great significance in the oocyst and sporozoite stages of Toxoplasma gondii.2.The role of mitochondrial pyruvate transporters in the growth,development and metabolic homeostasis of T.gondii.Potential MPC1 and MPC2 proteins of the T.gondii genome were discovered through sequence alignment.Tg MPCs were reversely expressed into the yeast MPC mutants and demonstrated that Tg MPCs could compensate for the growth defects of ScΔmpc1 and ScΔmpc2 due to the deletion of endogenous MPC.The result verified the transport functions of Tg MPC1 and Tg MPC2.Knocking out Tg MPC1 and Tg MPC2,either alone or in combination,can cause the same degree of growth defects in the tachyzoites.Similar to the growth phenotype produced when mitochondrial pyruvate is blocked to form acetyl-Co A.The metabolic flux of the parasite was detected by the isotope tracer method.It was demonstrated that the central carbon metabolic flux of TgΔmpc1 strain was reduced,and the level of acetyl-Co A was significantly decreased.Clinically,Toxoplasma without SS shows reduced proliferation in mice,decreased virulence,and reduced brain cyst formation.These results indicated that the mitochondrial pyruvate transporter of Toxoplasma gondii was not essential for the growth of tachyzoites.However,it was vital for maintaining central carbon metabolism homeostasis,rapid proliferation and chronic infections.3.Effect of mitochondrial citrate synthase(CS)on the growth of T.gondiiThree potential citrate synthase-expressing genes encoded by the Toxoplasma gondii genome were identified based on bioinformatics methods.Through multi-sequence comparison analysis,evolutionary development analysis,and 3D protein structure simulation and molecular docking,we initially believed that these three proteins belonged to type I citrate synthase.They were named CS1,CS2,and Prp C according to their sequence characteristics.Subcellular localization of the three proteins by genetic means revealed that CS1 and Prp C were located in the mitochondria,while CS2 was located in the cytoplasm.Through the analysis of enzyme activity mechanics experiments in vitro,we were able to demonstrate that both CS1 and Prp C can synthesize citrate from acetyl-Co A and oxaloacetic acid.However,Prp C’s affinity with substrate was significantly lower than that of CS1.In order to investigate the biological effects of mitochondrial citrate synthase,mutants of CS1 and Prp C have been created,either separately or in combination.Compared with the wild type,the growth rate of Δcs1 strain was remarkably decreased.On this basis,further deletion of Prp C leads to more serious defects in growth.However,Prp C is dispensable for the rapid growth of tachyzoite.Additionally,the virulence of these strains was evaluated in mice.We discovered that the absence of mitochondrial CS did not affect the virulence of parasite.These results suggest that mitochondrial CS is important but not essential for virulence.In summary,the study found that although SS,MPC and CS play role in maintaining central carbon metabolism but they are not essential for the growth of T.gondii in vitro.SS played a significant role in the activation of bradyzoite in vitro,and MPC contributed to chronic infection.These results provide a theoretical basis for understanding the physiological significance of glucose storage and metabolism at different stages.