Functional And Phylogenetic Analyses Of Apicoplast-localized Glycolytic Enzymes Of Toxoplasma Gondii

Toxoplasma gondii is able to invade and survive in all nucleated cell types of the hosts,becoming a widespread parasitic pathogen.Its hosts can be almost warm-blooded animals,including livestock and poultry.T.gondii has a complicated life cycle and diverse transmission forms.Although Toxoplasmosis has the capacity of severely impacting on human health and hindering the development of animal industry,there is no effective vaccine against T.gondii and no efficient medicine against tissue cysts.Apicoplast is a unique organelle present in most apicomplexans as it is derived from two independent endosymbiotic event.While no longer photosynthetic,the apicoplast is a center of metabolic activity harboring the major anabolic pathways.Because no plastid exists in human beings,the apicoplast is supposed to become a potential drug target.All glycolytic enzymes,from phosphofructokinase up to pyruvate kinase,are present in the T.gondii genome as duplicates.Among these enzymes,isoforms of pyruvate kinase(PYK2),phosphoglycerate kinase(PGK2)and triose phosphate isomerase(TPI2)were found to localize to apicoplast.PYK2 was predicted to catalyze phosphoenolpyruvate to produce pyruvate as well as ATP for the apicoplast,playing an important role.Besides,PGK2 was considered to utilize 1,3-diphosphoglycerate to produce 3-phosphoglycerate and ATP,providing energy for the apicoplast.In an attempt to elucidate the mechanism of apicoplast metabolism,we will dissect the function and evolution of these apicoplast-localized glycolytic enzymes.In this study,the most recently used technology was performed to observe the localization of a series of glycolytic enzymes.Following,PYK2 single-knockout strain,PGK2 single-knockout strain together with a double-deletion strain Δpyk2Δpgk2 were produced to evaluate their functions.The phylogenetic analyses of these apicoplast-localized glycolytic enzymes were carried out in the end.The main work of this dissertation including the following aspects:(1)Localization of glycolytic isoenzymesBecause the protein localization method used in the previous study has some defects,this study used endogenous tagging to re-observe the localization of glycolytic enzyme of T.gondii.Since studies have clearly shown that EON1 and ENO2 are expressed in the cytoplasm and nucleus and ALD1 is a cytoplasmic marker while ALD2 has no expression,this study focused on the subcellular localization of other 14 glycolytic enzymes by CRISPR/Cas9-mediated site specific insertion.Then IFA was used to observe the localization of these 14 fusion proteins.Our results clearly showed that PGK2,TPI2 and GAPDH2 were localized in the apicoplast,PYK2 targeting to the apicoplast as well as other organelles.(2)Construction and phenotype of PYK2 single-knockout strain,PGK2 single-knockout strain and a double-deletion strain Δpyk2Δpgk2Since PYK2 was expected to be critical for the supply of pyruvate and energy for the apicoplast,this study firstly focused on this gene.Through CRISPR/CAS9 directed gene editing technology,PYK2 was successfully knocked out on RHΔHX strain.Plaque assay and replication assay demonstrated that PYK2 had no impairment on the growth of tachyzoites in vitro.Parasite virulence in mice suggesting that the virulence of PYK2 mutant has no significant role in vitro.Because PYK2 has no impact on the growth of T.gondii,PGK2 was considered to perform a major energy supply for the apicoplast.Following,this study aimed to investigate the function of PGK2.PGK2 was also deleted in RHΔHX strain by the same method.A series of phenotypic experiments showed that PGK2 did not affect the normal growth of T.gondii in vivo and in vitro.Considering that both PYK2 and PGK2 could catalyze ATP production,PYK2 was further disrupted on the basis of PGK2 single-knockout strain.As a result,the virulence of Δpyk2Δpgk2 strain was not attenuated in laboratory mice and the mutant had slight growth defects in vitro when compared with wild strain.These findings indicate that PYK2 and PGK2 localized in the apicoplast are not essential for the growth of T.gondii under normal culture conditions.(3)Phylogenetic analysis of glycolytic isoenzymesThe little impact of PYK2 and PGK2 on the growth of T.gondii led to a hypothesis that the significance of glycolytic enzymes may be related to their phylogenies.Previous study showed that TgPYK1 was most closely related to plant/algal enzymes while TgPYK2 had a proteobacterial origin.In this study,the phylogenetic analyses of PGK,TPI and GAPDH were performed.From the phylogenetic trees we constructed,TgPGK2 is closely related to cyanobacteria/algae PGKs while TgPGK1 has high homology with fungi/yeast PGKs.At the same time,TgGAPDH2 in the apicoplast was closer to algal plastid/bacterium enzymes while TgGAPDH1 in the cytosol has high homology with fungi enzymes.However,TgTPI1 and TgTPI2 do not like TgPGK and TgGAPDH,having no obvious phylogenetic divergence.In fact,TPI2 was tried to be inactivated by gene replacement via double homologous recombination but failed,suggesting that TPI2 may be critical for the growth of T.gondii.As a consequent,we proposed that a part of glycolytic enzymes in the apicoplast of T.gondii were remained through secondary endosymbiosis which is the origination of the apicoplast.These enzymes have high homology with that of prokaryote.On the other hand,T.gondii possesses a whole set of glycolytic enzymes originated from eukaryotes.These prokaryote-derived and apicoplast-localized enzymes generally do not play a key role for parasite growth.To summary,this study clarified the localization of 14 glycolytic enzymes in T.gondii,and revealed that PYK2 and PGK2 in apcioplast have little influence on the growth of T.gondii,but their functions depend more on their phylogenies.