Fig 1.
Toxoplasma expresses two PFKs that are PPi- and ATP-dependent.
A, Conserved domains in Toxoplasma PFK1 and PFK2, as determined by BLAST analyses. PFK: phosphofructokinase, PFP: PPi-dependent phosphofructokinase. The shaded region (644–1110) in PFK2 is a pseudo-PFK domain that lacks key residues for activity. B, Sequence alignments of two types of PFKs from selected species. The “GGDD/G” motif that specifies the substrates is boxed in gray. C-D: ATP- and PPi-dependent activities of recombinant PFK1 and PFK2 when assayed under different F6P concentrations. Each assay was repeated three times independently. E, Enzymatic activity of the N (PFK2-N, 1–595) and C (PFK2-C, 597–1225) terminal domains of PFK2 in the presence of 4 mM F6P. PBS (Blank) and heat inactivated PFK2 (Heated PFK2) were included as negative controls. ***P < 0.001, paired student’s t-test (n = 3).
Fig 2.
Generation and characterization of a Toxoplasma mutant without PFK1.
A: Schematic diagram illustrating the replacement of PFK1 in the RH strain with the selection marker DHFR* through CRISPR/Cas9-assisted homologous recombination. PCR1/2/3 denote the diagnostic PCRs for positive clone identification. B: Diagnostic PCRs on a Δpfk1 clone. C: Plaque assay comparing the overall growth of the Δpfk1 mutant to that of the wild-type strain RH. D-E: Numbers and sizes of plaques from C. These experiments were independently repeated twice, each with three replicates. F: Intracellular replication of parasites in HFF cells, as determined by the number of parasites in each parasitophorous vacuole (Tg per PV). Means ± SEM of three independent experiments. G: Virulence test in mice. The tachyzoites of RH or the Δpfk1 mutant were used to infect ICR mice (100 parasites/mouse, n = 10 mice for each strain) by intraperitoneal injection, and the survival of the mice was monitored daily.
Fig 3.
Construction of a PFK2 depletion strain and a corresponding complementation strain.
A: The strategy used to construct the conditional depletion strain iPFK2, which had a mAID-HA tag fused to its C-terminus at its endogenous locus. The red bar indicates the CRISPR targeting site. B: Diagnostic PCRs of an iPFK2 clone. The WT is the parental strain RH/Tir1. C: Western blots checking the degradation of PFK2 over time (from 0 to 240 minutes) in the iPFK2 strain after IAA treatment. PFK2 was detected by an HA antibody, whereas ALD was included as a loading control. D: Immunofluorescent staining examining the expression of PFK2 in the depletion and complementation strains. E: Diagram showing the strategy to construct the PFK2 complementation strain ComPFK2, which had a second copy of PFK2 expressed from the UPRT locus. The red bar indicates the CRISPR targeting site. F: Diagnostic PCRs of a ComPFK2 clone.
Fig 4.
PFK2 is essential for tachyzoite growth.
A: Plaque assay of the indicated strains in the presence or absence of IAA. B-C: Numbers and sizes of plaques from A. The experiments were repeated twice independently, each with three replicates. D: Intracellular replication of parasites in HFF cells. The means ± SEM of three independent experiments. ***p ≤ 0.001, two-way ANOVA. E: Parasite burden in peritoneal fluids of mice 5 days post infection, n = 4 or 5. ***p < 0.001, unpaired student’s t-test. F: Survival of mice treated with or without IAA after being infected with indicated strains.
Fig 5.
Metabolic alterations associated with PFK2 depletion.
A: ATP levels in iPFK2 parasites with or without IAA treatment for 6 hours. B: Incorporation of 13C into the indicated metabolites in iPFK2 parasites, which were pretreated with or without IAA for 2 hours and then labeled with 13C6-glucose for 4 hours. The 13C inclusion in each metabolite was determined by LC-MS. M0-M7 denote the number of carbons in a given metabolite that was labeled with 13C. Means ± SD of three independent experiments, *P < 0.05, **P < 0.01, and ***P < 0.001, two-way ANOVA. Details of the statistical analyses are provided in S3 Table.
Fig 6.
PFK2 depletion causes PPi accumulation and reduced protein synthesis.
A: The pyrophosphate concentration in the indicated parasites with or without IAA treatment for 6 hours. Means ± SEM of four independent experiments, *P < 0.05, **P < 0.01, paired Student’s t test. B: Nascent protein synthesis rates in the indicated strains with or without 6 hours of IAA pretreatment. Means ± SEM of five independent experiments, *P < 0.05, ***P < 0.001, paired Student’s t test. Details for the statistical analyses are provided in S3 Table.
Fig 7.
Overexpressing soluble PPase in Toxoplasma partially rescues the growth defect of PFK2-depletion mutants.
A: Diagram illustrating the expression of Ppase, PFK1 or the N- or C-terminal parts of PFK2 in iPFK2 mutants from the UPRT locus. B: Immunofluorescence staining showing the expression of the introduced genes, as probed by the Ty tag. C: Intracellular replication of the indicated strains in the presence or absence of IAA. Means ± SEM of three independent experiments, **P < 0.01, ***P < 0.001, two-way ANOVA. D: Plaque assays for the indicated strains grown with or without IAA for 8 days or the iPFK2/PPase strain grown in the presence of IAA for 11 or 14 days. E: The sizes of plaques from D. ***P < 0.001, student’s t test.
Fig 8.
A model for PFK2 in maintaining PPi homeostasis and balanced catabolism and anabolism in Toxoplasma.
The biosynthesis of macromolecules such as proteins, DNA and RNA generates PPi, which is then converted to Pi by PPase and the PPi-dependent phosphofructokinase PFK2. The hydrolysis of PPi by PFK2 is coupled to the phosphorylation of F6P to FBP, the committed step of glycolysis. Through the activity of PFK2, PPi is hydrolyzed, which otherwise would inhibit biosynthetic reactions. In addition, glycolysis is activated to produce more building blocks for macromolecule synthesis as well as energy. As such, PFK2 serves as a key factor in coordinating the parasites’ catabolic and anabolic activities. PFK2-deficient parasites suffer from increased levels of PPi, reduced macromolecule biosynthesis and decreased glycolysis. PFK1 may be able to rescue in part the defects in glycolysis caused by PFK2 depletion, but it would not be able to reverse the defects in PPi homeostasis and macromolecule synthesis.