Fig 1.
Toxoplasma gondii encodes its pentose phosphate pathway within 2 subcellular locations.
(A), Schematic representation of the pentose phosphate pathway in Toxoplasma parasites. (B), Gene ID of the PPP genes. (C), Representative images of immunofluorescence assay. Different enzymes of PPP tagged at the C terminus with smHA epitope by CRISPR-Cas9-mediated homologous recombination in the RHΔku80 strain. Subsequently, the parasites of testing strains were fixed, permeabilized and stained with mouse anti-HA and rabbit anti-TgALD, which were detected by Alexa 488- and Alexa 594-conjugated secondary antibodies, respectively. TgALD and Hoechst were used as the cytoplasm and cell nucleus markers, respectively. Bar = 5 μm.
Fig 2.
TgG6PDH1, TgG6PDH2, Tg6PGDH1, TgRuPE and TgTAL are dispensable for parasite growth and virulence, while TgG6PDHs are required for defending against oxidative stress.
(A), Plaque assay comparing the growth of Δg6pdh1, Δg6pdh2, Δ6pgdh1, Δrupe and Δtal tachyzoites in vitro to that of wild-type strain RHΔku80. (B), Relative sizes (pixel size calculated by photoshop) of plaques. Means ± SD of >60 plaques (n = 3). (C), Representative Plaques image showing the comparative growth of Δg6pdh1Δg6pdh2 and RHΔku80 strains. (D), Graph presentation of plaque sizes of Δg6pdh1Δg6pdh2 and RHΔku80 strains. Means ± SD of >60 plaques (n = 3). (E), Virulence tests of indicated strains in ICR mice (100 parasites per mouse, 10 mice for each strain). (F), Intracellular replication assay comparing parasite proliferation under standard culture conditions. Freshly egressed tachyzoites of RHΔku80, Δg6pdh1, Δg6pdh2 and Δg6pdh1Δg6pdh2 parasites were allowed to infect HFF monolayers for 1 h, and invaded parasites were cultured at 37°C with 5% CO2 for another 24 h. Percentiles of the parasitophorous vacuole (PV) containing 1, 2, 4, 8, 16, or more parasites were determined and plotted. Means ± SEM from three independent experiments (n = 3). (G), Freshly egressed tachyzoites of indicated strains were pre-treated with 500 μM H2O2 medium for 3 h, and then subjected to intracellular replication assay (without H2O2) for 24 h. Means ± SEM from three independent experiments was graphed. Two-way ANOVA, *, p <0 .05, ***, P < 0.001. (H), RHΔku80 tachyzoites were purified, total RNA was extracted and reversed into cDNA. Transcript levels for TgG6PDH1 and TgG6PDH2 in each sample were analyzed by quantitative real-time PCR, using β-tubulin as an internal reference. Means ± SEM of four independent experiments (n = 4). Student’s t-test, ***, P<0.001. (I), Parasites (approximately 3×107) were purified by 3 μm membrane filtration, washed with cold PBS and extracted with extraction buffer. Relative NADPH levels in RHΔku80 (WT), Δg6pdh1 and Δg6pdh2 were determined by the NADPH Assay kit. Means ± SEM from three independent experiments (n = 3). **, P<0.01; one-way ANOVA.
Fig 3.
Tg6PGDH2 is important for parasite survival.
(A), Diagram showing strategy to construct the conditional knockout strain Tg6PGDH2-cKD, using the LoxP- Cre system in the DiCre strain. (B), Diagnostic PCRs on a representative Tg6PGDH2-cKD clone. PCR1 and PCR2 examined the integration of homology templates at the 5’ and 3’ end of Tg6PGDH2, whereas PCR3 confirmed the deletion of the endogenous Tg6PGDH2 locus. (C), Immunofluorescence staining for TgALD and YFP expression in Tg6PGDH2-cKD parasites treated with rapamycin for 2 d. (D), Western blotting for checking the expression of Tg6PGDH2 in Tg6PGDH2-cKD parasites treated with or without rapamycin for 4 days. TgALD was included as a loading control. (E), Competition assay comparing the growth of Tg6PGDH2-cKD parasites treated with rapamycin for 24 h to that of untreated parasites. (F), Plaque assay showing the defect in the Tg6PGDH2-cKD parasites after 4 days of rapamycin treatment. (G), Tg6PGDH2-cKD parasites pretreated for 4 days formed smaller plaques than their parental strain, and untreated Tg6PGDH2-cKD parasites formed plaques similarly to DiCre. (H), Intracellular replication assay comparing parasite growth in vitro. Tg6PGDH2-cKD parasites were treated with rapamycin for 4 days, and then they were allowed to infect fresh HFF cells and grown for 24 h, subsequently, the number of parasites in each PV was checked by IFA. Results are means ± SEM for n = 3 independent experiments. (I), Survival curves of mice infected with tachyzoites of indicated strains. Tg6PGDH2-cKD and Δ6pgdh2 mutants were used to infect ICR mice (100 parasites/mouse, n = 10 mice for each strain) by intraperitoneal injection, and the survival of mice was followed for 30 days. *, p <0 .05, Gehan–Breslow–Wilcoxon tests.
Fig 4.
Depletion of TgRPI results in severe growth defects in vitro and in vivo.
(A), Diagram showing strategy to construct the conditional knockout strain TgRPI-cKD. (B), Diagnostic PCRs on a representative TgRPI-cKD clone. PCR1 and PCR2 checked the integration of homology templates at the 5’ and 3’ end of TgRPI, whereas PCR3 confirmed the deletion of endogenous TgRPI locus. (C), Immunofluorescence staining for TgALD and YFP expression in TgRPI-cKD parasites treated with rapamycin for 24 h. (D), Western blotting for analyzing the expression of TgRPI in TgRPI-cKD parasites treated with or without rapamycin for 4 days. (E), Competition assay comparing the growth of TgRPI-cKD parasites treated with rapamycin for 24 h to that of untreated parasites. (F-G), Deletion of TgRPI showed severe lytic cycle defects by plaque assay and quantification of plaque sizes. Means ± SD of more than 60 plaques for each strain was graphed. (H), Intracellular replication assay comparing parasite growth in vitro. TgRPI-cKD parasites were treated with rapamycin for 4 days, and then they were allowed to infect fresh HFF cells for 24 h, subsequently, the number of parasites in each PV was checked by IFA. Results are means ± SEM for n = 3 independent experiments. (I), Invasion assay where freshly egressed parasites were used to invade HFF monolayers for 20 min. Efficiencies of invasion, as determined by two-color staining to distinguish invaded vs non-invaded tachyzoites (means ± SEM, n = 3 assays), ***, P<0.001; Student’s t-test. (J), Parasite loads in the peritoneal fluids of ICR mice. ICR mice were infected with TgRPI-cKD and Δrpi tachyzoites (104 tachyzoites/mouse) by intraperitoneal injection (n = 5 for each group), and parasite loads in peritoneal fluids 5 days post-infection were estimated by qPCR. (K), Virulence tests of indicated strains in ICR mice (100 parasites per mouse, n = 10 mice for TgRPI-cKD strain, n = 20 mice for Δrpi mutants), ***, P<0.001; Gehan–Breslow–Wilcoxon tests.
Fig 5.
Trypanosoma brucei RPI complementation restored the growth defects of TgRPI depletion mutants.
(A), Schematic diagram showing the insertion of a Trypanosoma brucei RPI expressing mini gene into the UPRT locus of the Δrpi strain by CRISPR/Cas9 mediated site-specific integration and selection with 10 μM FUDR. (B), Diagnostic PCRs on a selected compTbRPI clone. PCR4 examined the successful insertion of Trypanosoma brucei RPI on the UPRT locus, whereas PCR5 confirmed the deletion of the endogenous UPRT locus. (C), Expression of the complementing Trypanosoma brucei RPI as shown by IFA using rabbit anti-HA. (D-E), Plaque assays comparing the growth of TgRPI depletion strain before and after Trypanosoma brucei RPI complementation. Means ± SD of more than 60 plaques for each strain was graphed. Student’s t-test, ***, P < 0.001. (F), Intracellular replication rates of depicted strains (24 h post-infection). Means ±SEM from three independent experiments (n = 3), each with two replicates. ***, P < 0.001 by two-way ANOVA. (G), The invasion efficiency of the compTbRPI was compared to the TgRPI-cKD and Δrpi parasites. Means ±SD of more than 100 fields from three independent assays, ***, P < 0.001; one-way ANOVA.
Fig 6.
TgRPI depletion alters the global proteomic profile.
(A), Volcano plots showing the protein expression difference based on 4D label-free quantitative proteomic data of Δrpi mutants. The X-axis shows log2 (1.5-fold change) versus the TgRPI-cKD, and the Y-axis shows -log10 (P value) after ANOVA statistical test for n = 4 independent biological replicates. (B), Putative subcellular localization of differentially expressed proteins. (C), Enrichment and clustering analysis of the quantitative proteomics data sets based on gene ontology, only proteins that changed ≥1.5-fold in relative ratios (P<0.05) were considered. BP, biological process; CC, cellular component. (D-E), Heat map of differentially expressed ribosomal proteins, microneme proteins, rhoptry proteins and stage-specific proteins. Bright red indicates a 1.5-fold change (P<0.05). White indicates no change. (F), Four genes were selected for quantitative RT-PCR analysis, which examined their expression changes. The β-tubulin gene in parasites was used as an internal reference. Means ± SEM of three independent assays, *, p <0 .05, ***, P<0.001; Student’s t-test.
Fig 7.
Tg6PGDH2 and TgRPI are required to incorporate glucose-derived carbon into the pentose phosphate pathway.
(A-C), Tg6PGDH2-cKD (WT) and Δ6pgdh2 mutants were propagated with HFF monolayers cultured in glucose-free DMEM medium supplemented with 8 mM 1,2-13C2-glucose for 12 h. Subsequently, intracellular parasites were collected, metabolites were extracted from the parasites, and the relative abundance of isotopologues was determined by LC-MS. M0 means parental unlabeled. M1-M5 represents the number of carbons in a selected metabolite labeled with 13C atom. Values are means ± SEM from four independent experiments (n = 4). Student’s t-test, *, P<0.05; **, P<0.01; ***, P<0.001. (D), TgRPI-cKD (WT), and Δrpi mutants were cultured the same way as above. Metabolite from the intracellular parasites was determined by LC-MS. Values are means ± SEM from five independent experiments (n = 5). ***, P < 0.001 by two-way ANOVA. (E), Extracellular TgRPI-cKD (WT) and Δrpi parasites were incubated in a glucose-free medium containing 8 mM [U-13C] glucose for 4 h. Incorporation of 13C into Ru5P and Xu5P was determined by UHPLC-HRMS platform. M0-M5 represents the number of carbons in a selected metabolite labeled with 13C atom. Values are means ± SEM from five independent experiments (n = 5). ***, P < 0.001 by two-way ANOVA. (F), Freshly egressed tachyzoites (3×107) of TgRPI-cKD left untreated or pretreated with rapamycin for 4 days were collected, syringe released, and then incubated in medium containing 8 mM [U-13C] glucose for 4 h. Incorporation of 13C into glycolysis and PPP intermediates was determined by UHPLC-HRMS platform. M0-M7 represents the number of carbons in a selected metabolite labeled with 13C atom. Values are means ± SEM from five independent experiments (n = 5). *, P<0.05; **, P<0.01; ***, P < .001; all by two-way ANOVA.
Fig 8.
Proposed models for PPP metabolism under various conditions.
(A-B), Parasites can generate ribose-5-phosphate using the oxidative pentose phosphate pathway (TgG6PDH-Tg6PGDH2-TgRPI pathway) and non-oxidative pentose phosphate pathway (TgTKT-TgTAL pathway). (C), Upon disruption of TgG6PDH, TgG6PDH-dependent pentose synthesis is blocked but ribose-5-phosphate is still produced by the intact downstream oxidative pentose phosphate pathway (Tg6PGDH2-TgRPI pathway) which might utilize host-derived PPP intermediates. (D), In Δ6pgdh2 mutants, glucose imported from host cells cannot be fully catabolized to create ribose-5-phosphate. Even so, ribose-5-phosphate was still partly produced through the TgTKT-TgTAL pathway. (E), Similar to Δ6pgdh2 mutants, the Δrpi mutants also partly produced ribose-5-phosphate through the TgTKT-TgTAL pathway. (F), Mutants lacking TgRuPE rely on the TgTKT-TgTAL pathway for supplying Xylulose 5P. Notably, the ribose-5-phosphate can be generally provided by the oxidative and non-oxidative pentose phosphate pathway. (G), In Δtal mutants, the ribose-5-phosphate can be provided through the oxidative pentose phosphate pathway and TgTKT-TgSBPase pathway.