Mitochondrial metabolism of glucose and glutamine is required for intracellular growth of Toxoplasma gondii

Abstract

Toxoplasma gondii proliferates within host cell vacuoles where the parasite relies on host carbon and nutrients for replication. To assess how T. gondii utilizes these resources, we mapped the carbon metabolism pathways in intracellular and egressed parasite stages. We determined that intracellular T. gondii stages actively catabolize host glucose via a canonical, oxidative tricarboxylic acid (TCA) cycle, a mitochondrial pathway in which organic molecules are broken down to generate energy. These stages also catabolize glutamine via the TCA cycle and an unanticipated γ-aminobutyric acid (GABA) shunt, which generates GABA and additional molecules that enter the TCA cycle. Chemically inhibiting the TCA cycle completely prevents intracellular parasite replication. Parasites lacking the GABA shunt exhibit attenuated growth and are unable to sustain motility under nutrient-limited conditions, suggesting that GABA functions as a short-term energy reserve. Thus, T. gondii tachyzoites have metabolic flexibility that likely allows the parasite to infect diverse cell types.

Figure 1. Metabolomic analysis of intracellular and extracellular tachyzoites

(A) GC-MS chromatograms of polar metabolite extracts from intracellular (IT) and extracellular (ET) tachyzoites (2 × 10⁸ cell equivalents). Inserts shows a section of these chromatograms (10 to 14 min) with 10-fold expansion of the y-scale to show changes in γ-aminobutyric acid (GABA) levels in the two stages. Peaks corresponding to aspartate (Asp) and myo -inositol (Ins) that are either unchanged or decreased in extracellular tachyzoites are indicated. (B) Z-score plots of selected metabolites. Plotted z-scores show the mean and standard deviations of individual metabolites in replicate analyses following normalization to the intracellular tachyzoite sample set. Grey circles refer to metabolite levels in intracellular tachyzoites (which typically cluster within 5 s.d. of the mean) while blue circles refer to metabolite levels in extracellular tachyzoites. Metabolite levels that deviate from the mean by >5 s.d. are considered significant (Note that the z-score plots are truncated at 25 s.d. for clarity). See also Figure S1.

Figure 2. T. gondii tachyzoites catabolize glucose in a complete TCA cycle

(A) Infected HFF or egressed tachyzoites (ET) were suspended in medium containing either ¹³C-U-glucose or ¹³C-U-glutamine for 4 hr. Intracellular tachyzoites (IT) were isolated from host material prior to metabolite extraction. Incorporation of ¹³C into selected polar metabolites and fatty acids (derived from total lipid extracts) was quantified by GC-MS and levels (mol percent containing one or more ¹³C carbons) after correction for natural abundance are represented by heat plots. (B) Egressed tachyzoites were incubated in full medium containing either ¹³C-U-glucose (upper panel) or ¹³C-U-glutamine (lower panel) in place of naturally labelled glucose or glutamine, respectively. Culture medium was collected at 6, 12 and 24 hr and analysed by ¹³C-NMR. Rates of utilization of each carbon source are shown in black, while rate of secretion of lactate (Lac), CO₂ (detected as H¹³CO₃), glutamate (Glu), alanine (Ala), aspartate (Asp) and succinate (Suc) are shown in grey. See also Figure S2.

Figure 3. Complete TCA cycle and GABA shunt in T. gondii tachyzoites

Left panel: The diagram represents a reconstruction of a complete TCA cycle and GABA shunt in T. gondii tachyzoites inferred from isotopomer analysis. Acetyl-CoA generated from ¹³C-glucose is used to synthesize citrate. Grey boxes indicate the fate of carbons in the incoming acetyl group in early intermediates of the TCA cycle. Uniformly labelled citrate is generated through multiple rounds through the cycle. Inputs of 5-carbon and 4-carbon skeletons from glutamate or GABA comprise the major anaplerotic influxes. NaFAc leads to inhibition of the TCA enzyme, aconitase. Right panel: Abundance of different TCA cycle isotopomers after labelling of egressed tachyzoites with ¹³C-U-glucose (red graphs) or ¹³C-glutamine (blue graphs) for 4 hr. Parasites were treated with or without NaFAc at the initiation of labelling. The numbers on the x-axis indicate the number of labelled carbons in each metabolite. The y-axis indicates the fractional abundance of each mass isotopomer. Data are represented as mean +/− SEM, where n = 5. See also Figure S3.

Figure 4. T. gondii tachyzoites require a functional TCA cycle for intracellular growth

(A–D) HFF were infected with freshly-egressed tachyzoites (10³) in the presence or absence of 0.2 mM NaFAc. (A) Infection in the absence of NaFAc. (B & C) Tachyzoites were pre-treated with NaFAc 4 hr prior to infection and infected cultures maintained in medium containing NaFAc. In (C), the medium was supplemented with an additional 13 mM glutamine. (D) NaFAc was added 2 hr after initiation of infection. The appearance of plaques was monitored by crystal violet staining after 6 days. Bars represent 15 mm. (E) Schematic reconstruction of the proposed metabolic pathways and compartmentalisation of glycolysis, the TCA cycle and FASII fatty acid biosynthesis. (F) ¹³C-glucose and (G) ¹³C-acetate incorporation into tachyzoite fatty acids. Nomenclature Cx:y is shown where x is the number of carbons and y is the number of double bonds in the fatty acid chain. Error bars indicate standard deviation, where n = 6. Abbreviations: AcCoA, acetyl-CoA; αKG, α-ketoglutarate; Cit, citrate; G6P, glucose 6-phosphate; Glu, glutamate; Gln, glutamine; GABA, γ-aminobutyric acid; iCit, isocitrate; Lac, lactate; OAA, oxaloacetic acid; PEP, phosphoenolpyruvate; Pyr, pyruvate; Aco, aconitase; ICDH, isocitrate dehydrogenase. See also Figure S4.

Figure 5. Loss of GAD results in complete ablation of GABA biosynthesis and partial attenuation of parasite propagation

(A) Intracellular GABA and glutamate levels in parental and Δgad tachyzoites. (B) Cell-free extracts of T. gondii parental (filled circles) and Δgad tachyzoites (open circles) were incubated with 10 mM ¹³C-U-glutamate in the presence of 1 mM ATP. The synthesis of GABA at indicated time points was measured by GC-MS. Error bars represent standard deviation, where n = 3. (C) Parental and Δgad parasite lines are both capable of generating plaques in HFF monolayers. (D) Fluorescence growth assays showing similar growth rates (no significant difference) for parental (solid line, closed circles), and Δgad (dotted line, open squares) strains. Each data point represents the mean of 6 wells and the error bars indicate standard deviation where n = 6. (E) Equal numbers of wild type parasites (grey bar) expressing the dTomato fluorescent protein and Δgad mutant tachyzoites (black bar) were used to infect HFF. The recovery of fluorescent and non-fluorescent parasites was determined by FACS analysis of 2 × 10⁶ cells at indicated days. The Δgad parasite line was rapidly outcompeted by the WT line. (F) Swiss Webster mice were infected with Type-I parental and Δgad tachyzoites (10 parasites/mouse) and mouse survival observed over 20 days. See also Figure S5.

Figure 6. GABA can be used to sustain tachyzoite motility under nutrient limited conditions

(A) Wild type (RH) tachyzoites were suspended in medium containing glucose and/or glutamine as carbon source, PBS or PBS containing 2-deoxyglucose (DOG). Intracellular GABA levels were measured at the indicated time points over 4 hr. GABA was rapidly depleted in the absence of glutamine. Error bars indicate standard deviation, where n = 2 and results are representative of 4 biological replicates. (B) Parental and Δgad tachyzoites were suspended in medium lacking carbon sources and allowed to glide on poly-L-lysine-coated cover slips after addition of 2 µM ionophore to stimulate motility. Parasites were fixed and the resulting gliding trails were visualized using α-SAG1 antibodies. The Δgad mutant displays a clear defect in gliding motility under these conditions. (C) Parental and Δgad tachyzoites were incubated in HBSS-HEPES with or without NaFAc, and different carbon sources, as indicated. The average trail length/parasite was documented in 10 fields (>100 parasites). Counts are of representative frames from two independent experiments. Data are represented as mean +/− SEM. See also Figure S6.

Figure 7. Role of the TCA cycle in intracellular and egressed T. gondii tachyzoites

Intracellular tachyzoites co-utilize glucose and glutamine scavenged from the host and both carbon sources are catabolized in mitochondria via a canonical oxidative TCA cycle (left panel). Glucose is also used to fuel the FASII-dependent pathway of fatty acid biosynthesis in the apicoplast. Inhibition of tachyzoite aconitase (comprising mitochondrial and apicoplast isoforms) results in a reduction of FASII biosynthesis, providing evidence that a citrate shunt between the mitochondrion and apicoplast (brown lines) is at least partially required for regeneration of reducing equivalents for apicoplast fatty acid synthesis. Tachyzoites are likely exposed to elevated glucose levels following host cell egress (right panel). However, these stages continue to co-utilize glutamine and are dependent on operation of the TCA cycle for most of their ATP synthesis and normal gliding motility. The GABA shunt identified in this study involves at least three enzymes and a putative mitochondrial transporter (steps not shown). GABA accumulated under glutamine-replete conditions may function as a short-term energy reserve under nutrient limiting conditions. For Abbreviations see Figure 3.