<i>Toxoplasma gondii</i> BCKDH-complex is required for normal growth and virulence.

<p>(A) Schematic representation of pathways to produce acetyl-CoA in the mitochondrion. In green are pathways specific to <i>T. gondii</i> and in red pathways common to <i>T. gondii</i> and <i>Plasmodium</i> spp. (B) Total lysates from extracellular RH<i>ku80_ko</i> (RH) and Tg<i>e1a_ko</i> tachyzoites were analysed by Western blot. Expression of BCKDH-E1a was assessed using polyclonal anti-PfBCKDH-E1a antibodies. Detection of profilin was used as loading control. (C) Plaque assays were performed by inoculating HFF monolayers with RH or Tg<i>e1a_ko</i> parasites for 7 days. Plaques were revealed by Giemsa staining of HFFs. Scale bar represents 1 mm. (D) Intracellular growth of RH (blue) and Tg<i>e1a_ko</i> (red) was assessed after 24 h in complete media, media lacking glutamine, or glucose. Following 24 h of growth in glucose-depleted environment, glucose was added back to the media and rescue of the parasite’s growth was assessed. Data are represented as means ± SD from three independent biological replicates. (E) The apicoplast targeting sequence of TgPDH-E1a (aa 1–225, ABE76506) and mitochondrial targeting sequence of TgBCKDH-E1a (aa 1–73, XP_002366588) were replaced with the mitochondrial transit peptide of the superoxide dismutase 3 (SOD3) and myc-tagged <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004263#ppat.1004263-Pino2" target="_blank">[71]</a> to direct the expression of the fusion protein in the mitochondrion of Tg<i>e1a_ko</i> parasites for complementation (creating pTub8-SOD3mycPDHE1a and pTub8-SOD3mycBCKDHE1a respectively). Immunofluorescence assay shows localization of SOD3mycPDHE1a and SOD3mycBCKDHE1a in the single tubular mitochondrion (anti-myc (in green), anti-GAP45 (pellicle marker in red)). (F) Intracellular growth assay at 32 h post transient transfection of Tg<i>e1a_ko</i> with pTub8-SOD3mycPDHE1a, pTub8-SOD3mycBCKDHE1a and pTub8-mycNtGAP45 (negative control) in complete media or media depleted in glucose. Data are represented as means ± SD from three independent biological replicates. Only vacuoles containing parasites transiently expressing the transgene were taken into account. Over 200 vacuoles were counted per replicate. (G) CD1 mice were infected with RH (in blue) or Tg<i>e1a_ko</i> (in red) tachyzoites (∼15 parasites per mouse) and survival was assessed over 21 days. A challenge with ∼1000 wild-type RH tachyzoites was performed on mice that survived initial infection and survival followed for a further 10 days. Five mice were infected per condition.</p

Role of BCKDH during sexual development and in mosquito stages of <i>P. berghei</i>.

<p>(A) Asexual parasitaemia and male (micro-) and female (macro-) gametocytaemia in the peripheral blood of mice 3 days post infection with 1×10⁷ parasites i. p. Error bars show standard deviations from 3 mice. (B) Developmental capacity of gametocytes <i>in vitro</i> measured from the same infections shown in panel A. The relative ability of microgametocytes to release microgametes was assessed by counting exflagellation centres in a haemocytometer 15 min after addition to activating medium. The ability of activated macrogametocytes to become fertilised and convert to ookinetes was assessed by quantifying round and ookinete-shaped parasites following life staining of the surface marker P28. Colour code as in panel A; error bars show standard deviations. (C) Oocyst numbers on the midguts of individual <i>A. stephensi</i> mosquitoes on different days after feeding on three infected mice per mutant. Geometric means and 95% confidence intervals are also shown. (D) Sizes of individual oocysts from infected midguts at different days after infection. Black lines show geometric means and 95% confidence intervals. (E) Fluorescence micrographs of representative <i>A. stephensi</i> midguts dissected 14 days after feeding on wild type and mutant parasites expressing GFP. Scale bar = 0.5 mm. (F) Phase contrast images of representative oocysts. Scale bar = 10 µm. (G) Sporozoite numbers per mosquito as determined from 3 batches of 10 dissected salivary glands. Transmission from mosquito to mice was examined by measuring the prepatent period in 2 mice per group after bites from ∼20 mosquitoes or intraperitoneal injection of homogenates from 10 pairs of salivary glands. Data shown in all panels are representative of two independent experiments each performed with three infected mice per parasite line.</p

Proposed metabolic pathways, compartmentalisation and metabolic remodelling in <i>T. gondii</i> and <i>P. berghei</i>.

<p>(A) Schematic representation of the metabolism in <i>T. gondii</i> WT (left panel) and Tg<i>e1a_ko</i> (right panel) incorporating data from this study. (B) Scheme of the metabolism in <i>P. berghei</i> WT (left panel) and Pb<i>e1a_ko</i> (right panel). For both (A) and (B), the remodelling of the parasites metabolism upon ablation of BCKDH activity is highlighted in green. Dotted lines represent drops and disruption in the corresponding reactions. Abbreviations: AcCoA, acetyl-CoA; α-KG, α-ketoglutarate; Cit, citrate; Glc, glucose; Glu, glutamate; Gln, glutamine; Lac, lactate; Mal, malate; OAA, oxaloacetic acid; PEP, phosphoenolpyruvate; Pyr, pyruvate; Suc, succinate. Enzymes in red: BCKDH, branched chain keto acid dehydrogenase; ACL, ATP-citrate lyase; ACS, Acetyl-CoA synthetase; CS-II, second isoform of citrate synthetase.</p

<i>P. berghei</i> parasites lacking the BCKDH-E1a subunit exhibit a perturbed TCA cycle.

<p>Cultures containing ring/early-trophozoite WT and Pb<i>e1a_ko P. berghei</i>-infected RBCs were allowed to mature to schizonts and labelled with ¹³C-U-glucose or ¹³C-U-glutamine for 5 hr. Label incorporation was assessed by GC-MS. (A) Total ¹³C-glucose-derived label incorporation into central carbon metabolism metabolites in WT (blue) and Pb<i>e1a_ko</i> (red) <i>P. berghei</i>-infected RBCs, where label incorporation is the fraction of molecules of that metabolite containing one or more ¹³C carbons (after correction for natural abundance). Metabolites are colour-coded by metabolic pathway; central carbon metabolism, green; TCA cycle and associated amino acids, orange; PPP, purple; other, black. Error bars represent standard deviation (N = 4). Significance as determined by t-test is shown (corrected for multiple comparisons using the Holm-Sidak method), with significant (p-value&lt;0.05) differences indicated by an asterisk. † indicates metabolite not detected. (B) Mass isotopologue distributions of the TCA intermediates shown in Panel A. The x-axis indicates the number of ¹³C-atoms in each metabolite (‘M’ indicates the monoisotopic mass containing no ¹³C atoms). The y-axis indicates fractional abundance of each isotopologue when labelled with ¹³C-U-glucose (present in the culture medium at ∼50%). Error bars indicate standard deviation (N = 4). (C, D) As for A and B, but after labelling with ¹³C-U-glutamine (present in the culture medium at ∼98%).</p

BCKDH possesses PDH activity.

<p>(A) <i>In vitro</i> enzyme activity indicates a low level of classical branched chain keto-acid dehydrogenase activity and extensive pyruvate dehydrogenase activity in RH <i>T. gondii</i> cell lysates. Concentrations (mean ± SD) of acetyl-CoA (black), 2-methylpropanoyl-CoA (grey), and 3-methylbutanoyl-CoA (white columns), were measured following incubation of RH lysates with 2 mM pyruvate (black), 3-methyl-2-oxobutanoate (grey) or 4-methyl-2-oxopentanoate (white columns), respectively. (B) concentration of acetyl-CoA following <i>in vitro</i> incubation of RH or Tg<i>e1a_ko</i> extracts with 0.5 mM pyruvate (C–D) Relative abundance of acyl-CoA products following <i>in vitro</i> incubation of RH or Tg<i>e1a_ko</i> extracts with 0.5 mM (C) 3-methyl-2-oxobutanoate or (D) 4-methyl-2-oxopentanoate. (E–G) Relative abundance of hydroxyalkyl-TPP intermediates following incubation of RH or Tg<i>e1a_ko</i> lysates with 0.5 mM (E) pyruvate, (F) 3-methyl-2-oxobutanoate or (G) 4-methyl-2-oxopentanoate. Metabolite intensity (y-axis) is measured by LC-MS peak area (mean ± SD; n = 2). Significance as determined by t-test is shown, with p-values of &lt;0.05 and &lt;0.01 indicated by an asterisk and double asterisk, respectively.</p

BCKDH is required for conversion of pyruvate to acetyl-CoA and catabolism of glucose in the mitochondrion.

<p>Freshly egressed RH and Tg<i>e1a_ko</i> tachyzoites were labelled with ¹³C-U-glucose or ¹³C-U-glutamine for 4 h. Abundance and label incorporation were assessed by GC-MS and LC-MS. (A) Relative (%) abundance of selected metabolites in the Tg<i>E1a_ko</i> mutant parasites. Bars represent abundance of metabolites in Tg<i>e1a_ko</i> cells compared with a parental (RH) control. The dashed line refers the abundance of the metabolite in the parental control (‘100%’). 2HE-TPP refers to 2-hydroxyethyl-thiamine pyrophosphate, the stable intermediate specifically generated by pyruvate dehydrogenase activity. 2HE-TPP and acetyl-CoA were measured by LC-MS while other metabolites were measured by GC-MS (B) ¹³C-glucose and (C) ¹³C-glutamine incorporation into central carbon metabolites in RH (blue) and Tg<i>e1a_ko</i> (red) tachyzoites, where label incorporation is the fraction of molecules of that metabolite containing one or more ¹³C carbons (after correction for natural abundance). In A, B and C metabolites are colour-coded by metabolic pathway; central carbon metabolism, green; TCA cycle and associated amino acids, orange; PPP, purple; other, black. Error bars represent standard deviation (n = 3–6). Significance as determined by t-test is shown (corrected for multiple comparisons using the Holm-Sidak method), with significant (p-values of 0.05) differences indicated by an asterisk. † indicates metabolite not detected. (D) Mass isotopologue abundances of citrate generated in ¹³C-glucose and ¹³C-glutamine-fed RH and Tg<i>e1a_ko</i> tachyzoites. ‘M’ indicates the monoisotopic mass containing no ¹³C atoms. Error bars indicate standard deviation (n = 3). (E) Intracellular growth of RH (blue) and Tg<i>e1a_ko</i> (red) was assessed after 24 h in medium depleted of glucose complemented or not with 5 mM exogenous acetate. Data are represented as means ± SD from three independent biological replicates.</p

BCKDH is required for growth of <i>Plasmodium berghei</i> in mature RBCs.

<p>(A) Total lysates from a mixed population of parasitic stages for <i>P. falciparum</i> 3D7 (Pf), Pb wild type (WT) and Pb<i>e1a_ko</i> were analysed by Western blot. Expression of BCKDH-E1a (shown by the arrow) was assessed using cross-reacting anti-PfBCKDH-E1a antibodies. Profilin was used as loading control. (B) Parasitaemia was followed daily in mice infected with WT (blue line) or Pb<i>e1a_ko</i> (red line). Each line corresponds to the parasitaemia of one mouse. 5 mice were infected per condition. (C) Haematocrit was followed over the course of infection in mice infected with WT (blue) or Pb<i>e1a_ko</i> (red). Corresponding parasitaemia levels of this experiment are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004263#ppat.1004263.s003" target="_blank">Fig. S3B</a>. The dotted line represents the mean haematocrit level of uninfected control mice followed throughout the experiment. Data show mean ± SD from 4 mice per condition. (D) Parasitaemia was followed daily in mice pre-treated (full lines) or not (dotted lines) with phenylhydrazine to induce reticulocytosis 3 days prior infection with parasites. Mice were infected with 5×10⁶ WT (blue line) or Pb<i>e1a_ko</i> (red line) parasites. 5 mice were infected per condition and lines represent mean parasitaemia ± SD. (E) Invasion of Pb<i>e1a_ko</i> (red) compared to WT (blue). Vybrant Green-labeled purified schizonts were incubated with DDAO-SE-labeled purified normocytes or reticulocytes, and free merozoites were allowed to invade. Parasitaemia in the DDAO-SE-labeled target population was determined by flow cytometry. Invasion efficiency was determined as a percentage of the WT control parasites. Cytochalasin D (CD)-treated schizonts were used as a negative control. Data are represented as mean ± SD of three independent biological replicates. (F) Giemsa-stained blood smears showing normal development of WT, Pb<i>e1a_ko</i> and complemented Pb<i>e1a_ko</i>+Pfe1a parasites to the schizont stage in purified reticulocytes. Parasites were cultured <i>in vitro</i> for the times indicated. (G) Giemsa-stained blood smears showing development of the different strains within purified normocytes. WT parasites mature normally from ring to schizont stage while Pb<i>e1a_ko</i> degenerate rapidly. Complemented Pb<i>e1a_ko</i>+Pfe1a restored the ability of Pb<i>e1a_ko</i> to develop within normocytes. Parasites were cultured <i>in vitro</i> for the times indicated.</p