Metabolic Needs and Capabilities of Toxoplasma gondii through Combined Computational and Experimental Analysis

Abstract

Toxoplasma gondii is a human pathogen prevalent worldwide that poses a challenging and unmet need for novel treatment of toxoplasmosis. Using a semi-automated reconstruction algorithm, we reconstructed a genome-scale metabolic model, ToxoNet1. The reconstruction process and flux-balance analysis of the model offer a systematic overview of the metabolic capabilities of this parasite. Using ToxoNet1 we have identified significant gaps in the current knowledge of Toxoplasma metabolic pathways and have clarified its minimal nutritional requirements for replication. By probing the model via metabolic tasks, we have further defined sets of alternative precursors necessary for parasite growth. Within a human host cell environment, ToxoNet1 predicts a minimal set of 53 enzyme-coding genes and 76 reactions to be essential for parasite replication. Double-gene-essentiality analysis identified 20 pairs of genes for which simultaneous deletion is deleterious. To validate several predictions of ToxoNet1 we have performed experimental analyses of cytosolic acetyl-CoA biosynthesis. ATP-citrate lyase and acetyl-CoA synthase were localised and their corresponding genes disrupted, establishing that each of these enzymes is dispensable for the growth of T. gondii, however together they make a synthetic lethal pair.

Fig 1. Reconstruction workflow for ToxoNet1 summarized as a flowchart illustration.

Fig 2. Breakdown of the metabolic network.

A) By presence of gene-reactions association; B) By enzyme classes encoded in the genome of T. gondii (EC nomenclature, class 1: oxidoreductases, class 2: transferases, class 3: hydrolases, class 4: lyases, class 5: isomerases, class 6: ligases).

Fig 3. Both ACS and ACL are dispensable in the tachyzoite stage of T. gondii.

(A) Schematic representation of the two pathways to produce acetyl-CoA in the cytosol of T. gondii. Abbreviations: AcCoA, acetyl-CoA; α-KG, α-ketoglutarate; Cit, citrate; Glc, glucose; Lac, lactate; Mal, malate; OAA, oxaloacetic acid; Pyr, pyruvate; Suc, succinate. Enzymes in red: ACL, ATP-citrate lyase; ACS, Acetyl-CoA synthetase. (B) Scheme of the knock-in strategy used to introduce a 3Ty-tag in the endogenous loci of ACS, ACL and AT1. (C) Localization of endogenous ACS, ACL and AT1 C-terminally Ty-tagged (ACS-3Ty, ACL-3Ty and AT1-3Ty) in the cytoplasm, cytosol and endoplasmic reticulum respectively of intracellular parasites using anti-Ty as well as anti-GAP45 that stains the periphery and DAPI which stains the nucleus of the parasite. (D) Immuno-blot of total lysates from Ku80ko parasites expressing the C-terminally Ty-tagged endogenous ACS, ACL and AT1 proteins by Western blot using anti-Ty antibodies. Anti-Profilin (Prf) represents a loading control. (E) Schematic representation of the direct knockout strategy by double homologous recombination where ACS was replaced by the chloramphenicol resistance cassette and ACL by the HXGPRT selection cassette. The position of the primers used to confirm the integration and the length of the PCR products are indicated. PCRs performed on genomic DNA extracted from Ku80ko, ACSko and ACLko strains confirm the integration of the selection cassette and loss of the corresponding gene locus. The sequences of the primers can be found in S7 Table. (F) Plaque assays performed with Ku80ko, ACSko and ACLko parasite lines fixed after 7 days. No significant defect in the lytic cycle could be observed. (G) Intracellular growth assay performed on Ku80ko, ACSko and ACLko strains by determining the number of parasites per vacuole 24h post infection. Data are represented as mean ± SD from 3 biological replicates.

Fig 4. Generation of an inducible ACL knockdown in ACSko parasites.

(A) Schematic representation of the U1 snRNP-mediated ACL gene silencing with Cre-recombinase dependent positioning of U1 in Ku80ko wildtype and ACSko parasites. (B) PCRs performed on genomic DNA extracted from Ku80ko, ACL-lox, ACSko/ACL-lox validating integration of the pKI-ACL-3TyLox3’UTRLoxU1 construct to knock down ACL in the different strains. The sequences of the primers can be found in S7 Table (C and D). Immuno-blot of total lysates from ACL-lox, ACSko/ACL-lox where ACL-lox was integrated in the ACSko strain or ACS was knocked out in ACL-lox. Both independent lines show increased levels of ACL when ACS is absent. Western blot was performed using anti-Ty antibodies. Anti-TgProfilin (Prf) represents a loading control.

Fig 5. ACS and ACL are dually essential.

(A) Following transfection of ACL-lox and ACSko/ACL-lox with Cre recombinase, excision and repositioning of U1 is followed by genomic PCRs over several culture passages (every 48h) using primers P17/P18 as depicted in Fig 4A (P0, extracellular parasites 48h after Cre transfection; P1, extracellular parasites passaged once ~96h post transfection; P2, extracellular parasites passaged twice ~140h post transfection). (B) Histogram showing percentage of excised parasites over several passages (every 48h) in ACL-lox and ACSko/ACL-lox populations following transfection with Cre recombinase. Excised parasites were visualized by IFA looking for loss of ACL-3Ty signal. Due to fluctuation in transfection efficiency, data from one biological replicate is shown and represents mean ± SD from 3 technical replicates. 3 biological replicates were done and gave the same results. (P0, intracellular parasites 30h after Cre transfection; P1, intracellular parasites passaged once ~72h post transfection; P2, intracellular parasites passaged twice ~100h post transfection). (C) Immunofluorescence assay confirms the loss of ACL-3Ty and parasite pellicle integrity in a subset of vacuoles 30h following transfection of ACL-lox and ACSko/ACL-lox strains with a Cre recombinase expressing plasmid. IFAs were stained using anti-Ty, anti-GAP45 (Pellicle), anti-5F4 (mitochondrion) or anti-Atrx1 (apicoplast) antibodies and DAPI (nucleus). Arrowheads highlight nuclear and mitochondrial material lost in the vacuolar space and loss of the apicoplast due to loss of pellicle integrity.