Toxoplasma gondii requires its plant-like heme biosynthesis pathway for infection

Abstract

Heme, an iron-containing organic ring, is essential for virtually all living organisms by serving as a prosthetic group in proteins that function in diverse cellular activities ranging from diatomic gas transport and sensing, to mitochondrial respiration, to detoxification. Cellular heme levels in microbial pathogens can be a composite of endogenous de novo synthesis or exogenous uptake of heme or heme synthesis intermediates. Intracellular pathogenic microbes switch routes for heme supply when heme availability fluctuates in their replicative environment throughout infection. Here, we show that Toxoplasma gondii, an obligate intracellular human pathogen, encodes a functional heme biosynthesis pathway. A chloroplast-derived organelle, termed apicoplast, is involved in heme production. Genetic and chemical manipulation revealed that de novo heme production is essential for T. gondii intracellular growth and pathogenesis. Surprisingly, the herbicide oxadiazon significantly impaired Toxoplasma growth, consistent with phylogenetic analyses that show T. gondii protoporphyrinogen oxidase is more closely related to plants than mammals. This inhibition can be enhanced by 15- to 25-fold with two oxadiazon derivatives, lending therapeutic proof that Toxoplasma heme biosynthesis is a druggable target. As T. gondii has been used to model other apicomplexan parasites, our study underscores the utility of targeting heme biosynthesis in other pathogenic apicomplexans, such as Plasmodium spp., Cystoisospora, Eimeria, Neospora, and Sarcocystis.

Fig 1. Toxoplasma gondii encodes its de novo heme biosynthetic pathway within 3 subcellular locations.

a, The working model of the de novo heme biosynthesis in Toxoplasma parasites. The enzymes catalyzing the de novo heme biosynthesis are distributed within three subcellular locations in the parasites, whereas they are only localized in the mitochondria and cytoplasm in mammals. b, Determination of the expression of the heme biosynthetic genes in Toxoplasma during its acute infection and their subcellular locations by endogenous gene tagging with 3xHA or 3xmyc epitopes. A subunit of Toxoplasma mitochondrial ATPase (TgF1β) and an apicoplast-associated thioredoxin family protein (TgATrx1) were used as the mitochondrial and apicoplast markers, respectively. TgActin was used as a cytoplasm marker. Bar = 2 μm. ALA, 5-aminolevulinic acid; ALAS, 5-aminolevulinic acid synthase; Api, apicoplast; Copro III, coproporphyrinogen III; CPOX, Coproporphyrinogen III oxidase; FECH, Ferrochelatase; Gly, glycine; HMB, hydroxymethylbilan; IVN, intravacuolar network; Mito, mitochondria; PBG, porphobilinogen; PBGD, Porphobilinogen deaminase; PBGS, Porphobilinogen synthase; PPIX, protoporphyrin IX; PPO, Protoporphyrinogen oxidase; Protogen, protoporphyrinogen IX; PV, parasitophorous vacuole; PVM, parasitophorous vacuole membrane; SucCoA, Succinyl-CoA; Toxo, Toxoplasma gondii; UROD, Uroporphyrinogen III decarboxylase; Urogen III, uroporphyrinogen III; UROS, Uroporphyrinogen III Synthase.

Fig 2. Toxoplasma parasites principally rely on their de novo heme biosynthesis for intracellular growth and pathogenesis.

a, Growth comparison of the ALA-starved and non-starved Δalas::NLuc parasites in media containing or lacking ALA. The parasites were grown in confluent HFFs and their luciferase activities were measured every 24 h for up to 96 h. Data represent mean ± SEM of n = 3 biological replicates. b, Acute virulence determination of TgALAS-deficient parasites in a murine model. Ten mice of equal numbers of males and females were used for each strain. c, Evaluation of repression efficiency of TgFECH by ATc treatment via immunoblotting. TgFECH was endogenously tagged with a 3xmyc tag at its C-terminus for recognition by immunoblotting. The lysates were also probed against TgGRA7 as a loading control. d, Replication assessment of the TgFECH knockdown parasites. T7S4-TgFECH and its parental strains were pre-treated with ATc for the period described in the scheme before replication assay. Data represent mean ± SD of n = 3 biological replicates. e-f, Replication assay of Δcpox and Δppo parasites. Data represent mean ± SD of n = 3–4 biological replicates. g-h, Acute virulence measurement of Δcpox and Δppo parasites in a murine model. 5 male and 5 female mice were used for each strain. The statistical significance for each animal study in b, g, and h was calculated using the Log-rank (Mantel-Cox) test. Statistical significance in the rest of the studies was calculated by two-tailed unpaired Student’s t-test. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; n.s., not significant.

Fig 3. Toxoplasma harbors an active de novo heme biosynthetic pathway.

a, Complementation of Toxoplasma orthologs of heme biosynthetic genes in the corresponding S. cerevisiae heme-deficient knockouts. EV, empty vector; SD, synthetic defined medium; Ura, uracil. b, Total heme quantification in heme-deficient parasites. A protoporphyrin IX-based fluorescence assay was used to quantify the total heme. The total heme levels in transgenic parasite strains were normalized against the corresponding parental strains. Data represent mean ± SEM of n = 3 biological replicates with 3 technical replicates each. c, The average parasite replication or growth rates showed a positive correlation with the normalized heme abundances. Given the different genetic backgrounds of parental strains for individual knockouts, the plots were organized in different groups. The average numbers of parasites per PV for WT, Δcpox, ΔcpoxCPOX, Δppo, and ΔppoPPO were derived from Fig 2E and 2F. The average numbers of parasites per PV for WT::NLuc, Δalas::NLuc (starved) + ALA, Δalas::NLuc (starved)—ALA, and ΔalasALAS::NLuc were derived from S3A Fig. The average numbers of parasites per PV for TaTi-TgFECH (ATc-treated) and T7S4-TgFECH (ATc-treated) were derived from Fig 2D. The normalized growth rates of WT::NLuc, WT::NLuc + ALA, and WT::NLuc + SA at 48 h post-infection were derived from S7 Fig and S12 Fig. d, Chemical interference in heme production in the parasites requires a functional heme biosynthetic pathway in Toxoplasma. The TgFECH knockdown parasites, along with its parental strain, were treated with ATc for 144 h before a 48-h stimulation or repression of heme production by ALA or SA, respectively. The total heme abundances for each treatment per strain were normalized against the ATc-treated TaTi-TgFECH strain (left panel) or the same strain grown in ATc-containing, but ALA- or SA-lacking, media (right panel). Data represent mean ± SEM from n = 3 biological replicates. e, Partial restoration of TgFECH expression in ATc-treated T7S4-TgFECH parasites helped them respond to the growth stimulation by ALA treatment. Data represent mean ± SD of n = 3 biological replicates. Statistical significance in all of the studies listed in this figure was calculated by two-tailed unpaired Student’s t-test. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; n.s., not significant.

Fig 4. Chemical interrogation of Toxoplasma’s de novo heme production by oxadiazon and its derivatives reduced the intracellular growth of the parasites.

a, Chemical structures of oxadiazon and two derivatives. b, Efficacy determination of oxadiazon and its derivatives in the inhibition of WT Toxoplasma growth using a luciferase-based growth assay. The Δppo::NLuc, ΔppoPPO::NLuc, and WT::NLuc-pTub-TgPPO strains were included for evaluating the target specificity of the inhibitors to TgPPO. Pyrimethamine, an antibiotic targeting folic acid metabolism that is irrelevant to heme biosynthesis, was also included for assessing the specificity of these PPO inhibitors. Data shown in the table represent mean ± SEM of n = 3 biological replicates with 3 technical replicates each. The standard errors for individual IC₅₀s listed in the table were calculated from the IC₅₀s derived from 3 independent biological replicates for each inhibitor. The IC₅₀ values were obtained by curve fitting using the function of “normalized response” vs. “[inhibitor]”, under the “dose-response-inhibition” regression program embedded in GraphPad Prism software (8^th version). c, Heme levels were reduced in the parasites upon treatment with oxadiazon and its derivatives. Data shown here represent mean ± SEM of n = 5 biological replicates. d, Toxoplasma was incapable of taking up extracellular heme to support its intracellular growth. Data represent mean ± SD of n = 3 biological replicates. Statistical significance for the assays described in this figure was determined by two-tailed unpaired Student’s t-test. *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant.