Apicoplast and endoplasmic reticulum cooperate in fatty acid biosynthesis in apicomplexan parasite Toxoplasma gondii

Abstract

Apicomplexan parasites are responsible for high impact human diseases such as malaria, toxoplasmosis, and cryptosporidiosis. These obligate intracellular pathogens are dependent on both de novo lipid biosynthesis as well as the uptake of host lipids for biogenesis of parasite membranes. Genome annotations and biochemical studies indicate that apicomplexan parasites can synthesize fatty acids via a number of different biosynthetic pathways that are differentially compartmentalized. However, the relative contribution of each of these biosynthetic pathways to total fatty acid composition of intracellular parasite stages remains poorly defined. Here, we use a combination of genetic, biochemical, and metabolomic approaches to delineate the contribution of fatty acid biosynthetic pathways in Toxoplasma gondii. Metabolic labeling studies with [(13)C]glucose showed that intracellular tachyzoites synthesized a range of long and very long chain fatty acids (C14:0-26:1). Genetic disruption of the apicoplast-localized type II fatty-acid synthase resulted in greatly reduced synthesis of saturated fatty acids up to 18 carbons long. Ablation of type II fatty-acid synthase activity resulted in reduced intracellular growth that was partially restored by addition of long chain fatty acids. In contrast, synthesis of very long chain fatty acids was primarily dependent on a fatty acid elongation system comprising three elongases, two reductases, and a dehydratase that were localized to the endoplasmic reticulum. The function of these enzymes was confirmed by heterologous expression in yeast. This elongase pathway appears to have a unique role in generating very long unsaturated fatty acids (C26:1) that cannot be salvaged from the host.

FIGURE 1.

Metabolic labeling of intracellular tachyzoites with [U-¹³C]glucose and analysis of de novo fatty acid biosynthesis. A, T. gondii-infected fibroblasts were precultured in the presence or absence of ATc for 24 h and then labeled with [U-¹³C]glucose 24 h prior to parasite egress. After host cell lysis, extracellular tachyzoites were metabolically quenched in a dry ice/ethanol bath and separated from host cell debris by filtration prior to metabolite extraction. Examples of different labeling patterns (¹³C indicated in gray) and their interpretation are schematically shown for myristate. B, lipids from [U-¹³C]glucose-labeled tachyzoites were subjected to methanolysis and trimethylsilyl derivatization and analyzed by GC-MS. A portion of the mass spectrum containing the molecular ions of the methyl ester of myristate is shown. Unlabeled myristoyl methyl ester (M₀) has an m/z of 242 atomic mass units. Higher mass isotopomers (M_1–12) contain between 1 and 12 ¹³C atoms, corresponding to incorporation of ¹³C into the fatty acid (FA) biosynthetic pathways via [¹³C]acetyl-CoA. arb., arbitrary units. C, incorporation of ¹³C into the major fatty acids of intracellular wild type T. gondii tachyzoites (labeling is given as mol % of all labeled mass isotopomers (M_1–12) relative to M₀, after correction for natural abundance). The fatty acid notation Cn:m indicates the length of the fatty acid (n, carbon number) and degree of unsaturation (m, number of double bonds). Note that treatment of wild type parasites with ATc (black) does not result in any significant changes of the fatty acid labeling pattern when compared with untreated controls (white). D, intracellular tachyzoites of the T. gondii ΔACP/ACPi mutant carrying an inducible copy of FASII ACP were labeled with [U-¹³C]glucose in the presence or absence of ATc. Total lipids were extracted from the purified tachyzoites and released fatty acid methyl esters analyzed by GC-MS. The mass spectrum (molecular ion region from m/z 240 to 250) of the myristate fatty acid methyl esters from unlabeled parasites, and from [¹³C]glucose-labeled parasites cultured in the absence or presence of ATc is shown. Incorporation of multiple ¹³C₂H₄ units into myristate is observed in the absence of ATc (ACP active) and largely inhibited in the presence of ATc (ACP repressed). The spectrum is representative of three individual experiments. E, total ¹³C incorporation into fatty acids in ΔACP/ACPi tachyzoites labeled in the presence (black) or absence (white) of ATc. Error bars represent standard deviation where n = 3. Fatty acids for which significant changes in labeling were observed in the presence and absence of ATc using the Wilcoxon rank sum test (p values less than 0.05) are indicated with an asterisk. The absolute amount of incorporation can vary from experiment to experiment depending on the stage of the culture (no ATc in C and E). We therefore always directly compare ATc-treated samples with an untreated control from the same culture batch.

FIGURE 2.

Growth of ΔACP/ACPi parasites in media supplemented with myristic and palmitic acid. Growth of ΔACP/ACPi parasites stably expressing a dTomatoRFP transgene was evaluated by measuring parasite fluorescence daily in a 96-well culture format (35). Parasites were grown in normal growth media supplemented with fatty acid-free BSA (A) or media supplemented with 1 μm of each myristic and palmitic acid coupled to BSA (3:1 molar ration) (B). Parasites were grown in the presence (black) or absence (white) of ATc. The average of fluorescence intensity (arbitrary units) for three independent replicates is shown, and error bars represent the standard deviation for each data point. Continuous culture in BSA-conjugated fatty acids (or higher serum supplementation) did not lead to continuous growth of the ACP mutant in the presence of Atc. FA, fatty acids.

FIGURE 3.

T. gondii fatty acid elongation pathway is localized to the parasite endoplasmic reticulum. Transgenic parasite lines were allowed to infect coverslip cultures and were fixed and processed for immunofluorescence assay 24 h later. Transgenic proteins were marked with an HA (A–C) or a Myc (D–F) epitope tag detected with suitable antibodies (red channel). All strains carried a Der1GFP marker (green) previously shown to localize to the endoplasmic reticulum (17). DAPI and phase contrast images are shown for comparison. See supplemental Table S1 for reference to T. gondii gene models and “Experimental Procedures” for details on gene identification and cloning. Note that all six candidate fatty acid elongation enzymes are found to be associated with the endoplasmic reticulum. Tagged ELO-C produces a more restricted pattern at the apical side of the nuclear envelope.

FIGURE 4.

T. gondii genes complement yeast fatty acid elongation mutants. Yeast mutants with deletions in specific genes encoding fatty acid elongation enzymes were transformed with plasmids carrying T. gondii candidate genes as detailed under “Experimental Procedures.” The resulting strains were used to conduct spotting assays on YPGalGlu media and demonstrate complementation (panels on the left). Control experiments using the same yeast cultures were done on media that select against the yeast rescue plasmid and thus indicate that phenotypic complementation is due to presence of the T. gondii constructs only (5′FOA-GalGlu), or media that indicate presence of the yeast rescue plasmids only (ScUra-GalGlu) (center and right panels, respectively). Yeast cultures were progressively diluted prior to plating from left to right as indicated. A, complementation analyses for the yeast elongase ScΔelo2Δelo3 double mutant and the yeast (B) enoyl-CoA reductase (ScΔECR) and dehydratase (ScΔDEH) mutants, respectively. Note that under restrictive conditions (5′FOA-GalGlu) growth depends on the presence of the T. gondii genes. Only experiments that resulted in phenotypic complementation are shown here. See Table 2 for full detail on the genotype of all yeast strains.

FIGURE 5.

Conditional mutants lacking individual fatty acid elongases exhibit normal growth. We constructed conditional mutants for each of the three T. gondii ELO genes. A–F, immunofluorescence assay detecting the expression of the HA epitope-tagged ectopic allele for the indicated ELO genes when grown in the absence (−ATc) or presence (+ATc) for 24 h. Merge images also show DAPI staining of DNA. G–I, Southern blot analysis probing genomic DNA from RH wild type, a strain carrying both the native and the conditional allele, and the mutant carrying only the conditional allele (from left to right for each of the respective mutants as indicated). Probes detect the respective coding region of each gene and are detailed in supplemental Table S2. In each blot the expected size of the restriction fragment for the native locus (e.g. ELO-B) and the conditional locus (e.g. ELO-Bi) is indicated with an arrowhead. Note loss of native loci. J and K, Western blot analysis detecting ELO-Bi and ELO-Ci expression using an antibody against the HA tag. GRA8 is shown as a loading control. L, growth of the conditional ELO mutants was measured by plaque assay in absence and presence of ATc as indicated. ΔACP/ACPi serves as positive control.

FIGURE 6.

Radiolabeling analysis shows a reduction in long chain fatty acid synthesis in the T. gondii ELO-B and ELO-C mutant. T. gondii ELO mutants (A, ΔELO-A; B, ΔELO-B; and C, ΔELO-C) were metabolically labeled with [¹⁴C]acetate; lipids were extracted, and fatty acid methyl esters were analyzed by reverse phase thin layer chromatography, and representative autoradiographs for each mutant are shown. Each panel shows the parental strain carrying native and conditional locus (e.g. ELO-B/ELO-Bi) to the left and the mutant (ΔELO-B/ELO-Bi) to the right grown in the absence (−) or presence (+) of ATc.

FIGURE 7.

Loss of elongases results in selective reductions in the rate of synthesis and cellular levels of unsaturated fatty acids. Intracellular tachyzoites of the three ΔELO mutants were labeled with [U-¹³C]glucose in the presence (black) or absence (white) of ATc as detailed in Fig. 1. A, level of ¹³C incorporation into the major fatty acids (FA) of the isolated tachyzoites. Error bars represent standard deviation where n = 3–4. B, changes in the relative abundance of individual fatty acids in ΔELO tachyzoites in the presence of ATc as measured by GC-MS (values are given relative to the abundance of this fatty acid species in the no ATc control). Results are indicative of >3 individual replicates. Individual values for all experiments are listed in Table 4. Fatty acids for which significant changes in labeling were observed in the presence and absence of ATc using the Wilcoxon Rank Sum Test (p values less than 0.05) are indicated with an asterisk.

FIGURE 8.

GC-MS analysis of fatty acid methyl esters from S. cerevisiae complemented with T. gondii ELO-B. Fatty acids from yeast Δelo2/Δelo3 double deletion strain complemented by TgELO-B (see Fig. 5) were extracted and subjected to GC-MS analysis as described under “Experimental Procedures.” An overlay of the chromatogram of fatty acid methyl esters from this strain and the wild type is shown. A, enlargement of the region of the trace showing C22 and C24 methyl esters. B, region showing C26 methyl esters.

FIGURE 9.

Toxoplasma acquires fatty acids through a complex network of synthesis and uptake. A, T. gondii (pink) is an intracellular pathogen capable of fatty acid and lipid salvage from the host cell (blue). This process can intersect host cell import as well as synthesis routes. B, in addition, the parasite harbors three fatty acid synthesis pathways that are localized to different cellular compartments. C, apicoplast (green)-localized FASII pathway produces significant amounts of myristic and palmitic acid in addition to lipoic acid relying on cytoplasmic glycolysis for precursors. E, Toxoplasma also maintains an ER-associated elongase system that synthesizes very long chain monounsaturated fatty acids, subsequently using the activity of ELO-B and ELO-C. D, T. gondii FASI remains largely uncharacterized. Its stage-specific expression pattern and localization are not established. It is also unclear whether this megasynthase synthesizes fatty acids de novo like the FASI of humans or acts as an elongase for saturated fatty acids, as demonstrated for the FASI of C. parvum. Major products are highlighted in red. Des, desaturase; PEP, phosphoenolpyruvate; Mal, malonate; Ac, acetate; vlcFA, very long chain fatty acids.

FIGURE 10.

Total fatty acid composition of uninfected HFF host cells and RH tachyzoites. The total lipid extract of human foreskin fibroblasts (HFF) (A) and isolated purified tachyzoites (labeled in their host cells as detailed in the “Experimental Procedures”) (B) was subjected to solvolysis in methanolic-HCl and derivatized in trimethylsilyl reagent, and fatty acid methyl esters were detected by GC-MS. The mol % of the major fatty acids are shown (mean ± S.D.). The lower panels show same data set scaled to a maximum of 5% fractional abundance to display low abundance species.