The intracellular parasite Toxoplasma gondii depends on the synthesis of long-chain and very long-chain unsaturated fatty acids not supplied by the host cell - PubMed (original) (raw)

The intracellular parasite Toxoplasma gondii depends on the synthesis of long-chain and very long-chain unsaturated fatty acids not supplied by the host cell

Srinivasan Ramakrishnan et al. Mol Microbiol. 2015 Jul.

Abstract

Apicomplexa are parasitic protozoa that cause important human diseases including malaria, cryptosporidiosis and toxoplasmosis. The replication of these parasites within their target host cell is dependent on both salvage as well as de novo synthesis of fatty acids. In Toxoplasma gondii, fatty acid synthesis via the apicoplast-localized FASII is essential for pathogenesis, while the role of two other fatty acid biosynthetic complexes remains unclear. Here, we demonstrate that the ER-localized fatty acid elongation (ELO) complexes are essential for parasite growth. Conditional knockdown of the nonredundant hydroxyacyl-CoA dehydratase and enoyl-CoA reductase enzymes in the ELO pathway severely repressed intracellular parasite growth. (13) C-glucose and (13) C-acetate labeling and comprehensive lipidomic analyses of these mutants showed a selective defect in synthesis of unsaturated long and very long-chain fatty acids (LCFAs and VLCFAs) and depletion of phosphatidylinositol and phosphatidylethanolamine species containing unsaturated LCFAs and VLCFAs. This requirement for ELO pathway was bypassed by supplementing the media with specific fatty acids, indicating active but inefficient import of host fatty acids. Our experiments highlight a gap between the fatty acid needs of the parasite and availability of specific fatty acids in the host cell that the parasite has to close using a dedicated synthesis and modification pathway.

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Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Figure 1. The apicoplast localized fatty acid synthase type II pathway and the ER associated fatty acid elongation pathway interact in the synthesis of fatty acids in T. gondii

The enzymes of the type II fatty acid synthesis (FASII) pathway localize to apicoplast lumen (green). A starter molecule is synthesized by condensation from CoA activated precursors on acyl carrier protein (ACP). The molecule is sequentially reduced, dehydrated and reduced to yield a fully reduced fatty acyl chain. This process is repeated in multiple rounds to generate myristate or palmitate as the final products. The fatty acid elongation (FAE) pathway unfolds on the cytoplasmic face of the endoplasmic reticulum and its membrane bound enzymes catalyze a similar set of reactions. An elongase (ELO) condenses a malonyl-CoA molecule with a fatty acyl-CoA, for ELO-A the fatty acid is palmitate derived from the FASII pathway. T. gondii harbors three elongase enzymes, with catalytic specificity determined by length and saturation of the fatty acid. The products of all three elongases are then reduced, dehydrated and reduced by a ketoacyl-CoA reductase (KCR), hydroxyacyl-CoA dehydratase (DEH) and enoyl-CoA reductase enzymes (ECR). The enzymes that are the focus of this study are highlighted in red. Note that the carbon source for the two pathways is distinct, and that only the elongation pathway is efficiently labeled using acetate isotopes.

Figure 2. Parasite mutants in the dehydratase and enoyl reductase enzymes of the fatty acid elongation pathway exhibit severe growth defects

T. gondii conditional mutants for DEH and ECR were generated by insertion of a tetracycline regulatable promoter into the chromosomal locus of the respective gene by homologous recombination of a targeting construct (A). Mutants were identified by PCR. Clones that lack a diagnostic 750 bp product were identified as dehydratase mutants (iΔDEH) (B), The abundance of DEH mRNA transcript in iΔDEH parasites was determined in ATc treated (+) and untreated parasites (−) by quantitative PCR. Transcript levels for acetyl-CoA reductases (ACCase) another enzyme involved in fatty acid synthesis were measured to control knock down specificity (C). Clones that produced a new 2 kb product were identified as enoyl-CoA reductase (iΔECR) mutants (D). All clones were tested for the presence of genomic DNA samples using a control primer set which produces a 500 bp band (B, D). The growth of conditional mutants iΔECR and iΔDEH was evaluated by plaque assay in the absence or presence of ATc in the medium as indicated. The growth of ΔKu80/TATi, the parental line used to generate these mutants is unaltered by ATc treatment (E). Growth of iΔECR and iΔDEH (F) parasites stably expressing a transgene resulting in the expression of double Tomato red fluorescent protein was evaluated by measuring parasite fluorescence in 96 well cultures daily for nine days. The average fluorescence intensity (arbitrary units) for three independent replicates is shown and error bars represent the standard deviation for each data point. Parasites were grown in the absence (−) or presence (+) of ATc or under ATc after three days of pre-incubation in the previous passage (pre).

Figure 3. Analysis of fatty acid synthesis in mutant parasites by metabolic labeling using radioactive or stable isotope precursors

T. gondii conditional mutants for the hydroxyacyl-CoA dehydratase or enoyl-CoA reductase of the elongase pathway were labeled with [14C]-acetate and total cellular fatty acids analyzed by reverse phase TLC as their methylesters (the migration position of [14C]-palmitate methylester which was used as a standard in this experiment is indicated on the right hand side). Representative autoradiographs for the parental strain ΔKu80/TATi and the iΔDEH, mutant (A) and for the iΔECR mutant (B) are shown. The data shown is representative of 3 biological replicates for iΔECR, and more than three for iΔDEH. Parasites were grown in the absence (−) or presence (+) of ATc prior to the addition of 14C-acetate (48h for iΔDEH and 24h for iΔECR). Note that the TLC mobility of fatty acid methyl esters is inversely related to chain length. iΔDEH parasites were also subjected to comprehensive fatty acid analysis following stable isotope labeling with [13C]-glucose (C) or [13C]-acetate (D, E). Lipids were extracted and 13C-labeling of individual fatty acid methylesters determined by GC/MS. The level of incorporation of heavy isotope due to labeling is shown for parasites grown in the absence (white bars) or presence (black bars) of ATc in (C) and (D) respectively. Bars show the mean of three technical replicates and error bars represent the standard deviation of those measurements. (E) Changes in the overall abundance of fatty acids for [13C]-acetate labeled iΔDEH parasites depending on growth in the presence or absence of ATc. Please note that the absolute abundance of C24:1 is very low making the measurement of label incorporation in panels C,D and abundance in panel E susceptible to noise. Results are representative of 3 individual biological replicates. Average values for the representative replicate are listed in Table 1 and Table 2. 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 4. Repression of DEH leads to selective changes in tachyzoite phospholipid composition

iDEH parasites were cultivated in HFF in the presence or absence of ATc (48hr). Parasites were purified from host debris and major phospholipid classes analyzed by LC/MS. The relative abundance of selected PI (panel A), PE (panel B) and PC (panel C) species are shown. The precise acyl composition of selected molecular species was determined by MS/MS. Molecular species of PI (38:4) and PE (38:4) contained predominantly C18:1 and C20:4. Data are represented as mean +/- SEM for three replicate analyses derived from two independent experiments.

Figure 5. Chemical complementation of fatty acid elongation mutants depends on the enzyme lost and the specific fatty acid species provided

HFF were infected with T. gondii iΔDEH (A–F) and iΔECR (G) parasites stably expressing a dTomato-RFP transgene and cultivated in the presence of BSA-fatty acid conjugates. Intracellular parasite growth following repression of DEH or ECR in the presence of different fatty acids was monitored by fluorescence in 96 well cultures. Bars show percent fluorescence (mean of three measurements) compared to untreated controls included in the same plate after six days of culture. The concentration of fatty acids (conjugated to BSA) in the medium was 250 μM, unless stated otherwise. The BSA conjugates included individual fatty species or mixtures of saturated fatty acids (Sat FA; C18:0, C20:0, C22:0 and C24:0), unsaturated fatty acids (Unsat FA; C18:1, C20:1, C22:1 and C24:1) or combined fatty acids (FA; C18:0, C20:0, C22:0, C24:0, C18:1, C20:1, C22:1 and C24:1). Intracellular growth of i DEH parasites in HFF cultured in the presence of ATc and (A) the combined fatty acid mixture, (B) saturated fatty acids (Sat) or unsaturated fatty acids (Unsat) alone, or (C) individual saturated and (D) individual monounsaturated fatty acids. The effect of adding higher concentrations of FA on i DEH growth was also tested (E). Addition of unsat FA to iΔDEH infected HFF also restored the capacity of these parasites to form plaques (F). In contrast, growth of the iΔECR mutant was not restored when infected HFF were cultured in the presence of BSA-FA mix (G). Similar results were obtained with other FA mixtures (not shown). The results were analysed using an unpaired t-test. Differences with a P value < 0.05 were deemed statistically significant and are indicated by an asterisk.

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The intracellular parasite Toxoplasma gondii depends on the synthesis of long-chain and very long-chain unsaturated fatty acids not supplied by the host cell - PubMed (original) (raw)