The toxoplasma apicoplast phosphate translocator links cytosolic and apicoplast metabolism and is essential for parasite survival

Abstract

Apicomplexa are unicellular eukaryotic pathogens that carry a vestigial algal endosymbiont, the apicoplast. The physiological function of the apicoplast and its integration into parasite metabolism remain poorly understood and at times controversial. We establish that the Toxoplasma apicoplast membrane-localized phosphate translocator (TgAPT) is an essential metabolic link between the endosymbiont and the parasite cytoplasm. TgAPT is required for fatty acid synthesis in the apicoplast, but this may not be its most critical function. Further analyses demonstrate that TgAPT also functions to supply the apicoplast with carbon skeletons for additional pathways and, indirectly, with energy and reduction power. Genetic ablation of the transporter results in rapid death of parasites. The dramatic consequences of loss of its activity suggest that targeting TgAPT could be a viable strategy to identify antiparasitic compounds.

Figure 1. High frequency targeting of the parasite genome using modified cosmid clones

(A) Schematic representation of cosmid engineering using a gene replacement cassette as example, homologous recombination is guided by a 50 bp sequence added to the ends by PCR. (B) RsrII Restriction mapping of cosmid PSBYL85 prior (TgAPT, predicted restriction fragments are: 18,280, 11,852, 11,146, 2,635, and 2,490 bp), and after recombineering with cassette pHcG (TgAPT-HA, 18280, 11852,11146, 6028, and 2490 bp) and pICG (ΔTgAPT 22,407, 11,852, 11,146, and 2,490), respectively. Diagnostic restriction fragments for the parental cosmid (arrowhead) and modified cosmid (double arrowhead) are highlighted. Schematic representations of homologous recombination events in the T. gondii genome for cosmid-based genome tagging (C) or gene disruption (F). Diagnostic PCR products and restriction fragments for the native and modified TgAPT locus are highlighted. (D and G) PCR analysis of clones derived from a stable chloramphenicol resistant population after transfection with modified cosmid DNA. RH strain is shown as wild type (WT) control. (E and H) Southern blot analysis of parasite mutant clones along with RH, RH-HXGPRT⁻ and parental iTgAPT-HA strain (TATi strain with TgAPT minigene under control of the tet-regulatable promoter). Blots were probed with a 600 bp fragment of the TgAPT 5’ untranslated region as indicated. Maps shown in this figure are not to scale.

Figure 2. Genetic ablation of TgAPT results in loss of apicoplast FASII dependent PDH(E2) lipoylation

(A and B) Western blot analyses of TgAPT and ACP mutant after 0 to 4 days of ATc treatment (TgAPT mutant could only be analyzed for 3 days due to parasite death). The product of the regulated minigene is detectable in the absence of ATc (using an antibody against the HA epitope (A) or ACP (B)) and shows a severe reduction within 24 hours. Extracts of equal numbers of parasites (5 × 10⁶) were loaded and GRA8 serves as loading control. We also probed steady state levels of lipoylated PDH(E2) with a lipoylation specific antibody and found that levels are reduced in both mutants in response to ATc treatment. (C and D) Pulse-chase analysis of PDH(E2) lipoylation. Levels of newly synthesized lipoylated PDH(E2) are severely reduced in the TgAPT and ACP mutants after one or two days, respectively. Lipoylation of mitochondrial E2 enzyme subunits (mito-E2) is not affected in either mutant. Human PDH and mitochondrial E2 subunits (a contamination from the human host cell (Crawford et al., 2006; Mazumdar et al., 2006; van Dooren et al., 2008)) are marked by an asterisks. This experiment uses an antibody that recognizes all lipoylated proteins (we demonstrate that the 92 kDa lipoylated protein represents the apicoplast PDH-E2 through immunoprecipitation experiments using a specific antibody, see supplementary figure S2). P, pulse, C, chase.

Figure 3. Parasites lacking TgAPT show a more pronounced growth defect than parasites lacking apicoplast FASII

(A) Fluorescence growth assay of the TgAPT mutant in the presence and absence of ATc (a dTomato-RFP reporter was introduced by transfection and stable transformats were isolated by flow cytometry as previously described (van Dooren et al., 2008). Each data point represents the mean of six wells and the error bar gives the standard deviation). (B) Confluent host cell cultures were infected with 400 tachyzoites of various parasite strains (as indicated) and incubated for 10 days in the absence and presence of ATc. Cells were fixed and stained as described (Striepen and Soldati, 2007). Assays were performed in duplicate flasks and plaque areas were measured by image analysis after scanning. Areas are shown as whisker plots (C) providing the mean and 1.5 times the interquartile distance as the box. Statistical significance was evaluated using the unpaired t-test. No ATc white boxes, plus ATc, hatched boxes. No plaques were observed in the ATc treated APT mutant.

Figure 4. TgAPT but not ACP mutant shows dramatic loss of mitochondrial membrane potential

(A) Fluorescence microscopy of JC-1 labeled parasites shows accumulation of green fluorescent dye in the cytoplasm and the formation of red fluorescent J aggregates in the mitochondrion. This red fluorescence is abolished when parasites are treated with ionophores (valinomycin (+VM) is shown here). (B) TgAPT and ACP mutants were treated for 0 to 3 days with ATc, labeled with JC-1 and analyzed by flow cytometry. Untreated parasites show bright green and red fluorescence. Sustained ATc treatment leads to a pronounced decline in red fluorescence in the TgAPT mutant, but not the ACP mutant over the observation period.

Figure 5. Identification of lysine residues critical for TgAPT activity by mutational analysis and in vivo complementation assay

A ΔTgAPT/iAPT-HA mutant clone carrying the dTomato-RFP marker was transfected with the wild type APT coding sequence expressed from a constitutive promoter carrying an N-terminal Ty1 epitope tag (Ty-WT), the same transgene carrying in addition a FKB12 destabilization domain (DD-Ty1-WT), and three point mutations of the latter (DD-Ty1-K 67, 145 and 310 A respectively). (A–E) Immunofluorescence assays showing parasites cultured in the presence of 0.1 µM Shield1 for 36 hours and subsequently labeled with antibodies to the Ty-1 epitope tag (green) and the apicoplast resident protein Cpn60 (red, note that cytoplasmic red background fluorescence is due to residual dTomato-RFP autofluorescence after fixation). Transgenic parasite strains are as indicated. (F–I) Plaque assays of indicated strains grown in the absence (−ATc) or presence of 0.5 µg/ml ATc (+ATc), or the presence of ATc and Shield1 (+ATc +Shld1). (J–M) Fluorescene growth assays circles, −ATc; triangles, +ATc; squares, +ATc +Shld1 (each data point represents the mean of six wells and the error bar gives the standard deviation).

Figure 6. TgAPT links apicoplast metabolism to the parasite cytoplasm

Schematic representation of apicoplast key anabolic pathways (note that this is a highly simplified outline and that the number of arrows does not reflect the number of enzymatic steps needed). Note that TgAPT serves as a hub to supply carbon (through PEP and triose phosphate) as well as energy and reduction power through the triose shuttle. TgAPT is a phosphate antiporter and P_i is not shown for simplicity.