Posttranslational modification of TgHAD-2SCP-2 in wild-type T. gondii and transgenic parasites expressing HA-TgHAD-2SCP-2. Top, schematic representation of TgHAD-2SCP-2 showing the multidomains and their presumed length as indicated by the position of amino acids based on homology with various d-3-hydroxyacyl-CoA dehydrogenase and SCP-2 primary sequences. The dashed line and oversized Y indicate the antigenic fragment used to produce the anti-TgHAD-2SCP-2 antiserum. a–c, immunoblots of wild-type T. gondii (a) or parasites transfected with p1HA-TgHAD-2SCP-2 (b and c). Western blots analyses of the gel were probed with anti-TgHAD-2SCP-2 (a and b) or anti-HA (c) antibodies. The same blot has been used in b and c. Band A represents the full-length protein, whereas bands B, C, and D show fragments that are equivalent for each blot. The molecular weight markers in kilodaltons are at the left margin.

Localization and topology TgHAD-2SCP-2 and its cleavage products in transgenic parasites expressing various constructs derived from TgHAD-2SCP-2. (A) Parasites expressing HA-TgHAD-2SCP-2 labeled with anti-TgHAD-2SCP-2 antibodies for IFA (a) and immuno-EM (b), or with anti-HA antibodies (c). In fluorescence microscopy pictures, arrows and arrowheads show vesicles at the apical and basal ends of the parasite, respectively. In EM pictures, arrows pinpoint TgHAD-2SCP-2-containing structures. dg, secretory dense granule; n, nucleus; PM, plasma membrane. Bar, 0.5 μm. (B) Immunoblot of subcellular fractions of HA-TgHAD-2SCP-2–expressing parasites revealed first with anti-TgHAD-2SCP-2 and then with anti-HA antibodies. After parasite homogenization and clearance of the lysate of debris, the supernatant was collected to isolate vesicles by high-speed centrifugation. Vesicles from the pellet were either treated with protease inhibitors (P.I.) or subjected to mild proteolysis with proteinase K (prot. K) before Western blot analysis. The relative molecular weight of bands A and C′ is 69 and 35 kDa, respectively. The molecular weight markers in kilodaltons are at the right margin. (C) IFA on intracellular parasites stably expressing HA-Tg2SCP-2a, HA-Tg2SCP-2b, HA-Tg2SCP-2bΔSRL, or TgHAD-2SCP-2-YFP revealed by anti-HA antibodies (a–e) or anti-GFP antibodies (f). c and d, magnification of two single parasites that are representative of the dual localization of Tg2SCP-2b in vesicles (c) and in the cytosol (d).

SCP-2b–mediated lipid transfer and competition assays. The intervesicular transfer of BODIPY-PC from donor vesicles to acceptor vesicles was monitored in the presence of SCP-2b. PBS (control) or unlabeled lipids were added to the mixture at the indicated time (arrow), and their plots in black were compared with that of BODIPY-PC without addition of lipids (gray plots). Results shown are representative of three separate experiments. PE, phosphatidylethanolamine; PI, phosphatidylinositol.

Effect of SCP-2b on fatty acid and cholesterol uptake and incorporation into lipids in mammalian cells. (A) Lipid uptake assays. Confluent control ZF, SCP-2b–expressing ZF, control COS cells, TgHAD-2SCP-2–expressing COS cells, and TgSCP-2ΔSRL-expressing COS cells were incubated with complete MEM containing either [¹⁴C]oleate conjugated to BSA or [³H]cholesterol incorporated into LDL for 3 h at 37°C. After washing, the amount of cell-associated lipids was determined by radioactivity and expressed as cpm per milligram of cell protein. Values are means ± SD (n = 3), *p < 0.05 and **p < 0.01. (B) Lipid incorporation assays. Control COS cells, TgHAD-2SCP-2–expressing COS cells or TgSCP-2bΔSRL–expressing COS were used as described in A except that the incubation time with [¹⁴C]oleate-BSA was 20 min. After washing, cell protein concentration was determined, and lipids extracted to quantify the amount of radioactive phospholipids (PL), TAG, and cholesteryl oleate (CO) associated with the cells. Values are means ± SD (n = 3 or 4). *p < 0.05 and **p < 0.01.

Contribution of TgHAD-2SCP-2 in lipid uptake and formation in Toxoplasma. (A) Lipid uptake assays. After infection with control T. gondii (white symbols) or parasites expressing HA-TgHAD-2SCP-2 (black symbols), fibroblasts were exposed to [¹⁴C]oleate-BSA, [¹⁴C]palmitate-BSA, or [³H]cholesterol-LDL (chol) for 6 h at 37°C. After washing, parasites were isolated from host cells, and the amount of cell-associated lipids was determined by measuring radioactivity (expressed as cpm per milligram of cell protein). Data are means ± SD (n = 3), p < 0.01 for oleate and cholesterol uptake. (B) Lipid incorporation assays. Same parasite preparations and protocols were used as described in A except that the incubation time with [¹⁴C]oleate-BSA or [¹⁴C]palmitate-BSA was 60 min. After washing of infected cells and parasite purification, protein concentration was determined, and lipids were extracted to quantify the amount of radioactive phospholipids (PL), TAG, cholesteryl palmitate (CP), and cholesteryl oleate (CO) associated with the parasites. Values are means ± SD (n = 3 or 4). *p < 0.05 and **p < 0.01.

Sequence alignment of the predicted sequences of the SCP-2 domains of T. gondii and comparison with other organisms. (A) Sequence alignment of the two parasite SCP-2 domains by using the CLUSTALW program. Black and gray boxes indicate identical and similar amino acids, respectively. Amino acids in bold highlight the PTS1. Stars show residues that are important for the sterol transfer function (Stolowich et al., 2002). α1–5, α-helices 1–5; β1–5; β-sheets 1–5. (B) Multiple sequence alignment of individual T. gondii SCP-2 domain with the sequences from Homo sapiens SCP-2 (AAA03559), human SCP-2 domain at the C terminus of the DBP (P51659), Rattus norvegicus (P11915), Arabidopsis thaliana SCP-2 (NP_199103), Aedes aegypti (AAQ08007), Caenorhabditis elegans SCP-2 domain at the C terminus of the short chain dehydrogenase (AAK68187), Neurospora crassa SCP-2 (XP_960876), and Rhodobacter sphaeroides SCP-2 (YP_352482).

Subcellular distribution of TgHAD-2SCP-2 in wild-type Toxoplasma in comparison with mammalian cells transfected with TgHAD-2SCP-2. (A) Fluorescence assays on human fibroblasts stably expressing EGFP-catalase (b) and transfected with pmyc-TgHAD-2SCP-2 (a) or pTgHAD-2SCP-2-myc (d) before labeling with anti-myc antibodies. (B) a–d, IFA on wild-type intracellular parasites labeled with preimmune serum (a) or anti-TgHAD-2SCP-2 (b–d). e, immuno-EM labeling on cryosections of Toxoplasma-infected fibroblasts using anti-TgHAD-2SCP-2 antibodies revealing the morphology of a TgHAD-2SCP-2-containing vesicle (arrow). dg, secretory dense granule; n, nucleus; PM, plasma membrane. Bar, 0.5 μm.

Binding assays of lipids to SCP-2b. Fluorometry assays to monitor the interaction of the recombinant TgSCP-2b with fluorescently labeled lipids at different concentrations. This assay was performed by comparison of the fluorescence intensity in the absence and the presence of the protein. NBD-labeled cholesterol (A) or NBD-labeled PC (B) was incubated with or without SCP-2 (6.7 × 10⁻⁷ M) for comparison of fluorescence intensity. Data expressed in arbitrary units, are means ± SD of nine independent assays.

Influence of SCP-2b on transport of newly synthesized cholesterol to the plasma membrane in mammalian cells. (A) Assays on ZF stably expressing myc-TgSCP-2b. IFA has been performed on the transfected cells using anti-myc antibodies. Confluent ZF controls (mock transfected), transfected with TgSCP-2b, or NHFs) were pulsed with [¹⁴C]acetate for 7 min at 37°C and chased for the indicated times. Radioactive cholesterol incorporation in the plasma membrane was determined using the cholesterol oxidase assay. Results are the mean ± SD of three or four experiments and are expressed as percentage of radioactive cholestenone per 5 × 10⁶ cells. (B) Assays on COS cells transiently transfected with plasmids containing myc-tagged TgHAD-2SCP-2, HA-tagged TgSCP-2a, HA-tagged TgSCP-2b, or HA-tagged TgSCP-2bΔSRL. IFA performed on the transfected cells with the indicated constructs using anti-myc or anti-HA antibodies. The same protocol described in A was used to monitor the cholesterol transfer to the surface of control COS cells (mock transfected), COS cells expressing TgHAD-2SCP-2, TgSCP-2a, TgSCP-2b, or TgSCP-2bΔSRL. Values expressed as indicated in A are means ± SD (n = 3 or 4). *p < 0.05 and **p < 0.01.

Role of TgHAD-2SCP-2 in lipid distribution and parasite development. (A and B) Fluorescence assays on intracellular parasite either mock transfected as control or stably transfected with HA-TgHAD-2SCP-2. Parasites were incubated with filipin (A) or Nile red (B) to assess the distribution of membrane cholesterol or lipid bodies, respectively. Arrows pinpoint lipids accumulated inside the PV. (C) Quantitative distribution of the number of Toxoplasma per PV either wild-type parasites (black) or HA-TgHAD-2SCP-2–expressing parasites (black). Parasite development was monitored at the indicated time points p.i. for two independently infected monolayers.