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First published online March 12, 2004
doi: 10.1242/10.1242/jcs.00988

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Developmental-stage-specific triacylglycerol biosynthesis, degradation and trafficking as lipid bodies in Plasmodium falciparum-infected erythrocytes

¹ PRESTO, Japan Science and Technology Corporation, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan
² Sumitomo Chemical, 4-2-1 Takatsukasa, Takarazuka, Hyogo 665-0051, Japan
³ Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan
⁴ Department of Biomedical Chemistry, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyou-ku, Tokyo 113-0033, Japan
⁵ Department of Molecular Parasitology, Ehime University School of Medicine, Shigenobu-cho, Ehime 791-0295, Japan

View larger version (47K): [in a new window]   Fig. 1. Stage-specific incorporation of 14C-labeled oleic acid into TAG in P. falciparum-infected erythrocytes. Tightly synchronized cultures of Honduras-1 were labeled and total lipids associated with infected erythrocyte were analysed. (A) TLC of the extracted total lipid species for neutral (left) and polar (right) lipids. The positions corresponding to the authentic cold lipid species are indicated: CE, cholesteryl ester; DAG, diacylglycerol; FFA, free fatty acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; TAG, triacylglycerol. (B) Kinetics of the accumulation of various lipid species incorporated with radiolabeled oleic acid in P. falciparum-infected erythrocytes during intraerythrocytic development. At different time points, the distribution of lipid-associated radioactivity in TAG (filled circles), PC (open circles) and PE (open triangles) are shown. Values are the total radioactivity of each lipid from 5 ml of 3% hematocrit culture. At the top is shown the dominant parasite morphology at various sampling times. One of four independent experiments displaying similar profile is shown.

View larger version (31K): [in a new window]   Fig. 2. Degradation of the neutral lipid pool associated with P. falciparum-infected erythrocytes and its release into the culture medium during the later stages of intraerythrocytic cycle. Pulse-chase experiment using tightly synchronized cultures of Honduras-1 was performed. (A) TLC of extracted neutral lipids associated with cells and released into medium. The positions corresponding to the authentic cold lipid species are indicated: CE, cholesteryl ester; DAG, diacylglycerol; FFA, free fatty acid; TAG, triacylglycerol. (B) Kinetics of TAG degradation and the release of FFA into the culture medium. At different times, the distributions of lipid-associated radioactivity in cells (filled symbols) and medium (open symbols) are shown. Values are the total radioactivity of each lipid either from the cell or from the medium of 5 ml 3% hematocrit culture. Symbols: FFA, triangle; TAG, circle. On top is shown the dominant parasite morphology at different sampling times. A representative result is shown from two independent experiments displaying similar profiles.

View larger version (92K): [in a new window]   Fig. 3. Stage-dependent localization of lipid bodies in P. falciparum-infected erythrocyte. The erythrocytes infected with tightly synchronized Dd2 were stained with DAPI and the lipophilic dye, Nile Red (I) and Bodipy 493/503 (II) at the stages of (A) ring, (B) early trophozoite, (C) late trophozoite, (D) early schizont, (E) segmented schizont, (F) ruptured segmented schizont and (G) free merozoite. The first image in each set represents bright-field overlaid with DAPI (blue), the second is the Nile Red or Bodipy 493/503 staining (green) and the third is an overlay of lipid body fluorescence with DAPI signals. White areas denote regions of co-localization. Scale bar, 2 µm.

View larger version (114K): [in a new window]   Fig. 4. Electron micrographs of lipid bodies. Mature trophozoite (A) and early schizont (B) stages of P. falciparum-infected erythrocytes. The presumed lipid body structure (*), nucleus (N), food vacuole (FV) and rhoptry (R) are indicated. Scale bar, 500 nm.

View larger version (40K): [in a new window]   Fig. 5. Co-localization of lipid bodies with the PV marker protein SERA. (A) Fluorescent microscopic analysis of schizont-stage P. falciparum-infected erythrocyte stained with Sudan III and anti-SERA Ab. From left to right, panels represent bright-field overlaid with DAPI, Sudan III, SERA and overlay of Sudan III and SERA. Yellow areas denote regions of overlap. Single-stained cells (top two rows) exhibit virtually no fluorescence with the opposing filter. (B) Confocal microscopic analysis of lipid bodies and SERA co-localization in schizont (a) and segmented schizont (b). Three sections are collected through the center of the parasite by confocal microscopy. The planes on the first and third row of each set are 0.55 µm apart from the plane shown in the central row. The images, left to right, are: bright field overlaid with DAPI (blue), Sudan III (red channel), SERA protein (green channel) and merge images of red and green channels. Scale bar, 2 µm.

View larger version (70K): [in a new window]   Fig. 6. Inhibition in the trafficking of Nile Red fluorescent lipid bodies with BFA. Tightly synchronized cultures of 3D7 were treated with 5 µg ml-1 BFA or 0.1% ethanol (solvent control) for 0-26 hours (A) or 0-30 hours (B), or re-incubated for another 8 hours after BFA treatment (C). BFA inhibition of lipid body trafficking was visualized in live parasites (a) in comparison with the corresponding control culture (b). From left to right, panels represent bright-field images, DAPI signals (blue), Nile Red staining patterns (green) and merged images of DAPI and Nile Red. White areas denote regions of co-localization. Scale bar, 2 µm.

View larger version (57K): [in a new window]   Fig. 7. Effect of TFP on the formation of Nile Red fluorescent lipid bodies. (A) Tightly synchronized cultures of Dd2 were treated with 100 µM (a) or 500 µM (b) TFP, or 0.1% ethanol (c) for 3 hours. (B) Parasite cultures treated with 100 µM (a) or 500 µM (b) TFP, or 0.1% ethanol (c) were re-cultured for another 6 hours. Panels in each set are, from left to right, bright field overlaid with DAPI (blue), Nile Red staining patterns (green) and merged images of DAPI and Nile Red. White areas denote regions of co-localization. Scale bar, 2 µm.

View larger version (17K): [in a new window]   Fig. 8. DGAT activity in P. falciparum-infected erythrocyte ghosts and in isolated parasites. (A) Time dependence of TAG synthesis in P. falciparum-infected erythrocyte ghosts. TAG accumulation was monitored for 0-15 minutes from 50 µg infected erythrocyte ghost lysate at 20 mM Mg2+. (B) Dependence of DGAT activity on protein concentration. TAG synthesis rate using 0-50 µg infected erythrocyte ghost lysate at 20 mM Mg2+ was measured. (C) Mg2+ dependency of the DGAT activity associated with isolated parasite cells. DGAT activity at various concentration of Mg2+ (0-100 mM) using 6.0 µg lysate prepared from trophozoite/schizont-rich cultures by saponin treatment and N2 cavitation was determined. (A-C) The reaction mixtures with and without 1,2-DAG are indicated by filled circles and filled triangles, respectively; values are averages of triplicates; and s.d. for each data is indicated by error bar.

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