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Host cells: mobilizable lipid resources for the intracellular parasite Toxoplasma gondii

Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO 63110, USA

View larger version (123K): [in a new window]   Fig. 1. Selective diversion of host cell lipids. Coverslip-grown host cells were incubated with fluorescent probes alone (hosts), infected with T. gondii prior to the introduction of the probe (toxo+lipid), or labeled with the probe before infection with T. gondii (lipid+toxo). Images of live cells were immediately acquired by confocal microscopy. Single, 0.4 µm thick optical sections are shown. Cholesterol, NBD-cholesterol; PC, BODIPY-phosphatidylcholine; PA, BODIPY-phosphatidic acid; C4C9, C4-BODIPY-C9. Red emission in the C4C9 panels corresponds to excimer shift of highly concentrated BODIPY. Intracellular parasites in the PC lipid+toxo panel are indicated with asterisks. Bars, 20 µm (hosts); 5 µm (toxo+lipid, lipid+toxo).

View larger version (96K): [in a new window]   Fig. 2. Neutral lipid handling by intracellular parasites. The indicated fluorophore-conjugated probes were introduced to hosts alone (host and -ctl) or hosts infected with T. gondii before (TL) or after (LT) labeling. Lipids of hosts (host), isolated parasites (TL, LT), or contaminating membranes (-ctl) were resolved by thin layer chromatography. BODIPY-fatty-acid-labeled samples were resolved by developing TLC plates in hexane/ether/acetic acid (70:30:1), whereas NBD-cholesterol-labeled samples were resolved by developing first with hexane/ether/acetic acid (70:30:1), then with toluene/acetone (70:10), and finally with chloroform/acetic acid (96:4), allowing the plates to dry between each development. Lipids were detected by illumination of the plates with UV light. O, origin. The migration of the fluorescent probes NBD-cholesterol (chol.), BODIPY-C12 (C12), and C4-BODIPY-C9 (C4C9), as well as the cholesteryl ester standard cholesteryl BODIPY-C12 (chol. C12) are noted. Presumed cholesteryl esters are indicated with asterisks and metabolites of the BODIPY-fatty acids are indicated with arrowheads. The prominent species at the origin of the C12 and C4C9 panels is suspected to be a BODIPY-phospholipid on the basis of its migratory behavior in very polar solvents.

View larger version (46K): [in a new window]   Fig. 3. Storage of neutral lipids. Parasites grown in the presence of the fluorescent fatty acid C4C9 were added either to poly-L-lysine-coated coverslips (extracellular) or coverslip-grown host cells. After 1 hour (extracellular and short infection) or 6 hours (6h infection), coverslips were fixed and processed for immunocytochemistry using antibodies directed against MIC4, ROP2 or GRA2. Primary antibodies were detected by subsequent incubation with Cy5-conjugated anti-mouse or anti-rabbit antibodies. Additional coverslips were incubated with the lipophilic dye Nile Red or the nucleic acid stain TOPRO3. Images were acquired by confocal microscopy. Single, 0.4 µm thick optical sections are shown. The immunochemical staining of parasite organelles appears blue, whereas C4C9 is depicted in green. The two channels merged onto the brightfield image are shown to facilitate the comparison of labeling patterns. Bar, 5 µm.

View larger version (185K): [in a new window]   Fig. 4. The T. gondii lipid body. Parasites were grown in host cells exposed to the fluorescent probe NBD-cholesterol throughout the course of infection. Following host cell lysis, the purified, labeled parasites were allowed to infect fresh coverslip-grown host cells. After a 1 hour infection, coverslips were fixed, photoactivated in the presence of diaminobenzidine, and processed for electron microscopic analysis. An intracellular parasite is shown in low magnification in the top-left panel, and its prominent lipid body demarcated with electron-dense reaction product is shown in high magnification in the top-right panel. Two additional profiles of typical lipid bodies are depicted in the lower panels. The arrow in the top-left panel denotes an intravacuolar liposome. Bars, 500 nm (low magnification); 100 nm (high magnification).

View larger version (54K): [in a new window]   Fig. 5. Use of phospholipid precursors by T. gondii. The indicated 14C-labeled lipid precursors were introduced to hosts alone (host and -ctl) or hosts infected with T. gondii before (TL) or after (LT) labeling. Lipids of hosts (host), isolated parasites (TL, LT), or contaminating membranes (-ctl) were resolved by thin layer chromatography using the solvent system chloroform/methanol/acetic acid/water (25:15:4:2), and detected by autoradiography after visualizing standards. The migration of standards (stds.) is indicated: PA, phosphatidic acid; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine; PI, phosphatidylinositol; PC, phosphatidylcholine; SM, sphingomyelin. O, origin. Arrowheads denote the migration of radiolabeled precursors. Both within and between individual precursor label samples, spotting was normalized for protein concentration (with the exception of the host cell contaminant samples, which were spotted with a volume equal to that of the parasite samples). Because the host lanes were exposed to film for periods briefer than the -ctl, TL and LT lanes, the host lanes are separated from the others. All other lanes were exposed to film for equal time periods.

View larger version (53K): [in a new window]   Fig. 6. De novo synthesis of phospholipids by T. gondii. The indicated 14C-labeled phosphatidylcholine precursors were introduced to either untreated, purified extracellular parasites (EC) or intracellular (IC) parasites. Labeling was performed at either 2°C (2C) or 37°C (37C) before parasites were harvested for analysis by thin layer chromatography using the solvent system chloroform/methanol/acetic acid/water (25:15:4:2), and lipids detected by autoradiography after visualizing standards. The plates were exposed to X-ray film for identical time periods. The migration of standards (stds.) is indicated: PA, phosphatidic acid; PC, phosphatidylcholine; acet. acid, acetic acid. Asterisks indicate three clearly resolved phosphatidylcholine spots. O, origin.

View larger version (67K): [in a new window]   Fig. 7. Precursor requirements for T. gondii synthesis of phosphatidylcholine. Either CHO cells alone (CHO panel, H lanes), 89.1 cells alone (89.1 panel, H lanes), or hosts harboring T. gondii (CHO and 89.1 panels, T lanes) were pulse-labeled with [14C]choline. Following the removal of the label, the cultures were chased for the indicated time periods with label-free media. The cells were then harvested and the parasites in the T samples were isolated. Samples were normalized for protein concentration, resolved by thin layer chromatography using the solvent system chloroform/methanol/acetic acid/water (25:15:4:2), and lipids detected by autoradiography after visualizing standards.