Toxoplasma gondii exploits host low-density lipoprotein receptor-mediated endocytosis for cholesterol acquisition

Abstract

The obligate intracellular protozoan Toxoplasma gondii resides within a specialized parasitophorous vacuole (PV), isolated from host vesicular traffic. In this study, the origin of parasite cholesterol was investigated. T. gondii cannot synthesize sterols via the mevalonate pathway. Host cholesterol biosynthesis remains unchanged after infection and a blockade in host de novo sterol biosynthesis does not affect parasite growth. However, simultaneous limitation of exogenous and endogenous sources of cholesterol from the host cell strongly reduces parasite replication and parasite growth is stimulated by exogenously supplied cholesterol. Intracellular parasites acquire host cholesterol that is endocytosed by the low-density lipoprotein (LDL) pathway, a process that is specifically increased in infected cells. Interference with LDL endocytosis, with lysosomal degradation of LDL, or with cholesterol translocation from lysosomes blocks cholesterol delivery to the PV and significantly reduces parasite replication. Similarly, incubation of T. gondii in mutant cells defective in mobilization of cholesterol from lysosomes leads to a decrease of parasite cholesterol content and proliferation. This cholesterol trafficking to the PV is independent of the pathways involving the host Golgi or endoplasmic reticulum. Despite being segregated from the endocytic machinery of the host cell, the T. gondii vacuole actively accumulates LDL-derived cholesterol that has transited through host lysosomes.

Figure 1

Intracellular distribution of cholesterol in uninfected and T. gondii–infected fibroblasts. Uninfected fibroblasts (A) or infected with T. gondii (B, arrow) were cultivated for 24 h in medium containing 10% FCS, fixed, cytochemically stained with filipin for cholesterol detection, and observed by fluorescence microscopy. In B, the arrowhead shows the location of the PVM. Alternatively, freshly lysed-out and dividing parasites (C) were similarly processed for filipin-cholesterol labeling. In C, the arrows indicate the nascent apical complexes of the two progenies and the arrowhead, the residual body of the parent cell.

Figure 2

Parasite replication in the SSD mutant in the presence or absence of LP. (A) The distribution of PV sizes expressed in percent was determined at 48 and 60 h after infection of the SSD mutant or CHO cells with parasites in the presence 10% FBS in the culture medium. Data represent the averages for at least 50 randomly selected vacuoles in two different experiments. (B) At 12, 24, 48, 60, and 72 h, corresponding to incubation times of intracellular parasites in the presence or absence of LP, uracil incorporation was assayed using the SSD mutant infected every 12 h, as described in Materials and Methods. Results are expressed in counts per minute of [³H]uracil incorporated into parasites per milligram cell protein and are means of two separate experiments done in duplicate.

Figure 4

Comparison of receptor-mediated endocytosis and pinocytosis in uninfected and T. gondii–infected fibroblasts. Uninfected CHO cells (•) or infected with T. gondii (○) for 24 h were then incubated at 37°C in the presence of the indicated concentration of ¹²⁵I-LDL, ¹²⁵I-transferrin, or HRP for 2 h. After washing, the amount of cell-associated ligand was determined by radioactivity or enzymatic activity measurement and expressed as nanograms of ligand per milligram cell protein. Values are means ± SD of three separate experiments.

Figure 3

Influence of exogenous cholesterol and lipoproteins on parasite proliferation. (A) Uracil incorporation was assayed using CHO cells infected for 24 or 48 h in medium containing 10% LPDS, supplemented (or not) with LP. Data in percent are expressed relative to control (incubation in the presence of LP) taken as 100% ± SEM from four separate experiments done in triplicate. Differences between values of control and experimental groups were statistically significant (*P < 0.01). (B) The distribution of PV sizes expressed in percent was determined at 48 and 60 h after infection of HFF with parasites in 10% LPDS in the presence or absence of LP. Data represent the averages for at least 50 randomly selected vacuoles in four different experiments. (C) The CHO and CHO-7 cells infected with parasites (arrows) were cultivated for 24 h in medium containing 10% FCS or 10% LPDS, respectively, fixed, cytochemically stained with filipin for cholesterol detection, and observed by fluorescence microscopy. (D) Uracil incorporation was assayed using CHO cells infected for 24 h in medium containing 10% LPDS (solid bars), or supplemented with 10 μg/ml of water-soluble cholesterol (chol), or with LDL at the indicated concentrations. Data in percent are expressed relative to control (incubation in the presence of LPDS) taken as 100% ± SEM from three separate experiments done in duplicate. Differences between values of control and experimental groups were statistically significant (*P < 0.01; **P < 0.005). (E) The percent distribution of PV sizes was determined at 24 h after infection of HFF with parasites in medium containing 10% LPDS, supplemented or not with LDL at the indicated concentrations. Data represent the averages for at least 50 randomly selected vacuoles in two different experiments.

Figure 5

Kinetics of [NBD-C]-LDL acquisition in uninfected or T. gondii–infected cells. 24 h after infection in medium containing 10% LPDS, infected fibroblasts were pulse-labeled at 37°C with 0.1 mg/ml of [NBD-C]-LDL for 7 min (A), 12 min (B; fluorescent PVM is indicated by the arrow), or 60 min (C). Cells were washed and processed for fluorescence observation. In parallel, noninfected fibroblasts were labeled for 60 min with the fluorescent lipoproteins and treated under the same conditions (D). In one experiment, infected fibroblasts were pulse-labeled at 37°C with 0.1 mg/ml of [NBD-C]-LDL for 60 min, and then chased for 9 h in medium containing 10% LPDS (E).

Figure 6

Absence of [NBD-C]-LDL acquisition in pyrimethamine-treated or extracellular parasites. 24 h after infection in medium containing 10% LPDS, infected fibroblasts were preincubated for 20 h without (A) or with pyrimethamine at 1 (B) or 10 (C) μM. As observed in B′ and C′, the pyrimethamine-treated cells contain vacuoles with dying or dead parasites (arrows), having abnormal shapes. Cells were then pulse-labeled at 37°C with 0.1 mg/ml [NBD-C]-LDL for 60 min, washed, and processed for fluorescence observation. Extracellular tachyzoites were pulse-labeled at 37°C with 0.1 mg/ml of [NBD-C]-LDL for 60 min, washed, and directly observed by fluorescence microscopy.

Figure 7

Blockade of [NBD-C]-LDL acquisition by parasites with anti–LDL receptor antibodies or chloroquine. 24 h after infection in medium containing 10% FBS, fibroblasts infected by T. gondii were preincubated without (A) or with (B) 400 μg/ml anti–LDL receptor antibodies for 1 h at 37°C, and then for 30 min at 4°C, washed, pulse-labeled at 37°C for 10 min with 0.1 mg/ml [NBD-C]-LDL, washed, and processed for fluorescence observation. In another set of experiments, 24 h after infection in medium containing 10% LPDS, infected fibroblasts were incubated without drug (C) or with chloroquine either at 100 μM for 150 min (D) or at 200 μM for 30 min (E), pulse-labeled at 37°C for 30 min with 0.1 mg/ml of [NBD-C]-LDL, washed, and processed for fluorescence observation. Vacuoles containing parasites are illustrated by arrows.

Figure 8

Blockade and reversibility of [NBD-C]-LDL acquisition by parasites with progesterone and U18666A. 24 h after infection in medium containing 10% LPDS, fibroblasts infected by T. gondii (arrows) were preincubated without drug (A), with progesterone at 10 μg/ml for 24 h (B and C), with U18666A at 1 μg/ml for 90 min (D), or with progesterone at 10 μg/ml for 20 h, and then with U18666A at 0.1 μg/ml for 90 min. After washing, cells were pulse-labeled at 37°C for 10 min with 0.1 mg/ml of [NBD-C]-LDL, washed, and processed for fluorescence observation. A chase of 24 h in medium containing 10% LPDS was performed in the experiment in C before analysis of the fluorescence pattern.

Figure 9

Reduction of parasite replication and cholesterol content in the 2-2 mutant or in fibroblasts treated with progesterone. (A) Uracil incorporation was assayed using the 2-2 mutant or CHO cells as control infected for 24 or 48 h in medium containing 10% FBS. Data in percent are expressed relative to control taken as 100% ± SEM from three separate experiments done in duplicate. Differences between values of control and experimental groups were statistically significant (*P < 0.005). (B) The 2-2 mutant cells infected with parasites (arrows) were cultivated for 24 h in medium containing 10% FCS, fixed, cytochemically stained with filipin for cholesterol detection, and observed by fluorescence microscopy. (C) Uracil incorporation was assayed using fibroblasts infected for 24 or 48 h in medium containing 10% FBS plus 10 μg/ml of progesterone. Data in percent are expressed relative to control (infected fibroblasts without progesterone) taken as 100% ± SEM from three separate experiments done in duplicate. Differences between values of control and experimental groups were statistically significant (**P < 0.0005).

Figure 10

Hypothetical pathway for cholesterol acquisition from endocytosed LDL by intracellular T. gondii. This figure depicts the potential source of cholesterol for intracellular T. gondii after the binding and endocytosis of LDL via specific receptors as well as the trafficking of cholesterol effluxed from lysosomes to the PV containing four parasites. This pathway III can be blocked at several steps: (1) anti–LDL receptor antibodies block binding to LDL receptors, (2) chloroquine and sucrose affect the lysosomal function, abolishing LDL proteolysis, and (3) progesterone, U18666A, and NPC mutation impair cholesterol translocation from late endosomes/lysosomes, sequestering cholesterol inside these organelles. When blocked, the pathways II in the presence of LP, IIIb, and IIIc have no effect on T. gondii replication or cholesterol transport to intravacuolar parasites. The absence of host endo- and lysosomal markers detectable within the PV at any stage during infection excludes a direct access of host intravesicular LDL to the vacuole (pathway IV). No direct experimental evidence is yet available to support parasite cholesterol biosynthesis (pathway I). C, cholesterol; CE, cholesteryl ester; Cyto B, cytochalasin B; DOXP, 1-deoxy-d-xylulose 5-phosphate.