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© The Rockefeller University Press,
0021-9525/2000//167 $5.00
The Journal of Cell Biology, Volume 149, Number 1,
, 2000 167-180
Original Article |
Toxoplasma gondii Exploits Host Low-Density Lipoprotein Receptor-Mediated Endocytosis for Cholesterol Acquisition
keith.joiner{at}yale.edu
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.
Key Words: Toxoplasma gondii • parasitophorous vacuole • somatic cell mutant • LDL endocytic pathway • cholesterol transport
© 2000 The Rockefeller University Press
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Introduction |
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Small soluble molecules of <1,400 D are able to cross the PVM through functional pores (Schwab et al. 1994). Although devoid of transmembrane transporter/receptors of host cell origin (Porchet-Hennere and Torpier 1983), the PVM contains numerous secreted parasite proteins that might be implicated in metabolite transport (Sinai and Joiner 1997; Lingelbach and Joiner 1998). Within the vacuolar space is a tubulo-reticular network connected to the PVM, which likely increases the exchange surface between the host cytoplasm and intravacuolar parasites (Sibley et al. 1995).
Of importance, the PVM of T. gondii is tightly enshrouded by host mitochondria and endoplasmic reticulum (ER), the host cell lipid biosynthetic apparatus (Jones et al. 1972; Melo et al. 1992; Lindsay et al. 1993; Sinai et al. 1997). This organelle association has been postulated to play a role in lipid and possibly membrane scavenging from these host organelles to the intravacuolar parasite at sites of PVM-organelle association (Sinai et al. 1997). Indeed, T. gondii seems to be deficient in its ability to synthesize selected phospholipids de novo (Sinai, A.P., K.A. Joiner, and D.R. Voelker, unpublished observations).
Toxoplasma membranes contain cholesterol based on both biochemical and morphological criteria (Monteiro Cintra and de Souza 1985; Gallois et al. 1988; Foussard et al. 1991a, Foussard et al. 1991b). Cholesterol is concentrated in rhoptries, apical secretory organelles implicated in the extension of the PVM during invasion. Indeed, these organelles have a very high cholesterol/phospholipid molar ratio of 1.5 (Foussard et al. 1991a). In higher eukaryotic cells, cholesterol homeostasis is finely regulated by transcriptional, translational, and posttranslational mechanisms (reviewed in Goldstein and Brown 1990; Brown and Goldstein 1999). Cells have a number of options when it comes to the use of cholesterol for membrane biogenesis or synthesis of new molecules derived from cholesterol. This latter is synthesized in the ER via the key enzyme of the mevalonate pathway, the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Newly synthesized cholesterol is transported rapidly to the caveolae domains of the plasma membrane from where it constitutively cycles with the cell interior. Another important source of cholesterol is plasma low-density lipoprotein particles (LDL) that are internalized by specific receptors and delivered to late endosomes/lysosomes for hydrolysis. When cholesterol is effluxed from lysosomes, the bulk of cholesterol is transported to the plasma membrane probably by a Golgi-dependent pathway involving caveolae, while a portion is delivered to the ER by vesicular transport. Deposition of excess cellular cholesterol in the form of cholesteryl esters is catalyzed by the resident ER acyl-CoA:cholesterol acyltransferase (ACAT), leading to the biogenesis of lipid droplets (reviewed in Lange and Steck 1996; Liscum and Munn 1999).
Upon infection with T. gondii, the mammalian cell acquires a novel dynamic compartment, the PV, which contains live, dividing microorganisms. We have addressed the following questions, relating to the origin of cholesterol for the parasite: Can T. gondii synthesize its own cholesterol via the classical mevalonate pathway? Is the PV accessible to host cell cholesterol? If accessible, is it the cholesterol synthesized by the host cell or the exogenous cholesterol delivered by LDL endocytosis that can be transported into the parasite? If acquired exogenously from LDL, is cholesterol transported from lysosomes to the PV by a direct transfer, a Golgi-, or an ER-dependent pathway? Is the host cell altered in its cholesterol biosynthesis or LDL uptake in response to parasitization? Is the parasite capable of replication in host cells unable either to synthesize cholesterol de novo, or to use LDL-delivered cholesterol, or both?
Although the Toxoplasma PV remains segregated from vesicular trafficking through the endo- and exocytotic pathways in the host cell, the results presented here demonstrate that this parasite can actively intercept host LDL-derived cholesterol, in transit from the lysosomes to other cellular compartments.
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Materials and Methods |
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,2(n)-3H]cholesteryl oleate (sp act, 43 Ci/mmol), 3-hydroxy-3-methyl[3-14C]glutaryl coenzyme A (sp act, 61 mCi/mmol), RS-[2-14C]mevalonic acid lactone (sp act, 58 mCi/mmol), [5,6-3H]uracil (sp act, 45 Ci/mmol), and [methyl-3H]thymidine (sp act, 6.7 Ci/mmol). Human [125I]-diferric transferrin was from DuPont NEN Research Products. The monoclonal anti–low-density lipoprotein receptor antibody was obtained from Amersham Corp.
Preparation of LDL, Lipoprotein-deficient Serum, LDL Labeled with NBD-C, or with [3H]cholesteryl oleate–LDL
Human LDL (density 1.019–1.063 g/ml) was isolated from fresh plasma by zonal density gradient ultracentrifugation as described (Poumay and Ronveaux-Dupal 1984). Lipoprotein-deficient serum (LPDS) was prepared by ultracentrifugation of fetal bovine serum (Gemini Bio-Products, Inc.) after the density was increased to 1.215 g/ml with KBr (Havel et al. 1955). Total lipoproteins (LP) contained in the supernatant were used to reconstitute LPDS. The fluorescent lipid NBD-C was incorporated into LDL by mixing 50 µl of the lipid stock solution with 20 ml of filtered fresh human plasma. After incubation for 16 h at 37°C, LDL was isolated as described above. The association of the NBD-C to the LDL fraction was estimated by the specific uptake of the fluorescent LDL by microscopy. The fluorescence observed after uptake of labeled LDL in the presence of a 40-fold excess of nonlabeled LDL was negligible. Radiolabeling of LDL with [3H]cholesteryl oleate ([3H-CO]) was previously described (Coppens et al. 1995).
Cell Lines and Culture Conditions
The cell lines used in this study included: Chinese hamster ovary cells, primary human foreskin fibroblasts (HFF), and African green monkey (Vero) cells. Somatic mutants of CHO cells were generous gifts from Drs. M. Brown, J. Goldstein, and A. Nohturftt of University of Texas S.W. Medical Center (Dallas, TX): CHO-7 cells (Metherall et al. 1989), UT-1 cells (Luskey et al. 1983), and a sterol regulatory-defective (SRD) mutant, SRD-1 cells (Metherall et al. 1989). Dr. L. Liscum (Tufts University School of Medicine, Medford, MA) kindly provided the 2-2 mutant (Dahl et al. 1992, Dahl et al. 1993) and Dr. R. Simoni (Stanford University, Stanford, CA) generously gave a squalene synthase-deficient (SSD) mutant (Bradfute et al. 1992). All cell lines were grown as monolayers at 37°C in an atmosphere of 5% CO2 in -minimum essential medium (prepared at the Media Core Facility of the Department of Cell Biology, Yale University), supplemented with 2 mM L-glutamine and penicillin/streptomycin (100 U/ml per 100 µg/ml). This culture medium also contained variable % (vol/vol) of either FBS or LPDS that was reconstituted (or not) with LP, as indicated in each experiment. Specific requirements added in media for the mutants of CHO cells were: 40 µM compactin for the UT-1 cells, 1 µg/ml of 25-hydroxycholesterol for the SRD-1 cells, 0.2 mg/ml of geneticin for the SSD cells.
Parasite Culture and Purification
The RH strain tachyzoite of Toxoplasma gondii was used throughout this study, and was maintained by passage in the peritoneum of Swiss-Webster mice or by in vitro culture in Vero cells or HFF, as previously described (Roos et al. 1994). A purification scheme of intracellular parasites based on density gradient separation using Nycodenz and isopycnic centrifugation was developed. Confluent HFF or CHO cell monolayers were infected with T. gondii, which were further harvested from the culture supernatants. After two passages through a 27-gauge needle to disrupt any contaminating host cells, parasites were washed three times in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM NaH2PO4, 1.8 mM KH2PO4, adjusted to pH 7.4) by centrifugation at 1,000 g in a GRP tabletop centrifuge (Beckman Instruments, Inc.) for 10 min. Parasites were then resuspended in 10% Nycodenz before application at the top of a linear Nycodenz gradient from 30 to 10%. After centrifugation in a swing-out rotor at 2,000 g for 30 min at room temperature, fractions were collected and analyzed for density and protein content. Fractions containing parasites were equilibrated at a density of 1.09–1.11 g/ml of Nycodenz. They were then diluted 2.5-fold with PBS, centrifuged at 5,000 g for 30 min, and washed three times in PBS before use for lipid extraction. The purity of the parasite preparation was monitored by the comparison of the protein content in the 1.09–1.11-g/ml fractions after inoculation of the same number of parasites in either one or three dishes of cell monolayers. When parasites were purified from three dishes, no increase in protein concentration was observed in the parasite fractions, as compared with parasites from one dish.
Determination of Parasite Concentration, Replication, and Viability
Parasite concentration was determined by numeration using a Hausser counting chamber at 400x magnification. The analysis of the number of parasites per vacuole to estimate the parasite replication and the measurement of [3H]uracil incorporated into the parasites to evaluate their viability were determined as previously described (Nakaar et al. 1999). Parasite cultures were synchronized by removal of parasites that had not yet invaded 4 h after their inoculation into confluent cells.
Ligand Radiolabeling and Uptake Experiments
LDL was radiolabeled with 125I by means of ICl (McFarlane 1958) and specific radioactivity of 125I-LDL was 300–550 cpm/ng protein. 125I-transferrin was used at a specific radioactivity of 100–200 cpm/ng protein. For uptake experiments, confluent cultures of CHO cells in a six-well plate were inoculated with 106 freshly lysed-out parasites and incubated for 4 h at 37°C before washing and resuspension in culture medium containing either 10% LPDS (for LDL uptake experiments) or 10% FBS (for transferrin and horseradish peroxidase uptake experiments). The presence or absence of LP during the 24-h incubation is without influence on the subsequent transferrin or HRP uptake. 24 h post-infection, cultures were washed twice with PBS, incubated at 37°C for 2 h in culture medium (devoid of serum) containing 1% of serum albumin and different concentrations of 125I-LDL, 125I-transferrin, or HRP, and then chilled to 4°C and washed twice in PBS plus 5% FBS. Cell monolayers were further incubated for 60 min at 4°C in the presence of 0.1% pronase (wt/vol) to remove cell surface–bound ligand, washed twice in PBS and lysed in 0.01% Triton X-100. The pronase-resistant fraction was considered as internalized ligand. For comparison, noninfected CHO cells were used as controls, and processed identically. Cell-internalized radioiodinated ligand in the lysate was evaluated in a liquid scintillation system (LS 6000SC; Beckman Instruments, Inc.) and cell-internalized HRP was determined by measurement of the peroxidase activity in the lysate by the stopped colorimetric assay using ortho-phenylenediamine as a substrate, according to Steinman et al. 1976. Values of cell-internalized ligand in infected and uninfected cells were normalized to total cell protein.
Incubation of Infected Cells with [3H-CO]-LDL and Lipid Analysis
Synchronized infected cells were incubated in culture medium containing 10% FBS or 10% LPDS. After 24 h, cells were treated with the indicated inhibitor, and then pulse-labeled with 5 mg of [3H-CO]-LDL. After washing, parasites were isolated, their protein concentration determined, and lipids extracted to quantify the amount of [3H-C] associated with the sterol fraction, as described (Coppens et al. 1995). Results were calculated as counts per minute per milligram cell protein, and then expressed in percent control values corresponding to the amount of [3H-C] associated with the sterol fraction of infected cells that were incubated in the absence of any inhibitor.
Incubation of Infected Cells with LDL Labeled with NBD-C, Filipin, Lipid Droplet Dyes, and Fluorescence Microscopy
To visualize fluorescent cholesterol associated with T. gondii, synchronized infected cells were seeded on coverslips in a 24-well plate and incubated in culture medium containing 10% LPDS. After 24 h, cells were treated with the indicated inhibitor, and then labeled with 0.1 mg of [NBC-C]-LDL for various pulse-chase times. Coverslips with live cells were directly observed with an epifluorescence microscope (Microphot FXA; Nikon Inc.). Images were captured with a CCD camera (Photometrics), processed with Image-Pro Plus (Media Cybernetics), and their contrast was enhanced with Adobe Photoshop 5.0 (Adobe Systems Inc.). For cytochemical staining of β-hydroxysterols with filipin, infected cells seeded on coverslips were incubated in culture medium containing 10% FBS, washed in PBS, and then fixed in 3% paraformaldehyde for 30 min at room temperature and washed again with PBS. Coverslips with fixed cells were incubated with 25 µg/ml of filipin for 15 min, washed in PBS, and mounted with glycerol to be viewed by a fluorescence microscope using an excitation filter of 350–410 nm. For detection of parasite lipid droplets, extracellular T. gondii from cell cultures incubated in culture medium plus 10% FBS or 10% LPDS were fixed in paraformaldehyde and stained with either Oil Red O or Nile Red before observation by phase-contrast and fluorescence microscopy, as described (Greenspan et al. 1985; El-Jack et al. 1999).
Assay for HMG-CoA Reductase Activity
Confluent cultures of CHO cells in 10-cm plates were inoculated with 107 freshly lysed-out parasites and incubated for 4 h at 37°C before washing and resuspension in culture medium containing either 10% FBS or 10% LPDS. After 24 h of infection, the activity of HMG-CoA reductase was measured in situ on digitonin-permeabilized cells using [14C]HMG-CoA as substrate, as described (Leonard and Chen 1987; Geelen et al. 1991). In parallel, noninfected CHO cells were used as controls and similarly processed. Reductase activity was expressed as picomoles of radioactive HMG-CoA converted to radioactive mevalonate per minute per milligram detergent-solubilized protein.
Mevalonate and HMG-CoA Incorporation into Sterols
To study the biosynthesis of sterols in T. gondii, 500 nmol of [14C]mevalonic acid or 300 nmol [14C]HMG-CoA was added for 3 h at 37°C to confluent cultures of CHO cells in 10-cm plates infected with 107 freshly lysed-out parasites for 24 h in medium containing 10% LPDS. For labeling with HMG-CoA, infected cells were previously permeabilized with digitonin (Leonard and Chen 1987). After washing, parasites were isolated, their protein concentration determined, and lipids extracted to quantify the amount of [14C]cholesterol associated with the sterol fraction, as described (Coppens et al. 1995). An assay of radioactive mevalonate or HMG-CoA incorporation was performed on extracellular T. gondii in conditions where parasites are metabolically active, as described previously (Coppens et al. 1999).
Protein Determination
Protein content was determined by the bicinchoninic acid assay (Smith et al. 1985), using serum albumin as standard.
Statistical Analysis
For comparison of means, P was determined by analysis of variance against control (ANOVA 2).
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Results |
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Host Cholesterol Biosynthesis Remains Unchanged in Infected Cells
To assess the influence of parasites on host cholesterol production, we monitored the formation of mevalonate from [14C]HMG-CoA in parasitized or uninfected cells after permeabilization with digitonin. As illustrated in Table , a similar activity of HMG-CoA reductase was observed in control cells or in cells containing tachyzoites. The main contribution of the mevalonate production is from the host cells since parasites fail to use precursors from the mevalonate pathway, as demonstrated above in Table . The removal of LP from the culture medium induces an increase of the enzyme activity to the same extent in both conditions (Table ).
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The SSD mutant has a metabolic block at squalene synthase, converting farnesylpyrophosphate to squalene, the first enzymatic step committed solely to the biosynthesis of sterols, and is therefore incapable of endogenous cholesterol biosynthesis (Bradfute et al. 1992). As determined by enumeration of parasites per vacuole, T. gondii developed normally in SSD cells compared with CHO cells in medium containing 10% FBS (Fig. 2 A). The levels of uracil incorporation into newly synthesized parasite nucleic acids (99 ± 4% as control of uracil incorporated in parasites) as well as the filipin-cholesterol staining of these parasites maintained in these two types of cells were also similar (not shown). This suggests that host cholesterol production has no significant effect on parasite replication and that the bulk of the parasite cholesterol requirement can be satisfied by exogenous cholesterol.
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Extracellular LDL and Exogenous Free Cholesterol Stimulate Parasite Growth
We next asked whether parasite growth can be directly dependent upon exogenously supplied cholesterol. The replication rate of intracellular parasites incubated in culture medium devoid of LP or with an excess of exogenous cholesterol, either freely administrated or associated with LDL, was analyzed. Removal of any lipoproteins from the incubation medium resulted in a reduced incorporation of [3H]uracil into parasites by 10 and 16%, after 24 and 48 h, respectively, as compared with incubation in the presence of 10% FBS (Fig. 3 A). These lower values of radioactive uracil incorporation were coincident with the presence of smaller vacuoles containing fewer parasites when parasites were maintained without LP (Fig. 3 B). This slowing in parasite multiplication also caused a delay in the parasite-induced lysis of the monolayer of host cells as monitored by an increase in the parasite doubling time (10–13 h in LPDS vs. 6–8 h in FBS).
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The addition of exogenous cholesterol, particularly when incorporated into LDL in the incubation medium, has a stimulatory effect on T. gondii replication, measured by uracil incorporation into parasites (Fig. 3 D), as well as enumeration of parasites per PV in infected cells (Fig. 3 E). This growth stimulation was proportional to the concentration of LDL in the medium.
Altogether, these data indicate that an exogenous source of cholesterol, mainly provided by LDL, is required for optimal intracellular replication of T. gondii, although it is not essential for growth.
Receptor-mediated Endocytosis of LDL Is Specifically Increased in Infected Cells
To assess whether the presence of replicating tachyzoites in CHO cells resulted from an increased uptake of extracellular LDL as a source of cholesterol for the parasites, infected and noninfected cells were incubated in parallel with 125I-LDL, and the extent of radioactive ligand uptake was determined. Internalization of LDL was stimulated by two- to threefold in a ligand dose-dependent manner in infected CHO cells (Fig. 4). This uptake was specific for LDL as internalization of this ligand was inhibitable by an excess concentration of nonlabeled LDL, indicating that the enhanced endocytosis of LDL in infected cells did not result from receptor-independent fluid-phase endocytosis (not shown). Furthermore, the increased LDL uptake in infected cells was not a consequence of a global stimulation of endocytosis since uptake of transferrin as another receptor-internalized ligand, or the fluid-phase tracer HRP was unchanged after infection (Fig. 4).
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10 min, [NBD-C]-LDL is degraded to free NBD-C in lysosomal compartments, which subsequently is transferred to the plasma membrane via the Golgi apparatus (Craig et al. 1981; Ohashi et al. 1992; Fig. 5 D). Cholesterol that has reached the plasma membrane is then slowly transferred to cholesterol acceptors in the surrounding medium (serum proteins), contributing to a gradual loss of NBD-C, as shown by a decrease of the plasma membrane signal (Ohashi et al. 1992).
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A pulse (1 h)–chase (9 h) experiment performed on infected cells revealed a maintenance of fluorescence associated with the parasite plasma membrane, the apical structures, and the lipid droplets (Fig. 5 E); the intensity was slightly decreased due to a dilution of the NBD-C, concomitant with parasite division, while a diffuse labeling was observed throughout the host cell. The fluorescence initially associated with the PVM gradually disappeared with the chase time.
No specific fluorescence was detectable in infected cells or in parasites when incubated in medium containing 10% LPDS and NBD-C administrated as lipid alone, indicating a requirement for LDL in the acquisition of NBD-C by intracellular T. gondii (not shown).
LDL-derived Cholesterol Acquisition Requires Live Intracellular Parasites
The above data demonstrated that the PVs containing T. gondii gain access to exogenous NBD-C associated with LDL. To investigate whether this cholesterol uptake is induced by the parasite itself or resulted from a passive transfer to the PV, we infected cells for 24 h, treated for the next 20 h with the anti–Toxoplasma drug pyrimethamine, and then incubated with [NBD-C]-LDL. The reduction of parasite viability was confirmed by measurement of uracil incorporation, revealing <10 and <1% incorporation, compared with control without pyrimethamine, after exposure to 1 and 10 µM of drug, respectively. The fluorescent labeling in T. gondii was abrogated by pyrimethamine treatment in a dose-dependent manner.
The SDZ 215-918 cyclosporin A derivative is a particularly potent and rapid inhibitor of T. gondii invasion and replication (Silverman et al. 1997). Incubation of infected cells with SDZ 215-918 in conditions that inhibit the metabolic activity of intracellular parasites, before exposure to [NBD-C]-LDL, led to a decrease of fluorescence associated with the PV (not shown). This indicates that active parasite metabolism is necessary for cholesterol uptake (Fig. 6, A–C).
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Inhibitors of the LDL Endocytic Pathway Block Cholesterol Transport to the Parasite
We next wanted to elucidate whether delivery of exogenous cholesterol to the parasite involves the endocytic pathway of the host cell. The transport of LDL-derived cholesterol from the external medium was measured in the presence of agents known to block steps in LDL-cholesterol transport in mammalian cells. These treatments target LDL internalization and degradation, as well as cholesterol egress from lysosomes, by both vesicular and nonvesicular transport. Since the viability of parasites is required for cholesterol uptake, we monitored the levels of uracil incorporated into the parasites for each inhibitor tested, and found incorporation of 
94% of control without inhibitor.
Preincubation of infected cells with anti–LDL receptor antibodies inhibited LDL binding to the host cell and abrogated by 90% of control the delivery of radioactive cholesteryl esters to parasites after [3H-CO]-LDL endocytosis (Table ). This inhibition of accessibility of exogenous cholesterol by anti–LDL receptor antibodies was confirmed by the absence of fluorescent labeling in the host cell and in intravacuolar parasites after short pulse-labeling with [NBD-C]-LDL (Fig. 7A and Fig. B).
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We next tested the influence of U18666A, one of a number of hydrophobic amines that are reported to induce an accumulation of free exogenous cholesterol in late endosomes/lysosomes during the endocytic processing of LDL (Roff et al. 1991; Liscum and Faust, 1983; Kobayashi et al. 1999). Progesterone also sequesters cholesterol in lysosomes by an unknown and reversible mechanism (Butler et al. 1992). Progesterone and U18666A inhibited the transfer of tritiated cholesteryl oleate into the parasite sterol fractions after endocytosis of the reconstituted radioactive LDL (Table ); moreover, the effects of U18666A and progesterone are cumulative. This inhibition is coincident with a weak fluorescent pattern of parasites preincubated with these drugs, alone or in combination, followed by exposure to [NBD-C]-LDL (Fig. 8). After a 24-h chase in a progesterone-free medium, the free cholesterol pool leaves lysosomes to be redistributed intracellularly, as demonstrated by fluorescence throughout the host cell and parasites (Fig. 8B and Fig. C).
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Brefeldin A affecting the Golgi apparatus organization (Misumi et al. 1986; Klausner et al. 1992; Torii et al. 1995) had no significant effect on cholesterol transport to T. gondii, suggesting the use of a Golgi-independent pathway (Table ).
Parasite Replication Is Reduced in a Host Cell Line with Defective Mobilization of Cholesterol from Lysosomes
The cellular and biochemical consequences of the progesterone-induced block in lysosomal cholesterol transport strongly resemble the phenotypic manifestations of the Niemann-Pick type C (NPC) mutation (Pentchev et al. 1986; Sokol et al. 1988; Liscum et al. 1989). NPC fibroblasts exhibit lysosomal storage of unesterified cholesterol due to a single gene defect that affects the egress of LDL-cholesterol from late endosomes/lysosomes and LDL-mediated regulation of cellular cholesterol homeostasis (reviewed in Liscum and Klansek 1998). The somatic 2-2 mutant derived from CHO cells has the same biochemical phenotype of the NPC mutation (Dahl et al. 1992, Dahl et al. 1993).
A direct comparison of lysosomal cholesterol processing and transport to the parasite was carried out between the 2-2 mutant and progesterone-treated cells. Parasite growth in the 2-2 mutant is reduced by 15 and 25% in comparison with control after 24 and 48 h, respectively, as assessed by uracil incorporation (Fig. 9 A). Concomitant with the impaired parasite replication in these mutant cells, the parasite cholesterol content revealed by filipin is weak, indicating a lower cholesterol content due to the retention of cholesterol inside host lysosomes (compare Fig. 9 B with Fig. 1 B and 3 C for CHO cells). In addition, incubation of infected 2-2 mutant cells with [3H-CO]-LDL led to a lower incorporation of radioactive cholesteryl oleate into parasites (64 ± 6% of control). Incubation of fibroblasts with progesterone induced a more drastic decrease of parasite replication of 50 and 70% after 24 and 48 h, respectively (Fig. 9 C), as compared with untreated cells, probably consistent with a more profound block in cholesterol delivery to intracellular parasites (see Table ).
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The putative pathway underlying cholesterol-derived LDL acquisition by T. gondii is the pathway IIIa depicted in the schematic model in Fig. 10. First, a large contribution of the receptor-mediated endocytosis of LDL appears to be the primary mechanism as antibodies against LDL receptors can block the accessibility of LDL to intracellular compartments, including Toxoplasma vacuoles (Fig. 10, Fig. 1).
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Endocytosed lipoproteins are delivered to endosomes and their protein/phospholipid coat degraded in late endosomes/lysosomes, yielding free cholesteryl esters that will be hydrolyzed to cholesterol. In the presence of chloroquine or sucrose impairing lysosomal function and provoking an accumulation of intact LDL, minimal molecules of cholesterol reach the PV, implicating host LDL proteolysis in the parasite acquisition of this lipid (Fig. 10, Fig. 2). Incubation of T. gondii in host cells where cholesterol translocation from late endosomes/lysosomes (2-2 mutant or treated with progesterone and U18666A) is impaired leads to a dramatic reduction of cholesterol associated with the parasites, concomitant with a slowing of parasite replication (Fig. 10, Fig. 3). The higher inhibition of parasite growth by progesterone compared with the 2-2 mutant might be related to a direct toxic effect of the steroid on T. gondii. Alternatively, progesterone is known to sequester in lysosomes cholesterol derived not only directly from endocytosed LDL, but also from other existing sterol pools, such as lipid droplets or organelle membranes (McGookey and Anderson 1983; Butler et al. 1992), and could therefore also remove cholesterol from PV-containing parasites.
In mammalian cells, the mechanisms and precise routes of transport of cholesterol from late endosomes/lysosomes to the other cellular compartments are areas of intense investigation. From the cell biological point of view, the Toxoplasma vacuole may be considered as such a compartment in the infected cell and it could be used as a tool for analysis of intracellular cholesterol movement in mammalian cells. A portion of cholesterol is mobilized to the plasma membrane through the Golgi apparatus (reviewed in Liscum and Munn 1999). The pathway of cholesterol acquisition by T. gondii is clearly not altered by incubation of infected cells with Brefeldin A (no use of the pathway IIIb in Fig. 10), indicating a Golgi-independent route despite the location of mature PVs adjacent to the host cell Golgi. This absence of delivery of host lipids from the Golgi to the Toxoplasma vacuole, a matter of some controversy (Melo and de Souza 1996), has been recently confirmed by the observation of PV free of fluorescent lipids in experiments using NDB-ceramide loaded in fibroblasts before infection with parasites (Mordue et al. 1999). By comparison, the pathogen Chlamydia trachomatis, which similarly resides in a nonfusogenic vacuole, intersects with vesicles from the exocytic pathway since this bacteria can acquire endogenously synthesized sphingomyelin in transit from the host Golgi to the plasma membrane (Hackstadt et al. 1995, Hackstadt et al. 1996).
In mammalian cells, a portion of cholesterol effluxed from lysosomes is delivered to the ER by a pathway that is independent of the plasma membrane. For this vesicular transport of cholesterol, actin filaments, but not microtubules, appear to play a role. Cytochalasin B does not alter the cholesterol movement to the PV, suggesting that cholesterol originating from endocytosed LDL does not transit through the host ER to reach the Toxoplasma vacuole (no use of the pathway IIIc in Fig. 10). This observation suggests that the PVM-host ER association seen in infected cells (Jones et al. 1972; Melo et al. 1992; Sinai et al. 1997), does not participate in exogenous cholesterol trafficking, at least when LP are present.
Exogenous cholesterol, associated with LDL or freely administrated to cells infected by T. gondii, stimulates parasite growth and, in the case of large excess, induces the formation of parasite lipid droplets. Parasites incubated in the SRD-1 mutant, characterized by a high level of cholesterol due to a defect in sterol regulation, have a replication rate comparable with parasites grown in the parental CHO cell. Parasites do not take up cholesteryl esters packaged into LDL more avidly in spite of a high number of LDL receptors exposed at the cell surface of the SRD-1 mutant, suggestive of parasite regulatory mechanisms for sterol homeostasis. These mutant cells also show a high acyl-CoA:cholesterol acyltransferase activity that does not influence parasite growth. This is of interest since host esterification of cholesterol may be involved in the replication of T. gondii (Ernst et al. 1999).
Intracellular T. gondii do not modify the rate of host de novo cholesterol biosynthesis. Parasites are capable of replicating in cells altered in their sterol production such as the SSD mutant. These data indicate that parasites are not dependent upon host endogenous cholesterol as long as exogenous sterols are available. They do not intercept the transport of this lipid from the ER in transit to the plasma membrane. These results corroborate the absence of labeled cholesterol associated with the sterol fraction of parasites after incubation of infected cells with radioactive precursors of the mevalonate pathway (no use of the pathway II in Fig. 10). The survival of 30% of the parasite population in the SSD mutant in the absence of LP opens the question on the origin of other potential sources of sterols for T. gondii, such as cholesterol from cell debris, stored cholesteryl esters, probably present in their lipid droplets, or cholesterol biosynthesized by the parasite. However, the compactin insensitivity, as well as the absence of sterol synthesis from precursors of the mevalonate pathway in the parasites, suggests the absence of the classical mevalonate pathway functional for isoprenoid biosynthesis in T. gondii, as observed for Plasmodium falciparum, an apicomplexan parasite related to T. gondii. An alternative nonmevalonate pathway for the early steps in the biosynthesis of isoprenoids, the 1-deoxy-D-xylulose 5-phosphate (DOXP) pathway, has been proposed in the plasmodial apicoplast, a plastid acquired by members of the phylum Apicomplexa by secondary endosymbiosis of an alga (Jomaa et al. 1999; McFadden and Roos 1999). However, two lines of evidence suggest that sterol synthesis in the T. gondii apicoplast is not necessary for parasite replication in the presence of lipoproteins. First, parasites that cannot partition the apicoplast to daughter cells replicate normally in the primary vacuole (D. Roos, personal communication). Second, inhibitors of the DOXP pathway, which block plasmodial replication, do not inhibit T. gondii growth in presence of serum (D. Soldati, personal communication).
The accumulation of cholesterol from host lysosomes inside intravacuolar parasites suggests a largely unidirectional influx of this lipid and may be correlated to the rapid division rates of T. gondii. Alternatively, the isolation of the PV from the external medium results in the absence of transfer of cholesterol to extracellular cholesterol acceptors. The cholesterol transport pathway in cells infected with T. gondii clearly differs from those for the other cell organelles and is characterized by an absence of interaction with vesicles involved in export from the Golgi and ER, as well as those implicated in exchanges between the plasma membrane and the cell interior. The route of delivery of exogenous cholesterol from lysosomes to the PV might involve a sterol-binding protein, mediating a molecular transport of cholesterol towards the PVM. T. gondii may actively either divert a host sterol-carrier protein or synthesize such a protein since parasite viability is required for cholesterol acquisition. After crossing the PVM, cholesterol molecules must be translocated to the parasite. The elucidation of the mechanisms of these two sequential steps by generation of resistant mutants forms the basis for our future experiments.
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Acknowledgments |
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The work was supported by the National Institutes of Health grant AI-30060 and a Burroughs Wellcome Fund Molecular Parasitology Scholar Award to K.A. Joiner and an American Heart Association (CT affiliate) Postdoctoral Fellowship to A.P. Sinai.
Submitted: 20 December 1999
Revised: 16 February 2000
Accepted: 18 February 2000
Dr. Sinai's present address is Department of Microbiology and Immunology, University of Kentucky College of Medicine, Lexington, KY 40536.
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) 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.
