Bloodstream forms of Trypanosoma brucei are tsetsetransmitted pathogenic protozoa that live in the blood and body fluids of the mammalian host. In these parasites, the predominant nonpolar lipids are cholesterol, cholesteryl esters, fatty acids, and triglycerides. Among phospholipids, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin are the most abundant(1, 2) . Yet, bloodstream forms of T. brucei are incapable of de novo lipid synthesis(3, 4, 5) . Instead, they critically depend for their rapid growth on the presence in the culture medium of plasma low density lipoprotein (LDL) ()particles(6, 7) . LDL presumably provide essential lipid constituents, e.g. for membrane assembly.

Bloodstream forms of T. brucei acquire LDL particles by receptor-mediated endocytosis(8) . The LDL receptor of the parasite has been purified (6, 9) and shows extensive immunological cross-reactivity with the host LDL receptor(10) , but it possesses at least one specific epitope(11) . Upon endocytosis of LDL particles at the flagellar pocket, apoprotein B-LDL is rapidly degraded by thiol protease(s) acting at acidic pH(10) . Indirect evidence suggests that the receptor is recycled (10) and can be up-regulated upon sterol deprivation(12) .

While the fate of apoprotein B-LDL taken up by T. brucei is now well established, that of LDL-associated lipids remains to be characterized, in order to clarify the mode of lipid acquisition and to validate the hypothesis that these parasites avidly take up LDL particles to satisfy their lipid requirements.

In this paper, we have inserted [H]sphingomyelin and [H]cholesteryl oleate into LDL particles to study the mechanism of acquisition and the metabolic fate of these lipids. In order to confirm the role of LDL as source of exogenous phospholipids and cholesterol, we blocked receptor-mediated endocytosis of radiolabeled LDL by unlabeled LDL or by anti-LDL receptor antibodies, and we interfered with lysosomal degradation and lipid metabolism using chloroquine and the thiol protease inhibitor, leupeptin. The activity of key acidic hydrolytic enzymes was also measured. The ability to esterify sterols was also compared in the bloodstream forms and the culture-adapted procyclic form (insect stage) of T. brucei, which differ in their capacity of lipid biosynthesis.

EXPERIMENTAL PROCEDURES

Chemicals

Cell Culture and Incubation Media

Culture-adapted procyclic trypomastigotes of T. brucei (hereafter often referred to for convenience as procyclic forms) were cultured at 28 °C in medium supplemented with 10% (v/v) FCS(16) , collected from an exponentially growing population and seeded at 10 cells/ml in either lipoprotein-depleted FCS or lipoprotein-depleted rabbit serum. Rat-1 cells were seeded at 50,000/cm Petri dish and cultured in DMEM supplemented with 10% (v/v) FCS at 37 °C, under 5% CO.

All incubations were performed at 10 parasites/ml (about 10 cells, i.e. 1 mg of cell protein/assay). For metabolic labeling, bloodstream forms (37 °C), procyclic forms (28 °C), and Rat-1 cells (37 °C) were incubated with the radioactive precursor at the indicated temperature and then centrifuged and submitted to lipid extraction.

A polyclonal rabbit antiserum directed against the purified LDL receptor of bloodstream forms of T. brucei was prepared as described previously(6) . Both preimmune and immune rabbit sera were depleted of lipoproteins as above. The concentration of 10% (v/v) antiserum in the culture medium corresponded to an enzyme-linked immunosorbent assay titer of 1:2,000 against LDL receptor preparation. Synvinolin was added from stock solutions in dimethyl sulfoxide, with a final dimethyl sulfoxide concentration of 0.2% (v/v). Cholesterol was added from stock solutions in ethanol, with a final ethanol concentration of 0.1% (v/v). Controls contained identical solvent concentrations.

LDL Particles

Preparation of LDL-associated Lipids

[H]Cholesteryl oleate stored in toluene was similarly dried, resolubilized in 50 µl of chloroform/methanol (2:1), and mixed with 30 ml of plasma for 12 h at 37 °C. Specific radioactivity of LDL-associated [H]cholesteryl oleate was 0.9-1.2 dpm/ng of apoprotein B.

Association of LDL Particles to Cells

LDL-[H]Sphingomyelin and LDL-[H]Cholesteryl Oleate Metabolism

[C]Oleate Incorporation into Lipids

In Vitro Enzyme Assays

RESULTS

Acquisition by Bloodstream Forms of LDL-associated Sphingomyelin

Figure 1: Kinetics of binding of LDL-associated [H]sphingomyelin. Bloodstream forms were incubated at 4 °C in DMEM for the indicated times with 10 µg/ml of LDL labeled with [H]sphingomyelin, together with either 10% (v/v) lipoprotein-depleted preimmune serum (), 10% (v/v) lipoprotein-depleted preimmune serum with a 50-fold excess of unlabeled LDL (), or 10% (v/v) lipoprotein-depleted antiserum anti-LDL receptors (). After washing, cell-associated radioactivity was measured and expressed as ng of apoprotein/mg of cell protein. Results are means ± S.D. (, n = 4), or means of two experiments (, < 10% variation).

Figure 2: Comparison of binding and uptake of [H]sphingomyelin-LDL or I-labeled apoprotein B-LDL. Bloodstream forms were incubated in DMEM plus 10% (v/v) lipoprotein-depleted FCS, with the indicated concentrations of [H]sphingomyelin-LDL (A) or I-labeled apoprotein B-LDL (B) for 24 h, either at 37 °C () or 4 °C (). After washing, cell-associated radioactivity was measured and expressed as ng of apoprotein/mg of cell protein (means ± S.D., n = 3).

Metabolism of Sphingomyelin-LDL in Bloodstream Forms

When bloodstream forms were incubated for 24 h at 37 °C with LDL-associated [H]sphingomyelin, various radiolabeled metabolites were indeed detected (Fig. 3). The exogenous sphingomyelin was first split into ceramide (already seen within 2 h, not shown), and then fatty acids were generated that were further incorporated into phosphatidylcholine and cholesteryl esters. The proportion of the two latter increased with time, while the peak of ceramide remained fairly constant, as is the case for mammalian cells (19) . An acid sphingomyelinase activity could be detected in T. brucei extracts corresponding to 66 pmol of phosphocholine released/h/mg of cell protein in the bloodstream forms and to 6 pmol/h/mg of cell protein in the procyclics (means of duplicates). The breakdown of sphingomyelin was blocked when trypanosomes were incubated in the presence of lysosomal inhibitors, such as leupeptin (Fig. 3) or chloroquine (not shown). Inhibition by leupeptin strongly suggests that, despite the fact that sphingomyelin is inserted into the LDL shell, apoprotein B degradation is a prerequisite for sphingomyelinase to act on its substrate; inhibition by chloroquine indicates that both events take place in the lysosomal compartment.

Figure 3: Analysis of radioactive lipids after endocytosis of [H]sphingomyelin-LDL. Bloodstream forms were incubated in DMEM plus 10% (v/v) lipoprotein-depleted FCS supplemented with 15 µg/ml of LDL containing [H]sphingomyelin, in the absence () or in the presence of 50 µg/ml of leupeptin () for 24 h at 37 °C. After lipid extraction, the cellular content of [H]sphingomyelin, [H]phosphatidylcholine, [H]ceramide, and cholesteryl [H]esters were determined by TLC and identified by co-migration of the corresponding standards. The prominent peak represented nonmetabolized sphingomyelin. Data are from a representative experiment out of two, with < 5% variation.

Acquisition by Bloodstream Forms of LDL-associated Cholesteryl Oleate

Figure 4: Uptake of [H]cholesteryl oleate-LDL and effect of LDL-associated cholesterol on [C]oleate incorporation into cholesteryl esters. A, bloodstream forms were incubated at 37 °C in DMEM plus 10% (v/v) lipoprotein-depleted FCS supplemented with the indicated concentrations of LDL-associated [H]cholesteryl oleate for 24 h. After washing, cell-associated radioactivity was measured and expressed as ng of apoprotein/mg of cell protein. The experiments were repeated twice with <10% variation. B, bloodstream forms were incubated at 37 °C in DMEM plus 10% (v/v) lipoprotein-depleted FCS containing the indicated concentrations of LDL. After 17 h of incubation, 0.1 mM [C]oleate was added, and the cells were harvested 1 h later. After lipid extraction, the cellular content of cholesteryl [C]esters was determined. Experiments were made in duplicate with less than 15% variation. The 100% values corresponded to 1,029 ng/mg of cell protein.

Figure 5: Specificity of capture of [H]cholesteryl oleate-LDL. Bloodstream forms were incubated at 37 or 4 °C for 24 h in DMEM plus 10% (v/v) lipoprotein-depleted preimmune serum with 10 µg/ml of LDL-associated [H]cholesteryl oleate. Other trypanosomes were incubated at 37 °C for 24 h in DMEM with the same concentration of ligand, together with either 10% (v/v) lipoprotein-depleted antiserum anti-LDL receptor, the indicated molar excess of unlabeled LDL, or a 100-fold molar excess of BSA. After washing, cell-associated radioactivity was measured and expressed as ng of apoprotein/mg of cell protein (means ± S.D., n = 3).

Metabolism of Cholesteryl Oleate-LDL in Bloodstream Forms

To study the metabolism of cholesteryl esters included in LDL particles (70% of the cholesterol) by T. brucei, bloodstream forms were incubated at 4 °C or 37 °C, in the presence of LDL-[H]cholesteryl oleate. At 4 °C, a temperature preventing endocytosis, no radioactive cholesterol was produced by T. brucei from radioactive cholesteryl esters (Table 1). At 37 °C, free radioactive cholesterol was released, the proportion of which increased with time until about 6 h. Whereas the absolute uptake of [H]cholesteryl oleate increased with external concentration, the fraction of cholesterol released was independent of influx, indicating that lipase activity was not rate-limiting. Upon longer exposures, a constant fraction (25%) of esters was found. This constant fraction of cholesteryl oleate may either reflect an equilibrium between LDL endocytosis and degradation, or represent reesterification of [H]cholesterol into [H]cholesteryl esters.

Leupeptin or chloroquine totally abrogated cholesterol release, indicating that LDL proteolysis is a prerequisite and that hydrolysis of cholesteryl esters involves a lysosomal lipase (Table 1). An acid lipase activity was measurable in T. brucei extracts corresponding to 1,055 pmol of oleic acid released/h/mg of cell protein in the bloodstream forms and to 263 pmol/h/mg of cell protein in the procyclics (means of duplicates).

Synthesis of Cholesteryl Esters in Bloodstream and Procyclic Forms

Like free cholesterol, LDL provided substrate for cholesterol esterification in the bloodstream forms. Esterification of [C]oleate into cholesteryl esters leveled off at LDL concentrations that saturate the LDL receptors of T. brucei bloodstream forms (Fig. 4B) but was linear up to 100 µM of exogenously added cholesterol, the latter being able to freely diffuse across membranes (not shown).

Exogenous LDL-cholesterol, rather than endogenously synthesized sterol, stimulated the esterification of sterols in procyclic forms (Fig. 6). In contrast to the bloodstream forms, culture-adapted procyclics can grow in the absence of extracellular lipoproteins, but the incorporation of [C]oleate into steryl esters, was then nearly undetectable. On the other hand, the synthesis of steryl esters was readily detected when this form was cultured with LDL and was markedly inhibited by the addition of antibodies directed against the LDL receptors, confirming the requirement of LDL endocytosis. Leupeptin made cholesterol totally inaccessible as substrate for esterification. Synvinolin, a specific inhibitor of the 3-hydroxy-3methylglutaryl-coenzyme A reductase, which inhibits the sterol production in procyclics (12) only slightly reduced the formation of esters of sterol in procyclics grown in the presence of LDL-cholesterol. Taken together, these data strongly suggest that, also in procyclics, esterification depends on exogenous cholesterol delivered by receptor-mediated endocytosis.

Figure 6: Incorporation of [C]oleate into steryl esters in culture-adapted procyclic forms of T. brucei. Procyclic forms were incubated at 28 °C in medium containing 10% (v/v) FCS (control), 10% (v/v) lipoprotein-depleted nonimmune serum, 10% (v/v) lipoproteincontaining antiserum anti-LDL receptor, 10% (v/v) FCS plus 50 µg/ml of leupeptin, 10% (v/v) FCS plus 12.5 µM synvinolin. After 17 h of incubation, 0.1 mM [C]oleate was added, and the cells were harvested 1 h later. After lipid extraction, the cellular content of steryl [C]esters was determined (means ± S.D., n = 3). The 100% value corresponded to 770 ng/mg of cell protein.

DISCUSSION

The incapacity of bloodstream forms of T. brucei to synthesize sterol and fatty acids implies uptake from plasma, more precisely from lipid-protein complexes, since sterols and fatty acids do not circulate in a free form in the plasma. The avid uptake of LDL particles by trypanosomes (clearance of 3 orders of magnitude higher than for fluid endocytosis)(8) , further indicates that their contribution to lipid supply must be prevailing. Indeed, the level of receptor-mediated uptake of mammalian LDL by T. brucei bloodstream forms, at the concentrations found in the host plasma, appears sufficient to fulfill the cholesterol requirement of the parasite(12) . In addition, the growth rate of bloodstream forms is directly proportional to the extracellular concentration of lipoproteins, especially of LDL, and is completely arrested in medium devoid of lipoproteins(6, 7) . A central role of LDL in supporting cell growth does not exclude that plasma albumin and high-density lipoprotein (HDL) particles may also contribute to lipid delivery. Receptors for HDL have been identified on T. brucei and lead to receptor-mediated delivery of HDL to lysosomes(26, 27) . Interestingly, a HDL subclass at very high density (d = 1.24 g/ml) is associated with an unique trypanolytic factor that is activated in acidic organelles(27) .

The interaction of trypanosomes with plasma lipoproteins is controversial. Gillett and Owen (28) have previously shown that the specific uptake of human LDL in T. brucei is capable of inducing a burst of cholesteryl ester synthesis, an observation that is fully confirmed and extended by our results. In opposition, Vandeweerd and Black (29) did not observe any specific interaction of LDL particles with trypanosomes. It is not clear whether this discordance results from a different handling of LDL particles or from another unexplained reason. Based on their results, Vandeweerd and Black suggested that lipid requirement was met by desorption of lipids from plasma proteins and diffusion into parasites without endocytosis or degradation of LDL inside these parasites. Such a mechanism was actually demonstrated in Plasmodium falciparum-infected red blood cells, where phosphatidylcholine was transferred from HDL particles bound to infected erythrocytes without any need to endocytose the lipoprotein(30) . However, the different modes of acquisition of LDL-cholesterol in T. brucei and of HDL-phospholipid in P. falciparum-infected erythrocytes can be easily explained by the fact that phospholipids are at the surface of HDL particles, and thus are easily exchanged with membranes, in contrast to cholesteryl esters, which are sequestered within the core of LDL and thus not readily released without disruption of the particle. Moreover, erythrocytes do not endocytose.

In the present paper, we demonstrate that the acquisition by T. brucei bloodstream forms of both the phospholipids inserted into the amphipathic shell of LDL particles and the cholesteryl esters included in their core relies on receptor-mediated uptake and intracellular degradation of LDL. To draw this conclusion, we have incubated the trypanosomes with LDL labeled with H-labeled lipids. The parasites acquire LDL-associated sphingomyelin or cholesteryl oleate through the following sequence of events.

First, LDL-associated H-labeled lipids bind to the same surface-LDL receptors that are involved in endocytosis of native LDL particles. Indeed, binding kinetics and endocytosis of labeled lipid-LDL and I-labeled apoprotein-LDL are comparable. Uptake specificity is also demonstrated by competition and receptor antibody blocking experiments. Second, LDLassociated H-labeled lipids are endocytosed, as evidenced by the difference in uptake at 4 and 37 °C. Third, [H]sphingomyelin and [H]cholesteryl oleate are released from LDL. Indeed, these lipids remain intact when degradation of apoprotein B, the keystone of the LDL particles, is blocked by leupeptin or by chloroquine(10) . The latter experiments clearly demonstrate that the acquisition of sphingomyelin and cholesterol from LDL by T. brucei cannot be accounted for by desorption and passive transfer to the parasite membrane.

Fourth, [H]sphingomyelin is cleaved by an acid, presumably lysosomal sphingomyelinase, yielding ceramide that is split up into sphingosine and fatty acids. The latter can, in turn, be incorporated into phosphatidylcholine, the prominent phospholipid in T. brucei, into triacylglycerols, or into cholesteryl esters. The metabolism of sphingomyelin in trypanosomes is thus comparable with that found in mammalian cells, suggesting a similar downstream enzymatic equipment. Similarly, [H]cholesteryl oleate is hydrolyzed into free cholesterol by an acid lipase, presumably lysosomal.

Although the specific activities of acid sphingomyelinase and acid lipase in T. brucei are lower than in cultured mammalian cells, these values may not reflect the effective metabolic activity in living cells. Indeed, while the specific activity of acid sphingomyelinase is about 50 times lower in human lymphoblast than in skin fibroblast extracts, the effective half-time of degradation of sphingomyelin by lysosomal sphingomyelinase in intact cells is similar in both cell types(19, 31) .

Fifth, free cholesterol is able to cross the lysosomal membrane and thus becomes available for either membrane assembly or reesterification. Evidence that cholesterol can be reesterified by fatty acids, such as oleate, suggests the existence in T. brucei of an acyl-CoA:cholesterol O-acyltransferase for which the substrate can be provided by LDL-cholesterol. When T. brucei procyclics are deprived of cholesterol by growth in the absence of LDL, no incorporation of radiolabeled oleate into steryl esters can be detected. However, when cholesterol has accumulated in both bloodstream and procyclic forms through the uptake of LDL, the requirement for membrane synthesis is fully met, and excess cholesterol is stored as steryl esters. In this respect, both forms of trypanosomes behave like mammalian cells, in which the binding of LDL particles to the LDL receptor appears to initiate and regulate a cellular process leading to esterification of cholesterol derived from extracellular LDL (25, 32) .

Taken together, our studies clearly demonstrate that bloodstream forms of T. brucei acquire lipids associated with LDL particles through receptor-mediated endocytosis and lysosomal proteolysis of apoprotein B-LDL. They utilize these lipids or produce metabolites by processes similar to those existing in mammalian cells. However, the crucial dependence of T. brucei bloodstream forms on lipoprotein-derived lipid for membrane assembly and other functions (12) , together with their incapacity to synthesize cholesterol and fatty acids, makes these cells particularly vulnerable to a limited supply of lipoproteins in their environment and, more interestingly, to pharmacological interference with lipoprotein delivery and intracellular degradation. Conversely, lipoproteins can be used for the targeting of trypanocidal agents, such as suramin(33) , or trypanolytic factors(27) .

Although pathways leading to the utilization of lipoprotein-associated lipids appear to be similar to those found in its mammalian hosts, the possibility that key enzymes show parasitic-specific properties, including unique sensitivity to pharmacological inhibitors, deserves to be explored. Indeed, unique features in the digestive machinery of T. brucei have already be found. First, digestion may start in a peculiar extracellular compartment, the flagellar pocket. This small and almost closed space contains specific acidic hydrolases, the activity of which correlates with the parasite life cycle(34) . It is elevated in blood-dwelling trypanosomes that depend on the uptake of intact plasma proteins and is considerably attenuated in the procyclic stage, during which parasites develop in the midgut of tsetse fly where nutrient breakdown is largely made by the insect(35) . Second, lysosomal degradation in trypanosomes depends on a variety of peptidyl-hydrolases, that are variably expressed according to the parasite life cycle(36) . Third, the activity of the lysosomal activity of phospholipase A is much higher in bloodstream forms of T. brucei than in unrelated organisms(37) . This enzyme also markedly differs from the mammalian one in pH dependence, ionic requirement, and sensitivity to inhibitors(38) .

In conclusion, we have previously shown that the LDL receptors play an important role in the multiplication of bloodstream forms since growth is arrested in lipoprotein-depleted serum, restored by purified LDL, and slowed down by antibodies anti-LDL receptors(6) . Here, we provide direct evidence that the LDL receptors are involved in the lipid supply for T. brucei. Interference with the various events underlying receptor-mediated endocytosis of LDL particles, lysosomal degradation, and the subsequent events leading to utilization of lipid metabolites could open new approaches to fight against these parasites.

FOOTNOTES

*

This investigation was supported in part by Grants 2.4549.88, 3.4570.88, 2.4547.91, and 1.5246.91 of the Belgian National Fund for Scientific Research, as well as by the Belgian Federal Office for Scientific, Technical, and Cultural Affairs (concerted actions, Grant 88), by the Framework of Interuniversity Attraction Poles (Grant 44), by the ``Tournesol'' Franco-Belgian Scientific Collaboration Programs, and by Merck, Sharp, and Dohme. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Supported by special grant from the Belgian National Fund for Scientific Research.

¶

To whom correspondence should be addressed: ICP-CELL 75.41, 75, avenue Hippocrate, B-1200 Brussels, Belgium. Tel.: 32-2-764-75-69; Fax: 32-2-762-75-43.

()

The abbreviations used are: LDL, low density lipoprotein; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; TLC, thin layer chromatography; HDL, high density lipoprotein.

ACKNOWLEDGEMENTS

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