Exogenous and endogenous sources of sterols in the culture-adapted procyclic trypomastigotes of Trypanosoma brucei

MOLECULAR

ELSEVIER

Molecular and Biochemical Parasitology 73 (1995) 179-188

i%EMICAL PARASITOLOGY

Exogenous and endogenous sources of sterols in the culture-adapted procyclic trypomastigotes of Trypanosoma brucei Isabelle Coppens a7b,Pierre J. Courtoy a,ba* b International

a Cell Biology Unit, University ojLouvain Medical School, Louvain, Belgium Institute of Cellular and Molecular Pathology, avenue Hippocrate 75, 1200 Brussels, Belgium

Received 12 April 1995; accepted 29 June 1995

Abstract The growth of the culture-adapted procyclic forms of Trypunosoma brucei (procyclics) is accelerated by supplementation of the medium with low-density lipoprotein (LDL) particles. This effect can be attributed to receptor-mediated endocytosis of LDL, followed by utilization of lipids carried by the lipoproteins. Indeed, procyclics that normally contain ergosterol synthesized de novo, also incorporate exogenous cholesterol in their membranes. In turn, import of exogenous lipids down-regulates the isoprenoid biosynthetic machinery as measured by a approx. 3-fold decrease of [14C]acetate incorpora-

tion into sterols and a approx. 2-fold decrease of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, compared with cells grown in lipoprotein-depleted medium. Synvinolin, a specific inhibitor of HMG-CoA reductase that slows down the procyclic growth in vitro and decreases [14C]acetate incorporation into sterols, produces striking morphological modifications, including an arrest at cytokinesis and an extensive swelling of the kinetoplast-mitochondrion system. These cytotoxic effects are amplified in the absence of lipoprotein supply. In conclusion, procyclics may acquire sterols from both exogenous and endogenous sources. To a large extent, these two pathways compensate each other, illustrating Keywords:

adaptation of the parasites to survive in extremely Trypanosoma

different environments.

brucei; Sterol; Receptor-mediated endocytosis; LDL; HMG-CoA reductase; Synvinolin

1. Introduction The protozoan hemoflagellate Trypanosoma brucei, the causative agent of sleeping sickness in human and of nagana in lifestock, is an extracellular

Abbreviations: BSA, bovine serum albumin; FCS, foetal calf serum; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; LDL, low-density lipoprotein. * Corresponding author. Tel.: (32-2) 764-7569; Fax: (32-2) 762-7543; e-mail: [email protected] 0166-6851/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166-6851(95)00114-X

parasite that lives in the bloodstream and other body fluids of the mammalian host. Once ingested by the tsetse fly, the bloodstream form trypanosomes differentiate into procyclic (insect) forms. Like those of mammalian cells, T. brucei membranes contain sterols, the nature and the source of which vary according to the cycle stage. The bloodstream forms contain cholesterol [l] that is provided from an exogenous source, i.e., by receptor-mediated uptake of plasma low-density lipoprotein (LDL) partitles [2-41, lysosomal digestion [5] and release of

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cholesterol into the cytosol 161. In these rapidly dividing forms, cholesterol is not synthezised de novo [7] and the parasites do not survive in a medium devoid of lipoproteins [8]. By contrast, ergosterol is the major sterol that can be synthesized by the insect procyclic forms of T. brucei [7,9,10]. The procyclic forms of T. brucei can be easily cultivated in a semi-defined medium supplemented by foetal calf serum (FCS) [ll]. We have previously demonstrated that these culture-adapted procyclic trypomastigotes show an activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-COA) reductase, involved in the sterol synthesis [12]. This enzymatic activity can be blocked by specific inhibitors such as synvinolin, leading to a decrease in sterol production [12]. In this paper, we demonstrate that in addition to synthesize their sterols, procyclics can import exogenous cholesterol by LDL endocytosis through specific receptors and incorporate this lipid into their membranes. Major changes in the culture medium, such as supplementation with a large excess of LDL or conversely total removal of lipoproteins, as well as exposure to synvinolin, induce modifications in the rate of sterol biosynthesis and in the composition of membranes, as well as in the procyclic growth rate. Taken together, these data lead to the suggestion that procyclics can adapt to extremely different media, so as to maintain a regulated supply of sterols.

2. Materials and methods 2.1. Materials [3- “C]hydroxymethylglutaryl-CoA (55 mCi mmol-‘) and [‘4C]sodium acetate (45-60 mCi mmol-‘) were obtained from Amersham Intemational (Amersham, UK). Bovine serum albumin (BSA, fraction V) and cofactors for HMG-CoA reductase assays were obtained from Sigma (St. Louis, MO, USA). Silica gel 60 TLC plates were from Merck (Darmstadt, Germany). All solvents and standards were of the highest analytical grade. Synvinolin (simvastatin) was a gift from Merck, Sharp and Dohme. Suramin was from Bayer (Leverkusen, Germany).

73 (1995) 179-188

2.2. Cell culture Culture-adapted procyclic trypomastigotes of T. brucei strain 427 (hereafter often referred to for convenience as procyclics) were collected from an exponentially growing population, seeded at lo6 cells ml-’ and cultured at 28°C in a semi-defined medium SDM-99 which has been modified from SDM-77, supplemented with 10% (v/v) FCS [ll]. Motility of trypanosomes was routinely examined by phase-contrast microscopy. Growth of trypanosomes was monitored by counting cells in a haemocytometer. Trypanosomes were stained with May-Gtinwald-Giemsa for morphology by light microscopy. Statistical analysis was carried out using the x2 test. Lipoprotein-depleted serum was obtained by centrifugation after the density of the serum was increased to 1.215 g cme3 with KBr [13]. Synvinolin was added from stock solutions in dimethylsulfoxide, and final dimethylsulfoxide concentration in all media was 0.2% (v/v). Controls contained identical solvent concentrations. 2.3. Acetate incorporation into sterols. Sterol content and composition To study the biosynthesis of sterols in procyclics, 1 PCi of [‘4C]sodium acetate dissolved in 10 ~1 of ethanol/water (3:l) was added per ml of culture medium prior to inoculation. After 48 h, cells were harvested by centrifugation and washed thrice with phosphate-buffered saline. Unsaponifiable lipids were extracted from total lipids with petroleum ether, separated by thin-layer chromatography and their radioactivity measured by liquid scintillation counting [12]. The sterol content was quantitated in the extracts by reference to an internal standard [14]. For analysis of sterol composition, exponentially growing procyclics were collected after 2 days and washed 3 times in phosphate saline glucose (60 mM Na,HP0,/3 mM NaH,P0,/46 mM NaC1/55 mM glucose, pH 8). Cells were then homogenized at 4°C by sonication in 10 mM K2HP04/KH,P04 buffer pH 7.4 supplemented with 150 mM KCl/l mM EDTA/l mM EGTA, and cellular membranes were obtained as described [4]. After lipid extraction and

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and Biochemical Parasitology

separation of unsaponifiable material by chromatography, the plates were sprayed with 50% H,SO, and dried at 100°C. This colours cholesterol as a red spot, well-distinct from the brown spot of ergosterol. 2.4. Assay of HMG-CoA reductase activity 2 X lo6 procyclics in 100 ~1 were collected during the exponential or the stationary growth phases and added to 50 ~1 of the incubation mixture for the HMG-CoA reductase assay containing [ *4~]~~~CoA [12]. 2.5. Lipoproteins LDL and high-density lipoprotein particles were isolated from fresh human plasma as described [151. LDL were radiolabeled with 12’1 by means of iodine monochloride [16]. Specific radioactivity of 1251labeled LDL was measured in a y-counter (LKB) and was typically around 400 cpm (ng protein)-‘.

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2.7. Electron microscopy procedures A suspension of trypanosomes (10’ cells ml-‘) was fixed in 1.2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 30 min. Trypanosomes were collected by filtration [18] on a nitrocellulose filter (0.1 pm diameter pores, Millipore, Bedford, MA, USA). Postfixation was performed for 1 h at 4°C in a solution containing 1% (w/v> 0~0, and 2% (w/v) K,Fe(CN),. Samples were dehydrated in graded ethanol and embedded in an epoxy-resin mixture [19]. Sections were stained first with 3% (w/v) uranyl acetate in water, then with lead citrate [20] and examined with a Philips EM 301 microscope under 60 kV.

3. Results 3.1. Effect of availability nous lipids on procyclics

of exogenous and endoge-

2.6. Binding and uptake of [“‘I]LDL All incubations were performed at lo7 cells ml-’ (with about lo8 cells, i.e., 1 mg of cell protein). Procyclics were incubated at 28°C for 1 h (uptake experiments) or at 4°C for 6 h (binding experiments), with 45 nM [‘251]LDL in the culture medium containing 10% (v/v> lipoprotein-depleted FCS. Cells were washed at 4°C twice with a solution containing 137 mM NaC1/5.4 mM KC1/0.34 mM Na,HPO,/ 0.44 mM KH,PO,/3.6 mM CaC1,/0.8 mM MgSO, at pH 7.4 (PBS-Ca2+ ) containing 1% (w/v) BSA, then twice with PBS-Ca2+ alone. Washed cells were lyzed in 1 ml of 1% (w/v) sodium deoxycholate pH 11.3. Protein content [17] and 1251radioactivity were determined. Cell-associated LDL was expressed as the amount of apoprotein per mg cell protein. The number of LDL receptors (B,,,) and their affinity for LDL (K,) were estimated from the Michaelis-Menten equation by non-linear fitting based on the least-squares method (Systat, Apple). A polyclonal rabbit antiserum directed against the purified LDL receptors of bloodstream forms of T. brucei was prepared as described [4]. The concentration of 10% (v/v) antiserum in the culture medium corresponded to an ELISA titer of 1:2000, against the LDL receptor preparation.

Culture-adapted procyclic trypomastigotes of T. brucei, seeded at lo6 cells ml-’ in a semi-defined

L

I

I

012345 Days Fig. 1. Effect of LDL and synvinolin on trypanosome growth. Procyclics were. cultured in medium either with 10% complete FCS alone ( A ), or supplemented by 300 nM LDL (0 ), by 25 PM synvinolin (A), or by 300 nM LDL+25 PM synvinolin (0). Other trypanosomes were cultured in medium with 10% lipoprotein-depleted FCS alone (0) or containing 25 PM synvinolin (m 1. At the indicated times, viable trypanosomes were counted in an haemocytometer. The study was repeated 3 times with < 10% variation.

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Fig. 2. Effect of synvinolin on procyclics: light microscopy. Procyclics were incubated for 24 h in the presence of 10% (v/v) complete FCS with 12.5 PM synvinolin (a) or in the presence of 10% (v/v) lipoprotein-depleted FCS with 50 PM synvinolin (b). Note that cells do not respc lnd uniformly. Arrows indicate biflagellated cells. Lysed cells are obvious in panel b (arrowheads). Bars represent 20 pm.

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183

medium supplemented with 10% FCS, multiplied at 28°C with an exponential doubling time of 14 + 2 h [ll]. After reaching peak density (approx. 4 x lo7 cells ml-‘) after 5 days, parasites ceased to divide and died within the next 24-36 h. Cultivation of these forms in medium supplemented with a large excess of freshly isolated human LDL particles (300 nM, maximal competence for binding), accelerated the growth rate (doubling time of 10 + 0.5 h) and resulted in a higher maximal cell density (Fig. 1). This observation suggests that LDL could be a source of nutriments for these forms. The concentration of LDL in 10% frozen-thawed FCS (140 nM nominal concentration, unknown competence for binding) is thus not sufficient to support an optimal cell growth. Conversely, procyclics still multiplied in culture medium depleted of lipoproteins (Fig. 11, albeit at half the normal rate (doubling time of 28 f 3 h) and a lower peak density (- 30%). By electron microscopy, incubation for up to 5 days in lipoproteindepleted FCS caused no detectable ultrastructural alterations (not shown). This confirms that, in contrast to the bloodstream forms, procyclics are equipped with a lipid biosynthesis machinery and can grow independently of exogenous lipids provided by lipoproteins. As already reported in Ref. 12, growth of procyclics in 10% (frozen-thawed) FCS was slowed down 2-fold by 25 PM synvinolin, an HMG-CoA reductase inhibitor, but the drug effect was antagonized by the addition of 300 nM fresh LDL. Conversely, incubation with 25 JLM synvinolin in lipoprotein-depleted serum, arrested cell proliferation and led to death of the parasites (Fig. 1). These observations indicate that when the endogenous sup-

Fig. 3. Effect of synvinolin on procyclics: electron microscopy. Procyclics were incubated for 24 h with 12.5 PM synvinolin in the presence of 10% (v/v) complete FCS (a) or in 10% (v/v) lipoprotein-depleted FCS (b). Other trypanosomes were incubated in the presence of 10% (v/v) complete FCS with 50 FM synvinolin for 48 h cc). Note, in panel a the abundance of small vesicles in the cytoplasm (arrow) and a myelinic structure, probably produced by autophagy (asterisk); in panel b, an abundance of small vesicles within the flagellar pocket (arrowhead) and evidence of vacuolisation: in panel c, the swelling of the mitochondrion that occupies approx. 60% of the area of this sectioned cell, compared to approx. 25% in panel b. Bars represent 0.5 pm.

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ply is inhibited, uptake of exogenous lipids can compensate for the lack of lipid synthesis and becomes even crucial for parasitic survival. Synvinolin caused several cytopathic effects in procyclics (Figs. 2 and 3). After 24 h in the presence of complete FCS supplemented by 12.5 PM synvinolin, there was a significant increase in the percentage of cells bearing two flagella and two nuclei, indicating an arrest of cytokinesis (15 out of 270 treated parasites, or approx. 5%; 7 out of 520 control parasites, or approx. 1%; P < 0.0001). At higher synvinolin concentrations and/or longer incubation time, the drug led to cell lysis. At any concentration of synvinolin tested, these alterations appeared faster and were more conspicuous in trypanosomes cultivated in lipoprotein-depleted than in complete serum. By electron microscopy, the cytoplasm of parasites incubated for 24 h in complete FCS with 12.5 PM synvinolin contained autophagic vacuoles and an unusual abundance of small vesicles of unidentified nature (Fig. 3a). Other vesicles were filling the lumen of the flagellar pocket of parasites incubated for 24 h in the presence of lipoprotein-depleted serum containing 12.5 PM synvinolin (Fig. 3b). The volume of kinetoplast-mitochondrion complex, that normally occupies approx. 25% of the cell, was increased roughly 2-fold; its spherical contour and the dilatation of inner cristae suggested a loss of osmoregulatory properties (Fig. 3b,c). After 2 days of incubation with 50 PM synvinolin in the presence of 10% FCS and especially in lipoprotein-depleted serum, the remaining procyclics frequently showed disruption of the plasma membrane or were undergoing lysis. 3.2. Specificity of LDL endocytosis in procyclics An LDL receptor has been previously isolated from T. brucei bloodstream forms and procyclics as a 145-kDa glycoprotein [21]. The specificity of LDL receptors was determined by ligand blotting using human LDL. LDL receptors of the two forms share common epitopes [22,23]. Below we present evidence for receptor-mediated endocytosis of LDL in the procyclic form, as extensively reported for the bloodstream form [2-4,6,21]. Acquisition of LDL by procyclics at 28°C was much higher than at 4”C, indicating that endocytosis

73 (1995) 179-188

r

no

EDTA

4’C

suramin HDL

BSA no

anti-LDLR

28°C

LDL 10x LDL 100x LDL 500x 0

1000

2000

LDL [fmol.(mg cell

protein

3000 )_‘I

Fig. 4. Specificity of LDL acquisition. After cultivation in medium with 10% (v/v) complete FCS, procyclics were washed and resuspended at lo7 cells ml-’ in the culture medium containing 10% (v/v) lipoprotein-depleted FCS, for 6 h at 4°C or 1 h at 28”C, in the presence of 45 nM [“‘IILDL. Other binding experiments were performed with addition of 4 mM EDTA, 100 PM suramin, or a lOO-fold molar excess of HDL or BSA. Other uptake experiments were realized by incubation of the cells in 10% (v/v) lipoprotein-depleted anti-LDL receptor antiserum, or with the indicated molar excess of unlabeled LDL. After washing, cell-associated radioactivity was measured and expressed as ng apoprotein (mg cell protein)-’ (mean f SD, n = 5). For comparisons of means vs. control conditions (4°C or 28”C), Student’s t-test was used. * * P < 0.0005; * P < 0.005; NS, not significant.

of LDL took place (Fig. 4). Binding of LDL at 4°C was inhibited by the cation chelator EDTA or by the polyanionic compound suramin and was not affected by a large excess of non-related ligand such as high-density lipoproteins or BSA. Uptake of [lz51]LDL at 28°C was specifically blocked by antibodies directed against the LDL receptor of bloodstream forms of T. brucei and was competed for by unlabeled LDL in a concentration-dependent manner. We determined the affinity of the LDL receptor from procyclics, as well as the number of receptors per cell. From binding experiments performed by a 6-h incubation at 4”C, B,,, was estimated at 64 800 f 11650 and K,, at 7.4 X lop7 f 1.1 X 10m7 M (n = 6). These observations closely parallel those made for the specific binding and endocytosis of LDL in bloodstream forms [3] and in mammalian cells [24,25], suggesting that the LDL-binding site of procyclics shares a similar mode of interaction with its ligand.

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Table 1 Effect of LDL and synvinolin sterols Medium

10% + + + 10%

on [ l4 Clacetate

Molecular and Biochemical Parasitology

incorporation

into

[I4 Clacetate incorporation into sterols

Complete serum Synvinolin (5 PM) LDL (300 nM) LDL (800 nM) Lipoprotein-free serum

dpm (lo6 cells)- ’

n

24 340 rf: 1850 10120* 940 13820+ 760 11550+ 488 38444+3250

8 4’ 3 3 5

Procyclics were incubated with [14C]acetate in the indicated medium for 48 h. After washing, total lipids were extracted and radioactivity of unsaponifiable material was measured (mean+ SD). After comparison of means with control conditions (10% complete serum) using Student’s r-test, all differences are highly significant (P < 0.0005). * In good agreement with Ref. 12.

3.3. Adaptative changes of sterol content and biosynthesis in procyclics under various environments The content in endogenous ergosterol and exogenous cholesterol was evaluated in procyclics that were exponentially growing in various culture conditions. When parasites were cultivated in lipoproteindepleted serum, only ergosterol was detectable in sedimentable membranes (not shown). After a 2-day cultivation in complete serum, cholesterol was also detected in a proportion that increased roughly with

Table 2 Influence

of lipoproteins

the extracellular concentration of LDL, indicating a concomitant utilization of cholesterol imported by LDL and a lower rate of ergosterol biosynthesis. At the highest LDL concentration used (300 nM), ergosterol was still detectable. As expected, when procyclics were cultivated in complete serum with synvinolin, a time-dependent decrease of ergosterol similarly coincided with an increased content in cholesterol, whereas ergosterol remained detectable after 2 days in the presence of 25 PM synvinolin (not shown). We further explored to what extent the rate of sterol biosynthesis could be influenced by exogenous sterol supply. The addition of an excess of extracellular LDL (300 nM) led to a approx. 3-fold decrease of acetate incorporation in the sterol fraction in comparison with procyclics cultivated without lipoproteins (Table 1). We attribute this process to a down-regulation of the HMG-CoA reductase activity by exogenous cholesterol brought in by internalized LDL. In the presence of a very high LDL concentration (800 nM) in the incubation medium, no further reduction in the rate of acetate incorporation into sterols was observed, suggesting that the role of LDL import is limited by the saturation of its receptors, so that ergosterol biosynthesis cannot be totally abolished. Conversely, when procyclics were cultured in 10% of lipoprotein-depleted serum instead of 10% complete FCS, the activity of HMG-CoA reductase

and aging in culture on sterol content, HMG-CoA

reductase

activity and LDL clearance

in procyclics

HMG-CoA reductase activity (pm01 mevalonate (mg cell protein)- ’ mint)

LDL clearance ( ~1 (mg cell protein)-

11.3 + 0.8 a 16.7 f 2.1 a’

13.3 + 2.3 b 5.7 f 0.5 b’

44.5 + 5.2 ’ 59.9 * 8 ”

9.3 f 1.7 d 14.7 f 2.9 d’

21.8 f 1.8 e 11.9 + 2.7 ”

56.5 + 3.3 71.0 f 6.5

Sterol content ( pg (mg cell protein)-

Complete FCS Exponential phase Stationary phase Lipoprotein-depleted FCS Exponential phase Stationary phase

185

73 (1995) 179-188

’)

’ h- ‘1

’ f

Procyclics were seeded at lo6 cells ml-’ in medium supplemented with either 10% (v/v) complete FCS or 10% (v/v) lipoprotein-depleted FCS and harvested either after 72 h of culture (exponential phase), or after 108 h of culture (stationary phase). After washing, cells were processed for analysis of sterol content, HMG-CoA reductase activity and LDL clearance. The latter was calculated by substracting the difference between the amount of cell-associated LDL after 1 h of incubation at 28°C (total uptake) and after 6 h at 4°C (surface binding) to yield intracellular uptake (ng apoprotein (mg cell protein)-’ h- ‘1, and by dividing this value by the LDL concentration (25 pg ml-’ 1. Means f SD, n = 4. For comparison of means, Student’s r-test was used. P < 0.005 for a vs. a’, b vs. b’, e vs. e’; P < 0.01 for c vs. f, f vs. f’, P < 0.025 for c vs. c’, d vs. 6, P not significant for c’ vs. f’.

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and the rate of acetate incorporation into sterols were enhanced 2-fold, suggesting an inhibitory effect of cholesterol on the HMG-CoA reductase (Table 21, as reported for mammalian cells [26]. Incubation without lipoproteins also upregulated the abundance of surface LDL receptors [12]. Sterol content in a stationary population of procyclics having reached a high density was significantly higher than in exponentially growing parasites, irrespective of the presence of lipoproteins in the extracellular medium (Table 2). Similarly, the uptake of LDL was moderately increased in procyclics at the stationary phase, whether previously grown with or without lipoproteins, but the activity of HMG-CoA reductase was decreased approx. 2-fold upon aging in culture. Taken together, these observations suggest that, like many unicellular microorganisms exposed to different media, procyclics can adapt to major variation in their environments, such as availability of lipoproteins as well as to aging in culture, by modifying their sterol metabolism.

4. Discussion First, this study demonstrates that the growth of the insect forms of T. brucei (procyclic forms) in vitro is affected by the lipoprotein content in the medium, especially by LDL particles. Indeed, growth in LDL-enriched medium is accelerated and the maximal density of parasites is increased, whereas removal of lipoproteins slows down the growth and decreases the maximal population density. Presence of lipoproteins is however not critical for procyclic survival, since these forms have the capacity for fatty acid and sterol biosynthesis, in contrast to T. brucei bloodstream forms [7]. Growth inhibition by synvinolin, an HMG-CoA reductase inhibitor, can be antagonized by the addition of 300 nM LDL but cell densities do not reach those obtained with 300 nM LDL alone. This points to the fact that HMG-CoA reductase in procyclics is probably involved in the biosynthesis of vital isoprenoids other than sterols, such as ubiquinone and dolichol. Second, we show that procyclics, like bloodstream forms, internalize LDL by receptor-mediated endocytosis with the same avidity and utilize LDL-

73 (1995) 179-188

associated cholesterol [4]. Indeed, culture with LDL results in the occurrence in cellular membranes of cholesterol, a sterol these parasites do not synthesize [7]. This indicates that both exogenous and endogenous sterols contribute to procyclics grown in culture medium containing lipoproteins. Utilization of host sterols derived from lipoproteins is not rare in parasites, some of which possess specific receptors for lipoprotein endocytosis [lo]. The presence of LDL receptor in the insect stage of African trypanosomes is not so surprising, since insects synthesize lipophorins, one of which shows a striking homology with the receptor-binding domain of apoproteins [27]. Third, our data emphasize that procyclics in culture can detect environmental changes and modify their metabolism accordingly. When these parasites are grown in the presence of extracellular LDL, ergosterol biosynthesis is decreased as evidenced by measurement of HMG-CoA reductase activity and of acetate incorporation into sterols. We suggest that supply of host cholesterol down-regulate enzyme(s) involved in the sterol production. Fourth, we observe a significant increase of sterol content in aged culture of procyclics, a finding already reported for other aged parasites in culture and often associated with a concomitant decrease of phospholipid content, resulting into a more rigid architecture of membranes [28]. These phenomena might be related to a higher uptake of LDL in stationary procyclics, although a slower sterol catabolism upon aging cannot be excluded. Fifth, the ultrastructural alterations of T. brucei induced by synvinolin are similar to those reported in T. crud epimastigotes (proliferating in the digestive tract of the Reduviid vectors) when incubated with drugs blocking ergosterol biosynthesis at the level of cytochrome P-450-dependent 14a-demethylase, such as ketoconazole that produces an accumulation of 14a-methylated sterols [29]. The scenario leading to T. brucei disintegration upon synvinolin treatment is also similar to that reported in ketoconazole-treated T. cruzi. After a 48-h lag, during which the original ergosterol pool is presumably exhausted by dilution into dividing cells, the cells rather suddenly stop growing. Concomitantly, a marked swelling of the volume of the kinetoplastmitochondrion system rapidly develops, consistent wih a loss of function of this organelle. Although

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many drugs, with different modes of action (pentamidine, platinum, antibiotics) cause similar early changes in this organelle, it is interesting to note that ergosterol preferentially associates with the inner mitochondrial membrane of T. cruzi [30], and this seems to be a characteristic in trypanosomatids [29], in contrast with vertebrate and plant cells, where the inner mitochondrial membrane is devoid of sterols [311. So, both in T. cruzi epimastigotes and T. brucei procyclics, a key observation is the alteration in the structure and function of the single mitochondrion, the main energy-transforming organelle of these parasites. This could account for the loss of viability observed after total ergosterol depletion [32,33] since this sterol is considered to play an unique regulatory function [34]. In contrast, when T. brucei procyclics are incubated for 2 days with 25 PM synvinolin, the bulk of membrane sterol is cholesterol, but ergosterol remains detectable in membranes and could, thus, fulfill a regulatory role.

Acknowledgements We are indebted to Merck, Sharp and Dohme and to Rhbne-Poulenc Rorer for partial financial support to this project. This investigation was supported by grants 2.4547.91 and 1.5246.91 of the Belgian National Fund for Scientific Research (F.N.R.S.), as well as by the Federal Office for Scientific, Technical and Cultural Affairs (concerted actions) and Framework of Inter-university Attraction Poles. I.C. was supported by a special Grant of the F.N.R.S.

References [II Carroll, M. and McCrorie,

P. (1986) Lipid composition of bloodstream forms of Trypanosoma brucei brucei. Comp. Biochem. Physiol. 83B, 647-651. 121Gill&t, M.P.T. and Owen, J.S. (1987) Trypanosoma brucei brucei obtains cholesterol from plasma lipoproteins. Biochem. Sot. Trans. 15, 258-259. [31 Coppens, I., Opperdoes, F.R., Courtoy, P.J. and Baudhuin, P. (1987) Receptor-mediated endocytosis in the bloodstream form of Trypanosoma brucei. J. Protozool. 34, 465-473. 141 Coppens, I., Baudhuin, P., Opperdoes, F.R. and Courtoy, P.J. (1988) Receptors for the host low density lipoproteins on the hemoflagellate Trypanosoma brucei: purification and in-

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