Effects of sterol biosynthesis inhibitors on endosymbiont-bearing trypanosomatids

Abstract

Some protozoa of the Trypanosomatidae family have a close relationship with an endosymbiotic bacterium. As the prokaryote envelope has a controversial origin, a sterol 24-methyltransferase inhibitor (20-piperidin-2-yl-5α-pregnan-3β,20-diol; 22,26-azasterol) was used as a tool to investigate lipid biosynthetic pathways in Crithidia deanei, an endosymbiont-bearing trypanosomatid. Apart from antiproliferative effects, this drug induced ultrastructural alterations, consisting of myelin-like figures in the cytoplasm and endosymbiont envelope vesiculation. Concurrently, a dramatic reduction of 24-alkyl sterols was observed after 22,26-azasterol treatment, both in whole cell homogenates, as well as in isolated mitochondria. These effects were associated with changes of phospholipid composition, in particular a reduction of the phosphatidylcholine content and a concomitant increase in phosphatidylethanolamine levels. Lipid analyses of purified endosymbionts indicated a complete absence of sterols, and their phospholipid composition was different from that of mitochondria or whole protozoa, being similar to eubacteria closely associated with eukaryotes.

cellular evolution, endosymbiotic bacterium, lipid biosynthesis, mitochondrion, phospholipid composition, trypanosomatids

Introduction

In some trypanosomatids, endosymbiotic bacteria have coevolved with the host protozoan, making this a valuable model to understand the origin of organelles, such as the mitochondrion and the chloroplast. Several lines of evidence indicate intensive metabolic exchanges between both partners, this being an example of a mutualistic symbiosis (De Souza & Motta, 1999). Analysis of 16S rRNA gene sequence indicated that the endosymbiont present in all trypanosomatid species is phylogenetically associated with the genus Bordetella, which belongs to the β group of Proteobacteria (Du et al., 1994).

There is controversy about the origin of the symbiont envelope. For some authors, it is derived from the host plasma membrane (Chang, 1974), while others believe that it has a prokaryotic origin (Gutteridge & Macadam, 1971; Soares & De Souza, 1988; Motta et al., 1991). Previous studies revealed that the endosymbiont has a degenerated peptidoglycan layer (Motta et al., 1997a), and that during cell division the septum and the FtsZ ring are absent (Motta et al., 1991, 2004). Furthermore, ultrastructural analysis, using freeze-fracture techniques, demonstrated the presence of a number of intramembranous particles in the endosymbiont envelope, similar to those described for Gram-negative bacteria, but different from that found in the host trypanosomatid plasma membrane (Soares & De Souza, 1988; Motta et al., 1991).

No sterols are normally present in the lipid composition of bacteria, and the major phospholipid is phosphatidylethanolamine (PE), followed by phosphatidylglycerol (PG) and cardiolipin (Dowhan, 1997). Phospholipids play essential roles in bacteria, providing a permeability barrier, allowing for proper insertion and localization of proteins in the lipid bilayer, and are also involved in initiation of DNA replication (Cronan, 2003). On the other hand, phosphatidylcholine (PC) is the major phospholipid in eukaryotes, being an essential structural component of cell membranes and playing an essential role in signal transduction and the regulation of the cell cycle (Exton, 1994; Wright et al., 2004). However, in prokaryotes, phosphatidylcholine is present only in species closely associated with eukaryotes, either in symbiotic or pathogenic interactions with plants or animal hosts (Hindahl & Iglewski, 1984; Miller et al., 1990; Belisle et al., 1994; Abelo & Domenech, 1997; Rudder et al., 1997).

Eukaryotic organisms usually possess two alternative pathways for phosphatidylcholine biosynthesis. In the CDP-choline pathway, also known as the Kennedy pathway, free choline is converted to phosphatidylcholine via the intermediates choline-phosphate and CDP-choline and is the predominant pathway in mammalian cells (Kennedy & Weiss, 1956). In the methylation (Greenberg) pathway, phosphatidylcholine is formed by three successive methylations of phosphatidylethanolamine via a specific N-methyl transferase, and this is the predominant pathway in lower eukaryotes (Robson et al., 1990). Only the methylation pathway of phosphatidylcholine biosynthesis is thought to occur in prokaryotes (Vance & Ridgway, 1988). However, a novel pathway for phosphatidylcholine biosynthesis has been described, involving the enzyme phosphatidylcholine synthase, which condenses choline with CDP-diacylglyceride directly (Rudder et al., 1997, 1999). Furthermore, in symbiotic relationships, choline can be provided by the plant or animal hosts to the prokaryote, making the phosphatidylcholine synthase pathway an advantageous alternative route for phosphatidylcholine biosynthesis in the symbiotic bacterium (Sheard et al., 1986; Rudder et al., 1999).

Ergosterol is an essential component for many protozoa, yeasts and fungi that cannot be replaced by cholesterol, the main sterol present in the vertebrate hosts or common culture media (Beach et al., 1986; Goad et al., 1989; Urbina et al., 1991, 1995, 1996; Rodrigues et al., 2002). 20-piperidin-2-yl-5α-pregnane-3β,20-diol (22,26 azasterol) has been used in many studies of experimental chemotherapy against pathogenic trypanosomatids, as it has potent and selective biochemical, antiproliferative and ultrastructural effects on these protozoa (Urbina et al., 1995, 1996; Rodrigues ., 2002). This drug inhibits sterol 24-methenyl transferase (24-SMT), a key enzyme in the biosynthesis of ergosterol and other 24-alkyl sterols, not present in mammalian cells (Urbina et al., 1991, 1995, 1996). Ergosterol is an essential component for many protozoa, yeasts and fungi that cannot be replaced by cholesterol, the main sterol present in the vertebrate hosts or common culture media (Beach et al., 1986; Goad et al., 1989; Urbina et al., 1991, 1995, 1996). Furthermore, it has been found that 22,26-azasterol also affects the phospholipid composition in trypanosomatids, probably by indirect inhibition of phosphatidylethanolamine N-methyl transferase owing to the altered sterol composition of cell membranes (Contreras et al., 1997). Considering these antecedents, 22,26 azasterol is a valuable compound to investigate the lipid biosynthetic pathways in lower eukaryotes.

In this work, we investigated the effects of 22,26 on the lipid composition of Crithidia deanei, as well as that of purified mitochondria and endosymbionts. Ultrastructural studies were also performed to assess the effects of the altered lipid composition on subcellular structures. The mitochondrial was used as a comparative model in relation to the endosymbiotic bacterium, revealing new aspects concerning the symbiotic origin of organelles.

Materials and methods

Cell growth

Crithidia deanei used in this study was isolated from Zelus leucogrammus and corresponds to ATCC 30255. Cells were grown at 28°C for 24 h in Warren culture medium (Warren, 1960) supplemented with 10% fetal calf serum. 22,26-Azasterol was synthesized and purified as described before (Urbina et al., 1995). This drug was added to protozoa cultures from stock dimethyl sulfoxide (DMSO) solutions; the final DMSO concentration in the medium never exceeded 1% (volume in volume, v/v) and had no effect on cell proliferation. Protozoa were collected from the culture at 12 h intervals; part of the cells was fixed in 10% formaldehyde and the cells were counted in a Neubauer chamber. Another part of the culture was fixed in 2.5% glutaraldehyde and processed for transmission electron microscopy as described below.

Endosymbiont fraction

A modified version of the procedure described by Alfieri & Camargo (1982) was used to prepare endosymbiont-enriched fractions. Briefly, cells were grown to log phase (800 mL containing 10⁸ cells mL⁻¹) for 24 h in Warren medium. The culture was centrifuged at 4000 g for 10 min and washed twice in phosphate-buffered saline (PBS), pH 7.2. The pellet was resuspended in cold distilled water and left for 45 min on ice. Cells were then centrifuged at 4000 g, resuspended in 12 mL of 20 mM Tris-HCl, pH 7.6 containing 0.25 M sucrose and sonicated using a Ultrasonic disruptor GEX 600 (Sigma Chemical Company, St. Louis, MO) (three 15 s pulses at 10% amplitude). The volume was adjusted to 20 mL with 20 mM Tris-HCl, pH 7.6 containing 0.25 M sucrose, 2 mM CaCl₂, 10 mM MgCl₂ and 25 μg mL⁻¹ DNase type I (Sigma Chemical Company). Cells were incubated with this solution at 25°C for 30 min and the volume was increased to 30 mL with 20 mM Tris-HCl, pH 7.6 containing 0.25 M sucrose and 2 mM ethylenediamine tetra-acetic acid (EDTA). The homogenate was centrifuged at 5000 g for 20 min and the pellet obtained was resuspended in Tris-HCl/sucrose/EDTA buffer containing 0.5 mg mL⁻¹ of Pronase (protease type XIV from Streptomyces griseus, from Sigma Chemical Company). The homogenate was then immediately centrifuged at 4000 g for 20 min. The pellet was resuspended in 10 mL of Tris-HCl/sucrose/EDTA buffer and 2.5 mL aliquots were layered over 2.5 mL of 0.5 M sucrose in Falcon tubes (11.5 × 1.5 cm). After centrifugation at 550 g for 10 min, the upper layer was collected and the pellet, which mainly contained whole cells, was discarded. The upper layer was centrifuged at 4000 g for 10 min and then resuspended in 6 mL of 20 mM Tris-HCl, pH 7.6 containing 0.25 M sucrose. The resuspended material was layered (1 mL per tube) on top of six tubes (13 × 51 mm), each containing a two-step sucrose gradient consisting of 0.44 M (2 mL) and 0.88 M (1 mL) sucrose. After centrifugation at 1740 g for 30 min, the endosymbiont-containing pellet was collected.

Mitochondrial fraction

This procedure is described by Motta . (1997b). Briefly, cells (500 mL containing 10⁸ cells mL⁻¹) were centrifuged at 2000 g 10 min and rinsed twice in 0.1 M phosphate buffer containing 0.25 M sucrose, pH 7.2. Protozoa were resuspended in 12 mL of the same buffer, under the same conditions described for acquiring the endosymbiont fraction. The whole cell homogenate was sedimented at 270 g for 10 min at 4°C. The obtained supernatant was centrifuged at 12 000 g for 10 min at 4°C. The new supernatant obtained was centrifuged again at 12 000 g for 10 min, leaving a pellet that contained a pure mitochondrial fraction according to biochemical and ultrastructural analysis (Motta et al., 1997b).

Transmission electron microscopy

For routine transmission electron microscopy, protozoa were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2 for 1 h. After fixation, cells were washed in cacodylate buffer and postfixed for 1 h in 1% osmium tetroxide containing 0.8% potassium ferricyanide and 5 mM CaCl₂ in 0.1 M cacodylate buffer. The cells were then washed, dehydrated in acetone and embedded in Epon. Ultrathin sections were mounted on 300-mesh grids, stained with uranyl acetate and lead citrate and observed under a Zeiss 900 transmission electron microscope (Zeiss, Oberkochen, Germany).

Neutral lipid analysis

Total lipids from control and drug-treated cells were extracted and fractionated into neutral and polar lipid fractions by silicic acid column chromatography (Urbina et al., 1995, 1996; Liendo et al., 1998). The neutral lipid fraction was first analyzed by a thin-layer chromatograph (TLC) on Merck 5721 silica gel plates (Merck, Darmstadt, Germany), with heptane-isopropyl ether-glacial acetic acid (60 : 40 : 4, v/v) as the developing solvent. Then, neutral lipid fraction was analyzed by conventional gas-liquid chromatography using isothermic separation in a 4 m glass column packed with 3% OV-1 on Chromosorb 100/200 mesh (Advanced Minerals Corp., Goleta, CA), with nitrogen as the carrier gas at 24 mL min⁻¹ and flame ionization detection in a Varian 3700 gas chromatograph (Varian Inc., Palo Alto, CA). For quantitative analysis, neutral lipids were separated in a capillary high-resolution Ultra-2-columns; 5% phenylmethyl-siloxane in a Hewlett-Packard 5890 series II gas chromatograph (Agilent Technologies, Palo Alto, CA) equipped with an HP5971 mass-sensitive detector. The lipids were injected in ethyl acetate and the column (25 m × 0.2 mm × 0.33 μm) was kept at 50°C for 1 min. The temperature was then increased to 270°C at a rate of 25°C min⁻¹ and finally to 300°C at a rate of 1°C min⁻¹. The carrier gas (He) flow was kept constant at 1.2 mL min⁻¹. The injector temperature was 250°C and the detector was kept at 280°C. The polar lipid fraction, containing mostly phospholipids, was analyzed as follows:

Phospholipid extraction and analysis

Total lipids were extracted from C. deanei cells or from isolated symbiont or mitochondrion fractions obtained by fractionation as described above. Briefly, the extraction starts by adding 4 mL of chloroform : methanol : HCl (2 : 1 : 0.075, v/v) to the samples in glass tubes, as described by Horwitz & Perlman (1987). The addition of 0.5 mL 0.6 N HCl, followed by intense agitation and centrifugation (10 min, 600 g), permitted the isolation of the lower organic phase (containing the lipids). This resultant organic phase was dried under N₂ gas, quantified gravimetrically and then reconstituted in 90 μL of chloroform : methanol : H₂O (7.5 : 2.5 : 0.2 v/v). Sixty microliters of each sample were spotted onto heated-activated silica-gel 60 TLC plates and the chromatography was developed in chloroform : acetone : methanol : acetic acid : water (120 : 45 : 39 : 36 : 24, v/v). After 1 h, the TLC plates were developed in an iodine vapor atmosphere and the lipids of interest were identified comparing the relative mobility of the spots with the commercial standards used (from Sigma Chemical Company). After identification of the different phospholipid species, the spots corresponding to each one were scraped from the silica TLC plates and put into glass tubes for inorganic phosphate determination, as previously described (Bartlett, 1959). Results were compared with a standard inorganic phosphate curve. All the solvents and TLC plates used for lipid extraction and separation were from Merck.

Results

Antiproliferative effects

22,26 azasterol induced a dose-dependent inhibition of Crithidia deanei growth with a minimum inhibitory concentration (MIC) of 3 μM after 36 h of incubation (Fig. 1). Lipid analyses were performed using 1 μM of azasterol, which significantly decreased cell growth, but did not completely inhibit protozoa proliferation, thus providing ideal conditions to test the effects of this drug on lipid metabolic pathways.

1

 Effect of azasterol on the growth of Crithidia deanei. Results are representative of three independent assays.

Ultrastructural effects induced by 22,26 azasterol

When compared with control cells (Fig 2a), protozoa incubated in the culture medium with 1 μM 22,26 azasterol presented significant morphological alterations. One of the first indications of drug action was the blebbing and shedding of the plasma membrane (Fig. 2b), as well as myelin-like figures within the cytoplasm (Fig. 2c) and the flagellar pocket. Another typical morphological change observed after azasterol treatment was the presence of protrusions of the cell membrane (Fig. 2d), from which vesicles were released into the flagellar pocket (Fig. 2e). Interestingly, the endosymbiont was also affected by the action of the drug, showing convolutions and vesiculations of its envelope (Fig. 2f). The inner membrane of the mitochondrion was only slightly enlarged, even at the highest drug concentration (10 μM) (Fig. 2c). At times, the symbiotic bacterium and the mitochondrial fraction were seen surrounded by the endoplasmic reticulum profiles, indicating autophagic processes (not shown).

2

 Ultrastructure changes were observed in Crithidia deanei after azasterol treatment. (a) control cell; (b) an intense membrane shedding (arrow) was seen after 24 h of drug treatment with 1 μM azasterol; (c) myelin-like figures were observed in the cytoplasm of the host protozoan (arrow), – note a slight swelling of mitochondrial cristae (arrowheads) when cells were treated with 10 μM azasterol for 24 h; (d) protrusion of the flagellar pocket membrane (arrow and inset) was observed when cells were treated with 1 μM azasterol for 24 h, (e) such protrusions resulted in vesicles (arrows) released from the cell body, (f) the endosymbiont envelope showed membrane vesiculations (arrows) after treatment with 1 μM azasterol for 36 h. E, endosymbiont; FP, flagellar pocket; K, kinetoplast; M, mitochondrion; N, nucleus. (a) × 24 000, (b) × 24 000, (c) × 40 000, (d) × 16 300, inset: × 24 000, (e) × 26 700 and (f) × 63 000.

Effect of 22,26-azasterol on lipid composition

Sterols were extracted from control cells and from protozoa treated with 1 μM 22,26 azasterol for 36 and 48 h. Whole cells, as well as isolated and mitochondrial fractions, were submitted to gas-liquid chromatograph coupled to mass spectrometry as previously described, and the results are shown in Table 1. In control samples, the whole cell extracts and the mitochondrial fractions presented ergosterol (24-methyl-5,7,22-cholesta-trien-3β-ol) as the major sterol (>99%, considering that other sterols were found in trace quantities). The endosymbiont fractions had no detectable sterols, as expected. When C. deanei was grown in the presence of 1 μM azasterol (MIC) for 36 h, part of the endogenous sterol (42%) was replaced by cholesta-5,7,24-trien-3β-ol, indicating that blockade involved a reaction step dependent on 24-SMT. Incubation of protozoa for 48 h with the same drug concentration resulted in a slightly lower level of ergosterol (38%) and, conversely, in higher quantities of cholesta-5,7,24-trien-3β-ol (62%). The sterol composition of the mitochondrial fraction was less affected by the incubation of cells with the azasterol, as the level of cholesta-5,7,24-trien-3β-ol was very similar after treatment with 1 μM azasterol for 36 h (51%) or 48 h (47%).

Table 1

 Effect of 22,26 azasterol on sterol composition of Crithidia deanei where whole cell extracts and mitochondrial fractions were analysed

Compound	Molecular structure	Control cells	Cells+1 μM AZA 36 h	Cells+1 μmM AZA 48 h	Mitochondria	Mitochondria+1 μM AZA 36 h	Mitochondria+1 μM AZA 48 h
Ergosterol		100	58	38	100	49	53
Cholesta-5,7,24-trien-3β-ol		ND	42	62	ND	51	47

Results are expressed as percentage. Results are representative of three independent assays.

AZA, azasterol; ND, not detected.

Biochemical analysis revealed that the major phospholipid in C. deanei whole cells was phosphatidylcholine (31.2%), followed by phosphatidylethanolamine (25%), phosphatidylinositol (17.5%) and cardiolipin (14.5%) (Fig. 3a). When compared with whole cell extracts, mitochondrial fractions presented a phospholipid content with higher amounts of phosphatidylcholine (39%) and cardiolipin (19.5%) and minor amounts of phosphatidylethanolamine (19.7%) and phosphatidylinositol (13.9%) (Fig. 4a). In the endosymbiont fractions, the major phospholipid was cardiolipin (25.7%), followed by equal amounts of phosphatidylcholine (20.1%) and phosphatidylethanolamine (19%) and minor amounts of phosphatidylinositol (15.4%) (Fig. 5a).

3

 Effect of 22,26 azasterol on phospholipid composition of whole cell extracts of Crithidia deanei. (a) control cells; (b) after 48 h of drug treatment. It is worth mentioning that after 36 h of drug treatment, results were similar to that found in control cells. Results are representative of three independent assays. PI, phosphatidylinositol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; CL, cardiolipin.

4

 Effect of 22,26 azasterol on phospholipid composition of mitochondrial fractions obtained from Crithidia deanei. (a) control; (b) after 36 h of drug treatment; (c) after 48 h of drug treatment. Results are representative of three independent assays. PI, phosphatidylinositol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; CL, cardiolipin.

5

 Effect of 22,26 azasterol on phospholipid composition of endosymbiont fractions obtained from Crithidia deanei. (a) control; (b) after 36 h of drug treatment; (c) after 48 h of drug treatment. Results are representative of three independent assays. Results are representative of three independent assays. PI, phosphatidylinositol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; CL, cardiolipin.

Incubation of C. deanei with 1 μM azasterol for 36 or 48 h led to significant alterations of its phospholipids content (Fig. 3b). Treated cells presented a higher level of phosphatidylethanolamine (39%) when compared with untreated controls associated with lower levels of phosphatidylcholine (21.9%) and phosphatidylinositol (5.7%), but the level of cardiolipin (13.5%) was not affected. Regarding mitochondrial fractions, a trend similar to that of whole cells was observed: the levels of phosphatidylethanolamine increased after treatment with 1 μM azasterol (to 45.6% after 36 h and to 52.7% after 48 h), while the opposite was observed in relation to phosphatidylcholine (to 26.8% after 36 h and to 14.9% after 48 h). The levels of phosphatidylinositol and cardiolipin did not show significant alterations after the drug treatment, as seen in Figs 4b and c. Endosymbionts extracted from cells treated with 1 μM azasterol also presented an increase in the phosphatidylethanolamine content (to 25.2% in 36 h and to 37.5% after 48 h), but the levels of phosphatidylcholine were not significantly altered. A clear reduction in the content of phosphatidylinositol and cardiolipin was observed in the endosymbionts as a consequence of azasterol treatment of the host cell (Figs 5b and c).

Discussion

Drugs which affect lipid biosynthesis have been successfully used as antiproliferative agents in experimental chemotherapy studies against trypanosomatids. In this study, 22,26-azasterol, a bonafide 24-SMT inhibitor, was used to investigate the lipid metabolic pathways present in Crithidia deanei, an endosymbiont-bearing trypanosomatid. It was found that this protozoan was approximately 10 times more susceptible to 22,26 azasterol than the epimastigote (extracellular) forms of Trypanosoma cruzi (Urbina et al., 1996), but significantly less susceptible than the intracellular amastigote forms of T. cruzi and Leishmania amazonensis (Urbina et al., 1996; Rodrigues et al., 2002).

Regarding the effect of 22,26 azasterol on C. deanei ultrastructure, one of the first indications of morphological alterations was observed in the region of the flagellar pocket, which showed myelin-like figures and protrusions formed by the plasma membrane. Such morphological changes strongly suggest an intense alteration in membrane fluidity, probably as a result of the drug-induced alteration of their sterol content. In previous studies, the same type of ultrastructural modifications was observed in the promastigote forms of L. amazonensis and in the epimastigote forms of T. cruzi (Vivas et al., 1996; Rodrigues et al., 2002; Braga et al., 2004). On the other hand, the mitochondrion of treated cells had only minor ultrastructural modifications, in contrast to the situation of other trypanosomatids treated with azasterols or other ergosterol biosynthesis inhibitors, where an intense mitochondrial swelling was observed (Lazardi et al., 1990, 1991; Vannier-Santos et al., 1995; Vivas et al., 1996; Rodrigues et al., 2002). Azasterol treatment also promoted ultrastructural changes in the symbiotic bacterium, such as membrane convolutions and vesiculations, although no sterols were detected in these organisms.

It is known from previous studies on other trypanosomatid parasites that the depletion of endogenous sterols induces modifications on the phospholipid content, probably as a result of inhibition of phosphatidylethanolamine N-methyltransferase (Contreras et al., 1997). The data presented here revealed that the phospholipid biosynthesis in C. deanei was indeed affected by incubation with 22,26 azasterol. The most pronounced effect was the increased quantity of phosphatidylethanolamine during the drug treatment, with a concomitant reduction in the phosphatidylcholine content. This strongly suggested that the methylation pathway, which results in phosphatidylcholine biosynthesis through sequential methylations of phosphatidylethanolamine, was blocked.

The mitochondrion, an organelle of symbiotic origin, presented a sterol and phospholipid content similar to that of whole cells. During the evolutionary process, the mitochondrion lost or transferred to the host cell nucleus most of its DNA content. As a consequence, this organelle imports the majority of its proteins and lipids from the cytoplasm, which would explain the similarity in the lipid composition of the organelle and whole cells and the correlated changes observed as a result of treatment with the azasterol.

On the other hand, the endosymbiont had a marked difference in phospholipid content when compared with whole cells or mitochondria, and was devoid of sterols indicating that its lipid biosynthetic pathways were relatively independent from the host. However, significant levels of phosphatidylcholine were detected, which is unusual for prokaryotes, except in those that maintain a close symbiotic or pathogenic relationship with eukaryotic cells (Hindahl & Iglewski, 1984; Miller et al., 1990; Belisle et al., 1994; Abelo & Domenech, 1997; Rudder et al., 1997). One route for phosphatidylcholine biosynthesis in symbiotic bacteria involves the enzyme phosphatidylcholine synthase, which directly condenses choline with CDP-diacylglyceride (Rudder et al., 1997; López-Lara & Geiger, 2001). The symbiont can import choline from the host, making this route a valuable alternative to the methylation pathway for phosphatidylcholine biosynthesis (Sheard et al., 1986; Rudder et al., 1999). In this work, the levels of phosphatidylcholine in the symbiotic bacterium decreased after treatment with azasterol for 36 h, suggesting that some of the phosphatidylcholine in the symbiont is obtained from the host, via the methylation pathway. However, different from results obtained with the mitochondrion, the amounts of phosphatidylcholine in the symbiont did not diminish after 48 h of drug treatment. Conversely, the level of phosphatidylethanolamine in the symbiont increased as the azasterol treatment proceeded, indicating that the phosphatidylcholine synthase pathway is present in the symbiont. If true, this implies that the trypanosomatid host may function as the provider of choline for the endosymbiont.

Apicomplexan protozoa, such as Babesia bovis, can incorporate radiolabelled choline into complex lipids, especially phosphatidylcholine (Florin-christensen et al., 2000); also, a selective internalization of choline-containing phospholipids was demonstrated in Plasmodium falciparum parasitized erythrocytes (Simões et al., 1991). In Leishmania major, phosphatidylcholine biosynthesis via the CDP-choline or Kennedy pathway requires transport of the choline precursor from the host. This import into Leishmania is highly specific and is inhibited by phosphocholine analogs, such as miltefosine and edelfosine (Zufferey & Mamoun, 2002). Pseudomonas aeruginosa and Agrobacterium tumefaciens are parasite and symbiotic intracellular prokaryotes, respectively. Both Gram-negative bacteria are able to uptake and utilize choline from the medium for phosphatidylcholine biosynthesis (Sherr & Law, 1965; Abelo & Domenech, 1997). It has also been shown that Sinorhizobium meliloti, a prokaryote that develops a nitrogen-fixing symbiosis with roots of leguminous plants, uses exuded choline from the host to synthesize phosphatidylcholine, which is essential for the bacterial growth (Rudder et al., 1999). Furthermore, the presence of phosphatidylcholine in Bradyrhizobium japonicum membranes is essential for an efficient symbiotic interaction of this bacterium with its soybean host plant (Minder et al., 2001).

Another interesting finding concerning the lipid composition of the C. deanei endosymbiont is the significant amount of phosphatidylinositol. There are few reports showing the presence of phosphatidylinositol as a component of bacterial membrane or cell wall. It is well established that phosphatidylinositol is very important for the bacterial metabolism, being a precursor of important surface molecules including cardiolipin, as well as in cell-signaling pathways (Nigou et al., 2004; Haites et al., 2005). Moreover, for parasitic bacteria as well as for symbiotic ones it is not known if the phosphatidylinositol is only synthesized by the bacteria (Morita et al., 2005), if there is a subversion of the host machinery to obtain important lipids including phosphatidylinositol (Pizzaro-Cerda & Cossart, 2004) or if even both metabolic routes are present. Further experiments are underway to clarify this question, with regard to C. deanei endosymbiont. For other types of bacteria, the release of some phosphatidylinositol-containing molecules or phosphatidylinositol-related enzymes – such as the phosphatidylinositol-phospholipase C – leads to the activation of important pathways in the host cells, which in turn facilitates bacterial symbiont or parasite survival (Geisel et al., 2005; Poussin & Goldfine, 2005).

Taken together, these data suggest that in close symbiosis and parasitic relationships, microorganisms use similar strategies to obtain essential nutrients from the host cell. An intensive phospholipid trafficking between the host trypanosomatid and the intracellular symbiont is suspected, although its pathways are still unknown. In this way, assays with radiolabelled choline or phospholipids and characterization of enzymes involved in the phospholipid biosynthesis are essential to clarify important metabolic aspects of the endosymbiosis in trypanosomatids. The present study reinforces the idea that endosymbiont-harbouring trypanosomatids constitute an excellent experimental model to better understand the symbiotic origin of organelles.

Acknowledgements

We thank Danielle Cavalcanti and Thiago Manchester for technical assistance and David Graham Straker for English revision. This study was supported by CNPq, FAPERJ and FUJB, and by the Howard Hughes Medical Institute (Grant 55000620 to J. A. U.).