Effects of miltefosine on the proliferation, ultrastructure, and phospholipid composition of Angomonas deanei, a trypanosomatid protozoan that harbors a symbiotic bacterium

Abstract

Some trypanosomatids, such as Angomonas deanei formerly named as Crithidia deanei, present an obligatory intracellular bacterium, which maintains a mutualistic relationship with the host. Phosphatidylcholine (PC) is the major phospholipid in eukaryotes and an essential component of cell membranes playing structural, biochemical, and physiological roles. However, in prokaryotes, PC is present only in those species closely associated with eukaryotes, either in symbiotic or pathogenic interactions. In trypanosomatids, the endosymbiont envelope is composed by a reduced cell wall and by two membrane units that lack sterols and present cardiolipin (CL) and PC as the major phospholipids. In this study, we tested the effects of miltefosine in A. deanei proliferation, as well as, on the ultrastrucuture and phospholipid composition considering that this drug inhibits the CTP-phosphocholine cytidyltransferase (CCT), a key enzyme in the PC biosynthesis. Besides the low effect of miltefosine in cellular proliferation, treated protozoa presented ultrastructural alterations such as plasma membrane shedding and blebbing, mitochondrial swelling, and convolutions of the endosymbiont envelope. The use of ³²Pi as a tracer revealed that the production of PC, CL, and phosphatidylethanolamine decreased while phosphatidylinositol production remained stable. Mitochondrion and symbiont fractions obtained from protozoa treated with miltefosine also presented a decrease in phospholipid production, reinforcing the idea that an intensive metabolic exchange occurs between the host trypanosomatid and structures of symbiotic origin.

endosymbiosis, miltefosine, phospholipid biosynthesis, trypanosomatid protozoa, ultrastructure

Introduction

Phospholipids are essential components of biological membranes for playing roles in cell integrity, permeability, signaling, and growth (Dowhan, 1997). Phosphatidylcholine (PC) is known as the major phospholipid component in eukaryotic cells and also plays a role in signal transduction, especially through the generation of second messengers (Exton, 1994; Zeisel, 1997). In contrast only about 10% of all bacteria, those that live in close association with plant and animal hosts, present this phospholipid. In such cases, PC is essential to maintain the symbiotic and pathogenic interactions as well as the prokaryote virulence (Comerci et al., 2006; Wessel et al., 2006; Conover et al., 2008).

In higher eukaryotes, PC is mainly synthesized via Kennedy pathway, where free choline is converted to PC by intermediates of choline-phosphate and CDP = cytidine diphosphate-choline (Kennedy & Weiss, 1956). In bacteria and in lower eukaryotes, PC is mainly produced by three successive methylations of phosphatidylethanolamine (PE), which is catalyzed by phospholipid N-methyltransferase (Pmts), also called the Greenberg pathway (Kent, 1995; de Rudder et al., 2000). An alternative route that is exclusively found in bacteria comprises the direct condensation of exogenous choline and CDP-diacylglycerol in a reaction catalyzed by a PC synthase. This activity has been demonstrated in several symbiotic and pathogenic prokaryotes that maintain a close relationship with eukaryotic cells (Sohlenkamp et al., 2000; Martínez-Morales et al., 2003; Comerci et al., 2006; Wessel et al., 2006). In trypanosomatids, the phospholipid biosynthesis is better characterized in Trypanosoma brucei, where genes encoding all enzymes of the Kennedy pathway have been identified, including the CTP = cytidine triphosphate-phosphocholine cytidyltransferase (CCT), which catalyzes the limiting step of this main de novo PC biosynthesis pathway (Smith & Bütikofer, 2010).

Alkyl-lysophospholipids (ALPs) and their analogues are derived from naturally occurring phospholipids and have been widely used as anticancer and antiparasitic agents. These compounds exert their cytotoxic effect by interacting with lipid membranes, thus affecting essential cellular processes, such as signal transduction, phosphatidylinositol (PI)-phospholipase C, and phospholipase D activity (Seewald et al., 1990; Powis et al., 1992; Lucas et al., 2001) and especially PC metabolism (Vogler et al., 1996; Berkovic et al., 2002). In this group of compounds, miltefosine is the most well-characterized derivative. Different hypotheses try to explain the miltefosine mechanism of action, as a compound with nonspecific cytotoxic activity (Stafford et al. 1989), by inhibiting cell signaling via phospholipases (Powis et al., 1992; Berkovic et al., 1996; Van Blitterswijk & Verheij, 2008) or by its ability to interfere in phospholipid production, by modifying the CCT activity (Haase et al., 1991; Wieder et al., 1995), a key enzyme in PC biosynthesis via the Kennedy pathway. ALPs, such as edelfosine, ilmofosine, and miltefosine have been tested successfully in pathogenic trypanosomatids of Trypanosoma and Leishmania genera by inhibiting cell proliferation and differentiation, promoting ultrastructural changes, and affecting sterol biosynthesis and PC production (Lira et al., 2001; Croft et al., 2003; de Castro et al., 2004; Santa-Rita et al., 2004; Azzouz et al., 2005).

Some trypanosomatids such as Angomonas deanei (Teixeira et al., 2011) bear an intracellular bacterium, which maintains an obligate symbiotic relationship with the host protozoan, thus constituting an excellent model to study organelle origin and cellular evolution. According to rDNA sequences, symbionts of different trypanosomatid species are Gram-negative bacteria that present a common origin, being classified in the ß division of Proteobacteria, close to the genus Bordetella (Du et al., 1994). The symbiont is enclosed by two membrane units and presents a reduced cell wall, which is essential for bacterium division and morphological maintenance (Motta et al., 1997). In the symbiosis of trypanosomatids, intense metabolic exchanges occur between both partners: the bacterium contains essential enzymes that complete important biosynthetic pathways of the protozoa and in exchange receives suitable physical conditions and energy supply from the host (reviewed by Motta, 2010). As in most prokaryotes, sterols are absent in symbiont membranes that have cardiolipin (CL) as the major phospholipid, followed by similar amounts of PC and PE and a minor quantity of PI (Palmié-Peixoto et al., 2006). The endosymbiont enhances the protozoan phospholipid production and depends in part on its host cell to obtain PC (Azevedo-Martins et al., 2007). Organelles of symbiotic origin play important roles in the eukaryotic cell lipid biosynthesis. Significant levels of phospholipid production occur in mitochondria that synthesize phosphatidic acid (PA) and phosphatidylglycerol, which is used to produce CL, a lipid that is mainly found in prokaryotes and mitochondria, as well as PE (Van Meer et al., 2008).

In this work, we tested the effect of miltefosine on cell proliferation, ultrastructure, and phospholipid biosynthesis of A. deanei. The main proposal of miltefosine treatment on this nonpathogenic trypanosomatid species is to evaluate, if once the protozoan phospholipid production is affected, how does it influence the symbiotic bacterium and mitochondrion composition. Thus, it is worth considering that both structures have symbiotic origin and are related to the protozoan phospholipid metabolism.

Materials and methods

Cell growth

Angomonas deanei was grown at 28 °C for 24 h in Warren's culture medium (Warren, 1960) supplemented with 10% fetal calf serum. Miltefosine (Cayman Chemical) was used after dilutions of a 100 mM stock solution dissolved in absolute methanol. Cells (1.0 × 10⁶ mL⁻¹) were inoculated in culture medium and after 12 h (exponential growth phase) were submitted to different drug concentrations: 10, 25, 50, 75, and 100 µM. Protozoa were collected from the culture at 12 h intervals until 72 h of growth; then, part of the cells were counted in a Neubauer chamber, and the remainder was processed for transmission electron microscopy as described below. To verify the effect of miltefosine on phospholipid biosynthesis, cells were grown for 24, 36, and 48 h in Warren medium containing 4 µCi of ³²Pi, whereas for cell fractioning assays, protozoa were cultivated for 24 h in the presence of 10 µCi of ³²Pi.

Endosymbiont and mitochondrion fractions

Isolated symbionts and mitochondria were obtained by cell fractioning as established by Alfieri & Camargo (1978), with some modifications. Cells were disrupted using an ultrasonic disruptor GEX-600 (three series of 15-s pulses at 10% amplitude). The homogenate passed throws differential centrifugations and sucrose gradient, to obtain a rich fraction of endosymbionts and mitochondria. Then, fractions were resuspended in 1 mL of Tris-HCl 20 mM and sucrose 0.25 M to maintain the integrity of both endosymbiont and mitochondrion envelope. To normalize the number count of mitochondria and symbionts, a dilution curve was performed and the results obtained by Neubauer chamber counting were compared to the optical density (OD) on a wavelength of 600 nm. All the experiments were normalized to the medium efficiency by OD as 2.0 × 10¹⁰ for symbiont (OD = 0.9) and 4.5 × 10⁸ for mithocondrion fraction (OD = 2.5).

Transmission electron microscopy

Protozoa were washed twice in PBS and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2 for 1 h. After being washed again in 0.1 M cacodylate buffer, pH 7.2 cells where postfixed for 1 h in 1% osmium tetroxide containing 0.8% potassium ferrocyanide, 5 mM CaCl₂ in 0.1 M cacodylate buffer. Then, cells were washed, dehydrated in several crescent concentrations of acetone and embedded in Epon: first a mix of Epon: Acetone (1 : 1) and finally pure Epon. Ultrathin sections were obtained in an Ultracut Reichert Ultramicrotome and mounted on 400 mesh copper grids, stained with uranyl acetate and lead citrate. Samples were analyzed in a Zeiss 900 transmission electron microscope.

[³²Pi]-orthophosphate labeling and phospholipid extraction

Total lipids were extracted from A. deanei metabolically labeled after growth for 24, 36, and 48 h in the presence of [³²Pi]-orthophosphate or from endosymbionts and mitochondria obtained after cell fractioning of protozoa treated or not with miltefosine for 24 h. Samples were washed with PBS, and the pellet was used for lipid extraction as described below. The lipid extraction was performed as described by Horwitz & Perlman (1987). Subsequently, the organic phase, containing the phospholipids, was solubilized with 3 mL of CHCl₃ : CH₃OH : HCl (200 : 100 : 0.75 v/v), and the phases were separated by centrifugation after addition of 0.3 mL of 0.6 N HCl. To purify the phospholipid fraction, 0.5 mL of CHCl₃ : CH₃OH : HCl (3 : 48 : 47 v/v) was added to the organic phase and centrifuged. The pH was adjusted to 7.0 with 0.2 N NH₄OH in methanol before dry under N₂ gas.

Phospholipid separation and identification by thin-layer chromatography (TLC)

After lipid extraction, the protocol described by Einicker-Lamas et al. (1999) was used. Briefly, silica gel plates (Silica gel 60F254 Merck) were activated by heat, and the samples corresponding to the lipid extracts of A. deanei, control and miltefosine-treated cells, grown in the presence of ³²Pi, as well as lipid fractions derived from endosymbionts and mitochondria isolated from the host protozoan, were applied to the silica plates. The run of the samples was performed using a mobile phase (120 chloroform : 45 acetone : 39 methanol : 36 HCl : 24 H₂O), as described by the method of Horwitz & Perlman (1987), for 80 min. The TLC plates were dried and exposed to develop in an iodine vapor atmosphere. Control standards (Sigma) were used to determine the phospholipids composition in each sample. When lipids were labeled by ³²Pi, the TLC plate was sensibilized with ³²Pi radiation, which was detected in Molecular Dynamics Storage Phosphor Screen GP after 24 h of exposure. The bands of interest were identified in a Storm 860 (Molecular Dynamics). The phospholipids identified in samples were also quantified by densitometry using ImageQuant. The radioactivity of the bands of interest was determined by liquid scintigraphy in a TRI-CARB 2100TR (Packard Bioscience). Data were analyzed using the graphpad prism 5.0 software package (GraphPad Software Inc., San Diego, CA). One-way anova test and a posteriori of Tukey's were performed. P values ≤ 0.01 were considered significant.

Results

Effects of miltefosine on cell proliferation

Treatment of A. deanei with miltefosine resulted in a decrease in cell proliferation in a dose-dependent manner. The lower drug concentrations, 10, 25, and 50 µM, have no significant effect on proliferation when compared with control cells, which correspond to a growth reduction of 6%, 15%, and 13% after 12 h of treatment and 17%, 24%, and 21% after 24 h of treatment, respectively. Higher doses of miltefosine, such as 75 and 100 µM, provoked a reduction of 48% and 80% in cell proliferation after 24 h, respectively. The miltefosine activity was more pronounced after 48 h of protozoan cultivation in the presence of the drug, as this time corresponds to the climax of the exponential phase. Under this condition, the effect on cell proliferation was remarkable after treatment with 75 and 100 µM miltefosine that induced a decrease of 69% and 90%, respectively. The miltefosine 50% inhibitory concentration (IC₅₀) value in A. deanei is equivalent to 85 µM. Methanol, which was used as a vehicle to dissolve miltefosine, decreased the cell proliferation as the lower drug concentrations (Fig. 1).

Effect of miltefosine on the proliferation of Angomonas deanei. Results are shown as mean ± SEM of three independent assays. It is important to note that miltefosine was added to trypanosomatid culture only after 12 h of cell growth (indicated by arrow).

Effects of miltefosine on cell ultrastructure

The effect of miltefosine on the ultrastructure of A. deanei was evaluated by transmission electron microscopy to compare control (Fig. 2a and b) and treated cells, revealing which structures were affected by the drug treatment. This analysis was also important to establish the ideal conditions for cell fractioning in order to obtain well-preserved symbionts and mitochondria for subsequent biochemical assays. Miltefosine-treated protozoa exhibited ultrastructural alterations such as blebbing and shedding of the plasma membrane (Fig. 2c), as well as membrane profiles within the flagellar pocket (Fig. 2d), after treatment with 25 µM of the drug for 24 h. Swelled mitochondrion with enlarged cristae (Fig. 2e) and an intense cell vacuolization (Fig. 2f) were also observed, especially after longer treatments with high drug concentrations, such as 75 and 100 µM. Ultrastructural analysis showed that treatment with 10 µM of miltefosine for 24 h represents the ideal condition to obtain symbiont and mitochondrion fractions even if there is no significant effect in proliferation under these conditions. When protozoa were cultivated in higher drug concentrations, such as 25 µM, the symbiont envelope presented membrane detachment and convolution (Fig. 2g) and the mitochondrion structure was also affected (Fig. 2e). It is important to mention that methanol, used as a vehicle to dissolve miltefosine, did not promote alterations on protozoa ultrastructure.

Effects of miltefosine on Angomonas deanei ultrastructure. (a, b) Control cells (free from miltefosine and methanol) showing the typical ultrastructure of symbiont-bearing trypanosomatids, such as the close proximity between the symbiont and the host cell nucleus. Note the integrity of the endosymbiont envelope during its division process, when the bacterium presents a constricted format (b, arrows). Membrane blebbing and shedding (c, arrows) are observed in protozoa plasma membrane after treatment with 25 µM miltefosine for 24 h, as well as membrane profiles in the flagellar pocket (d, arrowhead). Longer treatments with high drug concentrations, such as 75 and 100 µM, promoted mitochondrial swelling (e) and intense vacuolization (f). Membrane detachment of the bacterium envelope was seen after treatment with 25 µM miltefosine for 24 h (g, arrow). FP, flagellar pocket; G, glycosome; K, kinetoplast, M, mitochondrion; N, nucleus; S, symbiont and V, vacuole. Scale bars = 1.2 µm in a, c, g and 1 µm in b, d, e and f.

Effects of miltefosine on the phospholipid biosynthesis of A. deanei and on the symbiont and mitochondrion fractions

Control cells, and protozoa incubated only with methanol, showed similar phospholipid production (Fig 3a–d). Miltefosine-treated cells presented an altered phospholipid biosynthesis, when compared to these cells. Protozoa incubated with 10 µM of drug for 24 h presented the highest reduction in PC biosynthesis, which is equivalent to 45% (Fig. 3d). Interestingly, PE production decreased after cultivation in the presence of 10 µM miltefosine in a time-dependent manner: 35%, 43%, and 53% in protozoa treated for 24, 36, and 48 h, respectively (Fig.3d–Fig.3f). Cell cultivation with higher drug concentrations, such as 25 µM, promoted an increase in PI biosynthesis as the treatment proceeded, reaching 61% after 48 h (Fig. 3g–i). The most significant increase in PC and PE biosynthesis was observed after protozoa treatment with 25 µM miltefosine for 36 h and is equivalent to 48% and 57%, respectively (Fig. 3h). However, when protozoa were treated with 25 µM miltefosine for 48 h, a slight increase in the PC production (7%) was observed, whereas the PE synthesis was reduced by 25% (Fig. 3i).

Effect of miltefosine on the phospholipid composition of whole-cell extracts of Angomonas deanei after different periods of drug treatment. (a–c) Control cells were compared with protozoa incubated only with methanol, the vehicle used to dissolve the drug. Data showed that methanol did not affect phospholipid production even after 48 h. (d–f) Protozoa treated with 10 µM of miltefosine for 24 h (a), 36 h (b), and 48 h (c). It is important to note the significant decrease in PC and PE production after 24 and 48 h of treatment. (g–i) Protozoa treated with 25 µM of miltefosine for 24 h (d), 36 h (e), and 48 h (f). A significant enhancement of PC, PE, and CL synthesis was observed after 36 h of treatment, whereas the maximum production of PI was noted after 48 h of protozoa cultivation in the presence of the drug. (j–l) Protozoa treated with 50 µM of miltefosine for 24 h (g), 36 h (h), and 48 h (i). The PC synthesis increased after 48 h, as well as the PI production, whereas PE levels decreased. Results are representative of three independent assays, and bars represent mean ± SEM. Asterisks represent statistic analyses between groups that were performed using one-way anova and Tukey's test (graphpad prism), where P < 0.001 is relative to each phospholipid control.

The effect of 50 µM miltefosine on PE production changed in a time-dependent manner, and thus reductions of 25%, 14%, and 13% were observed in protozoa treated for 24, 36, and 48 h, respectively (Fig. 3j–l). Values of PC biosynthesis enhanced in 19% after treatment with 50 µM miltefosine for 48 h, with a concomitant increase in PI levels (Fig. 3l). Taken together, these data showed that low doses of miltefosine (10 µM) employed for short periods (24 h) induced a reduction in the PC and PE synthesis (Fig. 3d). However, as the drug treatment proceeded, lower PE levels were observed when compared to PC and PI production (Fig. 3j–l). It is worth observing that PI synthesis never decreases during protozoa cultivation for different periods and drug concentrations (Fig. 3d–l). Furthermore, the values for CL production maintained constant during miltefosine treatment, except after cultivation with 25 µM for 36 h, when this phospholipid synthesis is significantly enhanced (Fig. 3h).

Symbionts and mitochondria obtained from host protozoa treated with 10 µM miltefosine for 24 h also presented alterations in phospholipid biosynthesis when compared to control isolates. In both fractions, a decreased synthesis was observed for all type of phospholipids analyzed, except for PE production by the symbiotic bacterium that was not affected (Fig. 4a and 4b). The symbiont synthesis of PC, PI, and CL was reduced by 42%, 68%, and 40%, respectively (Fig. 4a). The mitochondrial phospholipid production was also affected, as PC, PE, PI, and CL synthesis decreased by 77%, 71%, 80%, and 75%, respectively (Fig. 4b). It is important to mention that in all experiments the same amount of fractions was used, based on the OD of the samples.

Effect of miltefosine on the phospholipid composition of endosymbionts and mitochondria obtained from Angomonas deanei. (a) Symbiont and (b) mitochondrion after treatment with 10 µM miltefosine for 24 h. The production of all phospholipids was affected by this miltefosine, with the exception of PE synthesis by the symbiotic bacterium. Control cells correspond to protozoa free from miltefosine and methanol. Results are representative of three independent assays. Asterisks represent statistic analyses between groups that were performed using one-way anova and Tukey's test (graphpad prism), where P < 0.001 is relative to each phospholipid control.

Discussion

ALPs such as miltefosine, edelfosine, and ilmofosine have been tested as anticancer agents, promoting growth inhibitor of different cell lines. Such antitumoral activity is related to the blockage of cell proliferation and invasion, as well as the inhibition of PC synthesis (Berdel, 1991; Bergmann et al., 1994; Brachwitz & Vollgraf, 1995; Wieder et al., 1995; Berkovic et al., 2002; Giantonio et al., 2004). In trypanosomatids, ALPs present potent and selective antiparasitic activity, especially against Leishmania species and Trypanosoma cruzi, by inhibiting cell proliferation and promoting structural damage, as well as morphological alterations (reviewed by Lira et al., 2001; de Castro et al., 2004; Urbina, 2006; Santa-Rita et al., 2005).

Previous studies with T. cruzi epimastigotes have shown that ALPs affect the sterol and phospholipid composition, in this latter case by inhibiting PC biosynthesis via the Greenberg pathway, specifically at the level of PE N-methyltransferase (Lira et al., 2001). In the present work, miltefosine modified the A. deanei lipid composition after 24 h of treatment, when a significant reduction in the amounts of PC and PE were observed. However, as the treatment proceeded, the synthesis of PC increased, whereas the PI production enhanced considerably. In T. brucei, ablation of choline phosphotransferase activity of the Kennedy pathway also induced reduction in PC and PE levels and a protozoan proliferation arrest, induced by inhibition of nuclear division (Signorelli et al., 2008, 2009). The re-establishment of PC production in longer miltefosine treatments may be due to the fact that cell proliferation is not compromised, probably reflecting low levels of miltefosine in relation to the target enzyme. Furthermore, ultrastructural alterations, such as blebbing and shedding of the plasma membrane, in drug-treated cells is an indication that protozoa can eliminate part of the inhibitor by recycling its membrane components. The recovery of PC production in longer treatments also suggests that both de novo PC biosyntheses are present in A. deanei; thus, the inhibition of the Kennedy pathway by miltefosine treatment may induce alternative PC production via the Greenberg pathway. However, some authors have proposed that the methylation of PE to PC, which characterizes the Greenberg pathway, is absent in T. brucei (Signorelli et al., 2008; Gibelline et al., 2009; Serricchio & Bütikofer, 2011). It is worth observing that PI synthesis enhances after long treatment with miltefosine, suggesting that phosphoinositide turnover could be intensified, thus promoting an intense signaling response to bypass the harmful effects of the drug in PC production. Previous works have shown that ALPs associate with lipid rafts, thus altering signal transduction pathways that involve phospholipase C and protein kinase C, which are essential regulators of cell proliferation (Nishizuka, 1992; Malaquias & Oliveira, 1999; Wright et al., 2004).

The biochemical assays have shown that symbionts and mitochondria, obtained after cell fractioning of A. deanei treated with miltefosine, presented decreased biosynthesis of most types of phospholipids analyzed. Interestingly, the PE production was not affected in isolated bacteria, indicating that the symbiont maintained the biosynthetic route used for the formation of this phospholipid, which is usually the major one in the prokaryote envelope. This agreed with our previous works, which showed that PE is an essential constituent of the symbiotic bacterium membranes (Palmié-Peixoto et al., 2006). Once isolated from the protozoan, the symbiont is able to produce phospholipids, especially PE, independently of the host cell (Azevedo-Martins et al., 2007). However, it is noteworthy that the symbiosis in trypanosomatids is an obligatory relationship with extensive metabolic exchanges (reviewed by Motta, 2010) and that the bacterium may obtain part of PC or PC precursors from the host (Azevedo-Martins et al., 2007). This, in part, explains why the effect of miltefosine in the phospholipid biosynthesis of the host protozoan directly affected the phospholipid content of the symbiotic bacterium. The mitochondrion is an organelle of symbiotic origin that imports most of its proteins and lipids from the cytoplasm (Timmis et al., 2004). In mitochondrial fractions obtained from host protozoa submitted to miltefosine treatment, the production of all types of phospholipids was strongly affected. It is well established that mitochondria participates in the synthesis of different lipids, such as the PE, which is generated via PS decarboxylase that converts phosphatidylserine (PS) into PE (Van Meer et al., 2008). Thus, it is worth considering that phospholipid biosynthesis inhibition in mitochondrion may affect its bioenergetics owing to lipid membrane change that would in turn affect the host metabolism and consequently the symbiont.

Some aspects of lipid biosynthesis and composition were previously investigated in trypanosomatids using sterol biosynthesis inhibitors, such as 22,26-azasterol, that act on the methyltransferase (24-SMT), a key enzyme in the biosynthesis of ergosterol and other 24-alkyl sterols, which are absent in mammalian cells (Urbina et al., 1995, 1996). Such compounds also affect phospholipid production, by inhibiting PE and PC synthesis (Contreras et al., 1997; Urbina, 1997). When A. deanei was treated with azasterol, cells presented ultrastructural alterations as those reported in the present work. Furthermore, the sterol biosynthesis was blocked, and low rates of PC and increased levels of PE were observed, thus suggesting an inhibition of N-methyltransferase that converts PC into PE via the Greenberg pathway (Palmié-Peixoto et al., 2006). Interestingly, the PC content of the symbiotic bacterium was also reduced, reinforcing the idea that part of this phospholipid is obtained from the host cell (Azevedo-Martins et al., 2007). Taken together, our data reinforce the idea that an intensive metabolic exchange occurs between the host trypanosomatid and structures of symbiotic origin and indicate that the host phospholipid production is essential to maintain the obligatory symbiosis in trypanosomatid protozoa.

Authors' contribution

P.R.G.d.F.-J. and C.M.C.C.-P. contributed equally to this work.

Acknowledgements

Authors are grateful to David Graham Straker for English revision. This study was supported by CAPES, CNPq, and FAPERJ.