Toxoplasma gondii acyl-lipid metabolism: de novo synthesis from

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Biochem. J. (2006) 394, 197–205 (Printed in Great Britain)
197
doi:10.1042/BJ20050609
Toxoplasma gondii acyl-lipid metabolism: de novo synthesis from
apicoplast-generated fatty acids versus scavenging of host cell precursors
Cordelia BISANZ*1 , Olivier BASTIEN†‡, Delphine GRANDO§, Juliette JOUHET†, Eric MARÉCHAL†
and Marie-France CESBRON-DELAUW*
*Laboratoire Adaptation et Pathogénie des Micro-organismes, UMR 5163, CNRS-UJF, Grenoble, France, †Département Réponse et Dynamique Cellulaire, UMR 5168, CNRS-CEA-UJF,
Grenoble, France, ‡Gene-IT, 147 avenue Paul Doumer, Rueil-Malmaison, France, and §Laboratoire Bioinformatique et RMN Structurale, Pole Bioinformatique Lyonnais, UMR 5086,
CNRS-Université C. Bernard, Lyon, France
Toxoplasma gondii is an obligate intracellular parasite that contains a relic plastid, called the apicoplast, deriving from a secondary endosymbiosis with an ancestral alga. Metabolic labelling
experiments using [14 C]acetate led to a substantial production
of numerous glycero- and sphingo-lipid classes in extracellular
tachyzoites. Syntheses of all these lipids were affected by the
herbicide haloxyfop, demonstrating that their de novo syntheses
necessarily required a functional apicoplast fatty acid synthase II.
The complex metabolic profiles obtained and a census of glycerolipid metabolism gene candidates indicate that synthesis is probably scattered in the apicoplast membranes [possibly for PA
(phosphatidic acid), DGDG (digalactosyldiacylglycerol) and PG
(phosphatidylglycerol)], the endoplasmic reticulum (for major
phospholipid classes and ceramides) and mitochondria (for PA,
PG and cardiolipid). Based on a bioinformatic analysis, it is
proposed that apicoplast produced acyl-ACP (where ACP is acylcarrier protein) is transferred to glycerol-3-phosphate for apicoplast glycerolipid synthesis. Acyl-ACP is also probably trans-
ported outside the apicoplast stroma and irreversibly converted
into acyl-CoA. In the endoplasmic reticulum, acyl-CoA may not
be transferred to a three-carbon backbone by an enzyme similar
to the cytosolic plant glycerol-3-phosphate acyltransferase, but
rather by a dual glycerol-3-phosphate/dihydroxyacetone-3-phosphate acyltransferase like in animal and yeast cells. We further
showed that intracellular parasites could also synthesize most
of their lipids from scavenged host cell precursors. The observed
appearance of glycerolipids specific to either the de novo pathway
in extracellular parasites (unknown glycerolipid 1 and the plant
like DGDG), or the intracellular stages (unknown glycerolipid 8),
may explain the necessary coexistence of both de novo parasitic
acyl-lipid synthesis and recycling of host cell compounds.
INTRODUCTION
a plastid in apicomplexan parasites, which are responsible for
extremely serious diseases in humans (malaria and toxoplasmosis)
and livestock (coccidioses, theilerioses, babesioses etc.), therefore
raised hopes for the development of new innovative herbicidebased drugs [12].
Whereas the apicoplast’s function has been discussed since its
discovery [13], it became rapidly obvious that this organelle was
essential for parasite survival, at least in Plasmodium and Toxoplasma [12,14–16]. The hypothesized function of the apicoplast
is based on knowledge about metabolic pathways in non-photosynthetic plastids of plants such as FA (fatty acid), isoprenoid,
haem, starch and aromatic amino acid synthesis [9].
It has long been held that apicomplexan parasites were incompetent for de novo FA synthesis [17–19], but the recent record
of nuclear-encoded apicoplast-targeted genes for all enzymes of
the FA biosynthesis pathway provided strong arguments in favour
of a de novo FA biosynthesis in this organelle (for a review see
[20]). As in bacteria and plants, the T. gondii genome contains
the group of highly conserved proteins known as FAS II (Type II
FA synthase), with distinct enzymes for the different reactions
[21–24]. Incorporation of the precursor [14 C]acetate into FA
The unicellular eukaryote Toxoplasma gondii is an obligate intracellular parasite with an extremely broad host range, capable of
infecting virtually all types of nucleated cells from warm-blooded
vertebrate hosts [1]. It is an important opportunistic pathogen
of humans, causing severe encephalitis in immunocompromised
individuals and congenital birth defects when primary infection
occurs during pregnancy [2,3]. About a decade ago, a major breakthrough was reached in the understanding of the parasite’s evolution and biology, with the discovery that T. gondii and other
parasites of the Apicomplexa phylum harboured a relic plastidlike organelle, named the apicoplast, that derives from a secondary endosymbiosis with an ancestral alga [4–7]. Plastids are
semi-autonomous cellular organelles only described thus far in
plants and algae. In plants, the embryonic proplastid can differentiate into the photosynthetically active chloroplast, or into numerous non-green plastids (amyloplasts, chromoplasts, etioplasts,
leucoplasts etc.), which all have vital metabolic functions in
different organs [8,9]. These plastids are foremost on the list
of known targets for herbicides [10,11] and the discovery of
Key words: acyl-lipid metabolism, apicoplast, fatty acid synthesis,
glycerol-3-phosphate, Toxoplasma gondii, Type II fatty acid
synthase (FAS II).
Abbreviations used: ACC, acetyl-CoA carboxylase; ACP, acyl-carrier protein; ANS, 8-anilinonaphthalene-1-sulphonic acid; BODIPY® , 4,4-difluoro4-bora-3a,4a-diaza-s -indacene; DAG, diacylglycerol; DGDG, digalactosyldiacylglycerol; DPG, diphosphatidylglycerol; 2D-TLC, two-dimensional TLC;
ECL, enhanced chemiluminescence; FA, fatty acid; FAS II, Type II FA synthase; fop, aryloxyphenoxypropionate herbicide; GlcCer, glycosylcerebroside;
HFF, human foreskin fibroblast; HsTfR, human transferrin receptor; IF, immunofluorescence; LacCer, lactosylcerebroside; mAb, monoclonal antibody;
MGDG, monogalactosyldiacylglycerol; NEFA, non-esterified FA; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG,
phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; PV, parasitophorous vacuole; SQDG, sulphoquinovosyldiacylglycerol; TriHexCer,
globotriosylcerebroside.
1
To whom correspondence should be addressed, at Laboratoire Adaptation et Pathogénie des Micro-organismes, UMR 5163, Institut Jean Roget,
Domaine de la Merci, F-38700 La Tronche, France (email [email protected]).
c 2006 Biochemical Society
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C. Bisanz and others
chains by Plasmodium falciparum [24] and T. gondii [25] demonstrated that the de novo FA biosynthetic pathway was functional.
Furthermore, the existence of FAS II would explain the susceptibility of T. gondii to herbicides targeting plastid ACC (acetyl-CoA
carboxylase) [22,26] or FabI [enoyl-ACP (acyl-carrier protein)
reductase] [23,27]. This FAS II pathway is seen as a promising
drug target, mostly because it is structurally and functionally
distinct from the equivalent pathway in the vertebrate hosts, i.e.
FAS I [28]. A tempting hypothesis to explain the importance of
apicoplast FAS II would be that the produced FAs are used for
de novo syntheses of essential membrane acyl-lipids (glyceroand sphingo-lipids).
Parasite development is characterized by intense membrane
production and reorganization. Upon host cell invasion, T. gondii
resides and divides within a unique specialized compartment, the
PV (parasitophorous vacuole), which primarily derives from
the host cell plasma membrane but is rapidly modified by the
parasite [29,30]. The PV is devoid of host cell transmembrane proteins [31,32] and does not fuse with endocytic or exocytotic vesicles of the host cell [33,34]. It was hypothesized that the apicoplast
might provide material for some of the membranes necessary for
successful infection, the establishment and/or maturation of the
PV and/or for parasite cell division [21]. Using lipids conjugated
to fluorescent probes and radiolabelled precursors, Charron and
Sibley [25] provided evidence that part of the lipid material
required for membrane mass increase comes from scavenging of
some host cell lipids. Diversion of host cell material was mostly
of some phospholipids that were tentatively identified as different PC (phosphatidylcholine) species [25]. Lipid precursors
such as polar head groups, PA (phosphatidic acid) and small
FAs primarily introduced into host cells, are secondarily used to
manufacture PC. A production of PC by extracellular Toxoplasma
was also recorded after diffusion of radiolabelled acetic acid [25].
However, it remained unclear whether labelling was due to an
incorporation of acetate into the FAs owing to the FAS II pathway
or into other building blocks of the phospholipids, such as the
glycerol moiety or the polar head groups, owing to other metabolic
routes. Until now, all published analyses have concentrated on
some specific lipids [25,35–39] and no detailed global analysis
of T. gondii membrane acyl-lipid synthesis and metabolization is
available.
In the present study, we investigated the capacity of T. gondii for
de novo membrane glycero- and sphingo-lipid synthesis. Using
metabolic labelling with [14 C]acetate and a pharmacological knockout of the plastid FAS II, we showed that in extracellular
Toxoplasma the apicoplast-generated FAs were used to synthesize
numerous membrane acyl-lipids. The complex profile of de novo
synthesized lipids was analysed using 2D-TLC (two-dimensional TLC) and co-migration with characterized lipids from
Arabidopsis thaliana. Gene candidates for the corresponding
activities in T. gondii were inventoried by similarity searches
using A. thaliana genes as probes. Comparative analysis revealed
that, whereas most of the lipid classes were synthesizable owing
to either de novo synthesis or utilization of host cell compounds,
selective synthesis of some lipids was also observed. Therefore the
present study supports that a defect of the plastid FAS II pathway
compromises the selective production of some acyl-lipids, which
are possibly essential for the parasite and cannot be compensated
for by import of precursors from the host cell.
EXPERIMENTAL
Materials
Cell culture media and fetal bovine serum were obtained from Life
Technologies. The radiolabelled FA precursor [1-14 C]acetic acid
c 2006 Biochemical Society
sodium salt (55 mCi · mmol−1 ) was from Amersham Biosciences.
The herbicide haloxyfop and Merck silica gel 60 TLC plates were
purchased from Sigma. Protease inhibitor cocktail tablets
were from Roche Diagnostics, propidium iodide from Molecular
Probes and the ECL (enhanced chemiluminescence) system for
detection of Western-blot signals from Pierce Chemical. The primary antibody mAb (monoclonal antibody) H68.4 reacting with
the HsTfR (human transferrin receptor) was from Zymed Laboratories. Primary antibody mAb TG05-54 was used to detect the
Toxoplasma SAG1 surface protein (TgSAG1) and mAb TG17113 to detect GRA5 (TgGRA5), a protein of the PV membrane
[40]. The peroxidase-conjugated secondary antibody for detection
of proteins on Western blots and the BODIPY® (4,4-difluoro-4bora-3a,4a-diaza-s-indacene)-conjugated goat anti-mouse IgG for
IF (immunofluorescence) detection were purchased from Jackson
ImmunoResearch Laboratories.
Parasite culture
Tachyzoites of the RH strain were propagated under standard
procedures, by serial passage in HFF (human foreskin fibroblast)
monolayers in D10 medium (Dulbecco’s modified Eagle’s medium supplemented with 10 %, v/v, heat-inactivated fetal bovine
serum, 1 mM glutamine, 500 units · ml−1 penicillin and 50 µg ·
ml−1 streptomycin) at 37 ◦C and under 5 % CO2 .
Metabolic labelling
For metabolic labelling, freshly lysed parasites were purified
through a column of silicon-treated glass wool to eliminate host
cell debris and washed three times in PBS. Parasites were then
incubated with 5–10 µCi · ml−1 [14 C]acetic acid at 37 ◦C in D10
medium, subsequently collected by centrifugation, washed four
times with PBS to eliminate non-incorporated radioactivity and
used for lipid extraction (Figure 1). When required, a 1 h treatment
with 300 µM haloxyfop [22] preceded the metabolic labelling.
To determine lipid acquisition from host cells, HFF monolayers
were incubated in the presence of 5 µCi · ml−1 [14 C]acetic acid
for 8 h prior to tachyzoite infection. After an overnight infection,
intracellular parasites were collected by scraping the monolayers
and were mechanically released from host cells by sequential
passage through 20, 23, 25 and 27G needles. Parasites were further purified through a column of silicon-treated glass wool to
eliminate host cell debris prior to lipid extraction and chromatography (Figure 1).
Viability assay
Tachyzoite viability was routinely determined by propidium
iodide staining. Parasites were incubated for 15 min in the presence of 5 µg · ml−1 propidium iodide in PBS and observed with a
Zeiss Axioplan 2 fluorescence microscope. Non-viable parasites
take up the dye and show a red fluorescence.
IF microscopy
IF microscopy was used to determine if isolated parasites were
free of PV membrane fractions. Aliquots of parasitic preparations
collected before and after filtration through a glass wool column
were spotted on glass slides, dried and fixed in 2.5 % (v/v)
formaldehyde. The slides were further incubated with the mAb
TG17-113 and then with the BODIPY® -conjugated goat antimouse IgG. Slides were mounted in 50 % (v/v) glycerol and
examined with a Zeiss Axioplan 2 fluorescence microscope
using a × 200 magnification to provide a general view of the
preparations.
Fatty acid synthesis in the apicoplast
199
were blocked in 5 % (w/v) non-fat dry milk in PBS, incubated
with the appropriate primary antibody, rinsed and incubated with
the peroxidase-conjugated goat secondary antibody. Signals were
detected by using the ECL system.
Census of T. gondii gene candidates possibly involved
in acyl-lipid metabolism
Figure 1 Scheme for metabolic labelling of T. gondii extra- and intracellular tachyzoites
Extracellular Toxoplasma cells were labelled for 4–6 h in the presence of [14 C]acetate (left
panel) and the lipid profile was subsequently analysed. When required, a 1 h treatment with
the aryloxyphenoxypropionate herbicide haloxyfop (fop) preceded the metabolic labelling. HFF
cells were labelled in the presence of [14 C]acetate und subsequently infected by Toxoplasma
tachyzoites (right panel). Lipid analyses of intracellular parasites were then carried out to detect
diversion of labelled carbons from host cell compounds for the parasite acyl-lipid metabolism.
Lipid extraction and chromatographic analyses
Lipids from [14 C]acetic acid-labelled parasites or HFF cells were
extracted as described by Bligh and Dyer [41] and analysed by 2DTLC. To discriminate between synthesized glycerolipids (KOHsensitive) and sphingolipids (KOH-resistant), radiolabelled parasite- or HFF-synthesized lipids were incubated in 0.1 M KOH
in methanol/water (1:1, v/v) for 4 h at 50 ◦C so as to clear esterlinked FAs of glycerolipids. Hydrolysis-resistant sphingolipids
were subsequently resolved by 2D-TLC. Lipids were co-migrated
on silica gel plates along with an established mixture of total
A. thaliana lipids as an internal standard for identification. The
solvent system for the first dimension was chloroform/
methanol/water (65:25:4, by vol.) and chloroform/acetone/methanol/acetic acid/water (100:40:20:20:10, by vol.) for the second,
allowing the plates to dry between each development. Finally, after
a careful drying, TLC plates were sprayed with 0.2 % ANS (8anilinonaphthalene-1-sulphonic acid) in methanol and stained lipids were visualized with UV light. Radiolabelled Toxoplasma
lipids were detected by exposition of TLC plates with erasable
screens for 24–72 h. Screens were scanned with a phosphoimager
FLA 8000 (Fuji). The software used for reading screens and
further image analyses was ImageReader and ImageGauge (Fuji).
Radiolabelled Toxoplasma sphingolipids were identified by their
sensitivity to alkaline treatment and co-migration with ANSvisualized A. thaliana lipids. Glycosphingolipids from the HFF
lipid mixture were detected owing to their purple coloration after
α-naphtol staining and their resistance to alkaline treatment.
SDS/PAGE and immunoblot analysis
Proteins were separated by SDS/PAGE (13 % polyacrylamide)
and transferred to nitrocellulose by liquid transfer. Membranes
The list of acyl-lipid gene candidates from the Arabidopsis Lipid
Gene Database (620 entries, November 2004; [42]; http://www.
plantbiology.msu.edu/lipids/genesurvey/) was used as a probe to
seek gene candidates in the preliminary T. gondii genomic database via http://ToxoDB.org. These genomic data were provided
by The Institute for Genomic Research (supported by the NIH
grant no. AI05093) and by the Sanger Center (Wellcome Trust).
A first list of gene candidates was selected after comparison
between the Arabidopsis sequences and the Toxoplasma predicted
proteins (20878 entries, including possibly redundant annotations
provided by TigrScan, GlimmerHMM and Twinscan, November
2004) using both BLASTP [43] and the BLOSUM 62 similarity
matrix [44] implemented in the BIOFACET software package
[45]. A first criterion for a strong similarity was set with a Score
threshold of 100 and an E-value cutoff of 1 × 10−8 . The second
criterion for sequence selection was the ‘double-click’, i.e. confirmation of the best alignment with homologues of the
Arabidopsis probes in the best hits obtained after a comparison of
the Toxoplasma sequences with the Swiss-Prot molecular database (http://www.expasy.org/sprot/). Prediction of a cleavable
signal peptide (Sp) or signal anchor sequence (Sa) was analysed
using the SignalP method (version 3.0; [46]; http://www.cbs.
dtu.dk/services/SignalP/). In the absence of any bioinformatic
apicoplast-targeting prediction tool for Toxoplasma protein
sequences, prediction of a chloroplast-like transit peptide (Ctp)
downstream a signal peptide was sought using the ChloroP
method designed for a broad range of species (version 1.1;
[47]; http://www.cbs.dtu.dk/services/ChloroP/). Prediction of
a mitochondrial transit peptide (Mtp) was achieved using
the MitoProt method [48] (http://ihg.gsf.de/ihg/mitoprot.html).
Eventually, any sequence sharing homology with two Arabidopsis
isoenzymes localized in distinct organelles, was listed in the
compartment that corresponds to the target prediction. As a positive control of the method, we checked that all the apicoplast FAS
II pathway and ACP genes could be recovered.
RESULTS
Metabolic labelling strategies to dissect the 14 C-route from
provided acetate to Toxoplasma polar lipids
To investigate whether T. gondii tachyzoites were able to synthesize polar lipids from apicoplast-synthesized FAs, we analysed
lipid extracts obtained after metabolic labelling with the FA
precursor [14 C]acetate. Figure 1 summarizes the labelling and
analysis strategies to investigate the metabolic routes of the
labelled carbons from acetate to lipids. Haloxyfop was added
to block FAS II and to investigate whether the lipid labelling
was downstream of apicoplast FA synthesis. To analyse de novo
syntheses in extracellular parasites, tachyzoites were incubated in
the presence of [14 C]acetate for 4 h (labelling was linear for at least
6 h, results not shown) and washed thoroughly to eliminate nonincorporated radioactivity. These radiolabelled parasites were
then directly processed for lipid analysis. Addition of haloxyfop
[fop (aryloxyphenoxypropionate herbicide)] abolished the polar
lipid labelling (see below, and Figure 2). The herbicide sensitivity
indicated that a de novo synthesis of FAs, owing to the apicoplast
FAS II, was required for incorporation of radioactivity in complex
c 2006 Biochemical Society
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Figure 2
C. Bisanz and others
Identification of de novo synthesized lipids by T. gondii tachyzoites
Parasites were labelled with the FA precursor [14 C]acetate and extracted lipids were analysed by 2D-TLC along with total A. thaliana lipids as markers for identification. Lipids extracted from the
same number of parasites (6 × 108 ) were deposited on the chromatography plates. (A) Parasites were labelled for 4 h extracellularly prior to lipid extraction and TLC analysis. (B) Same labelling
conditions as in (A), but radiolabelled lipids were subjected to an alkaline treatment (KOH in methanolic phase) to identify sphingolipids amongst the parasite-synthesized lipids. (C) Same conditions
as in (A), but the labelling was preceded by a 2 h treatment with the herbicide haloxyfop; spots represent the radiolabelled lipids de novo synthesized by the parasite, broken line circled positions
correspond to the positions of the co-migrated Arabidopsis lipids identified by coloration with ANS, and solid line circled spots indicate positions of lipids not found in the Arabidopsis lipid mixture.
TAG, triacylglycerol; U1 –U7 , unknown lipids in the Arabidopsis lipid mixture. U3 was tentatively identified as PA; +, deposit point of lipids. FFA, NEFA.
lipids. Therefore the labelling was attributable to a 14 C-route from
acetate to the FA moiety of the produced lipids. Eventually, HFF
labelling with [14 C]acetate prior to T. gondii infection was also
carried out to investigate lipid synthesis that recycled host cell
precursors (Figures 1 and 4).
Metabolic profiles of extracellular Toxoplasma polar lipids
produced with apicoplast-synthesized FAs
In a first set of experiments, we analysed lipids metabolically
labelled after incubation of extracellular tachyzoites in the presence of [14 C]acetate. A representative lipid profile is shown in
Figure 2(A). Spots correspond to lipids synthesized by the parasites with radiolabelled acetate, while positions circled with
broken lines indicate the positions of ANS-visualized A. thaliana
lipids co-migrated as markers. The radiolabelled profile shows
that the parasite is capable of synthesizing the major phospholipids: PC, PE (phosphatidylethanolamine), PS (phosphatidylserine), PI (phosphatidylinositol) and, albeit to a lesser extent, PG
(phosphatidylglycerol). Concerning organellar lipids, the mitochondrial DPG (diphosphatidylglycerol or cardiolipid) was
weakly labelled (Figure 2A). Although the incorporation of
tritiated galactose from UDP-galactose into chloroplastic galactolipids was previously reported in Toxoplasma partly lysed cells
[49], no MGDG (monogalactosyldiacylglycerol) could be detected under our experimental conditions. However, a radiolabelled
glycerolipid co-migrated with Arabidopsis chloroplast DGDG
(digalactosyldiacylglycerol; Figure 2A). In addition, seven major
unidentified glycerolipids (U1 –U7 ) were synthesized. Their
characterization by MS was restricted by lack of biological
material. However, the failure to identify these lipids may be an
indication of rare or intermediary structures, U3 being tentatively
identified as a PA molecular species (according to the twodimensional Rf (retardation factor) position [50]). U1 is possibly
unique to the parasite (undetected in Arabidopsis standard lipids
or in HFF-synthesized lipids shown in Figure 4). Three series
of spots among the parasite-synthesized lipids were identified
as sphingolipids by their resistance to alkaline treatment:
GlcCer (glycosylcerebroside), LacCer (lactosylcerebroside) and
TriHexCer (globotriosylcerebroside) (Figure 2B).
c 2006 Biochemical Society
Metabolic labelling of Toxoplasma lipids was nearly completely
abolished after incubation of tachyzoites with haloxyfop (Figure 2C). The effect of this fop herbicide, which targets plastid
ACC, was investigated at a concentration previously shown to
affect Toxoplasma tachyzoites but not its host cells (300 µM;
[22]). A 1 h preincubation of extracellular tachyzoites with the
herbicide before [14 C]acetate addition was the minimal time
required to see an effect on lipid synthesis. When both components
were added simultaneously, [14 C]acetate incorporation started
before the herbicide could have reached its target (results not
shown). This very rapid incorporation is similar in isolated plant
chloroplasts, where free acetate is the most efficient substrate
for FAS II [51]. Thus, after a 1 h incubation with the herbicide
at 37 ◦C, 5 µCi · ml−1 [14 C]acetate was added and parasites were
incubated for another 4 h. During this time period, the herbicide
did not affect parasite viability, as monitored by propidium iodide
staining of parasites just before lipid extraction. Viability was
found to be higher than 90 % (results not shown) as in nonherbicide treated parasites. As shown in Figure 2(C), the fop herbicide abolished almost totally the FA and polar lipid biosyntheses
in Toxoplasma and we had to lengthen exposure of the chromatography over an additional 2 weeks to assess the labelling background. This collapse of polar lipid biosyntheses induced by
haloxyfop in viable cells demonstrates that the lipid production is
fully dependent on the FAS II pathway.
Metabolic profile of intracellular Toxoplasma polar lipids,
synthesized after mobilization of labelled precursors
from host cells
T. gondii is incompetent to divide in vitro in the absence of host
cells, indicating that the intracellular way of life not only protects
the parasite from extracellular immune attacks, but also provides
one or more essential factor(s) to the parasite. Previous studies
reported the acquisition and metabolization of host cell lipids by
Toxoplasma [25,35–39]; however, these studies did not provide a
detailed global analysis of the lipids produced. Furthermore, these
analyses used specific radioisotopic and/or fluorophore-conjugated lipids and were, most of the time, performed by microscopic
observations. But specific labelling of parasite compartments by
Fatty acid synthesis in the apicoplast
Figure 3
201
Purity control experiment of the intracellular parasite fraction
After an overnight intracellular growth within HFF cells, parasites were collected by scraping
the monolayer and were mechanically released from host cells by sequential passage through
20, 23, 25 and 27G needles. Parasites were further purified through a column of silicon-treated
glass wool. (A) Western-blot analysis of the parasite fraction before (−) and after (+) filtration;
detection of host cell membranes was done using the anti-HsTfR (α-HsTfR) mAb H68.4. Protein
extracts correspond to 107 (1), 106 (2) and 105 (3) parasites. The α-TgSAG1 antibody that is
directed against the T. gondii major surface protein SAG1 was used as an internal control of
protein load in each lane. (B) IF microscopy of the PV membrane protein GRA5 (α-TgGRA5) in
the same fractions before and after filtration.
exogenous, artificially labelled lipids does not necessarily reflect
the lipid scavenging and metabolization by the parasite. Therefore,
in a second set of experiments, HFF monolayers were labelled
with [14 C]acetic acid 8 h prior to infection with unlabelled tachyzoites. After an overnight intracellular growth within labelled HFF
host cells, parasites were purified and their radiolabelled lipid
profile was analysed. As the purity of this intracellular parasite
fraction is essential for the interpretation of our results, we conducted a non-radioactive control experiment under the same
conditions and analysed the parasites before and after glasswool filtration for the presence of contaminating host cell or
PV membranes. Western-blot analysis showed that the HsTfR
was not detected in the parasite preparation after glass-wool
filtration, whereas a positive staining was observed in the parasite
preparation before filtration (Figure 3A). In addition, microscope
examination of the same fractions revealed that the filtered
preparation was highly enriched in parasites and free of visible
membranous debris (phase contrast). IF microscopy showed that
the PV membrane protein GRA5 was only detected in the parasite
preparation prior to filtration (Figure 3B). These two control
experiments led us to conclude that our intracellular parasite
fractions are essentially free of contaminating PV and host cell
membranes.
Figure 4
Lipid acquisition from the host cell
HFF host cells were labelled with [14 C]acetate prior to infection with unlabelled parasites.
Intracellular tachyzoites were purified and their radiolabelled lipid content was analysed by
2D-TLC along with total Arabidopsis lipids as markers for identification. (A) HFF radiolabelled
lipids. (B) Toxoplasma radiolabelled lipids produced from 14 C-labelled host cell components.
U2 –U8 , unknown lipids in the Arabidopsis lipid mixture. U3 was tentatively identified as PA; +,
deposit point of lipids. FFA, NEFA.
Figure 4(A) shows HFF lipids synthesized after [14 C]acetate
labelling. The five major phospholipids PC, PE, PI, PS and PG
were detected as well as the four main cerebrosides: GlcCer,
LacCer, TrihexCer and globoside. The mitochondrial cardiolipid
(DPG) was also strongly labelled. Figure 4(B) shows the labelled
lipids that tachyzoites imported and/or manufactured using radiolabelled lipids or lipid precursors diverted from host cells. The
profile is far less complex than the one obtained after de novo
synthesis in free tachyzoites (Figure 2A). As reported by Charron
and Sibley [25], the major ubiquitous phospholipid is PC, but
it only represented about half of the labelled phospholipids in
our experiment; PE, PS and PI are also substantially produced.
PG was not detected. Charron and Sibley [25] also showed that
intracellular Toxoplasma did not mobilize fluorophore-conjugated PC from host cells; PC was therefore most likely generated
from metabolized precursors diverted from the host cell, rather
than directly imported. The radiolabelled NEFAs (non-esterified
FAs) we detected in our TLC analyses (Figure 4B, FFA) might
result from a direct import from host cells and/or from a
degradation/metabolization of other scavenged lipids. Some of
c 2006 Biochemical Society
202
C. Bisanz and others
Table 1 Polar lipid metabolic labelling profiles in extracellular and
intracellular Toxoplasma
Census of Toxoplasma gene candidates possibly involved
in acyl-lipid metabolism
Lipid assessment was based on 2D-TLC and co-migration with characterized lipids from A.
thaliana . Sphingolipids were identified by their resistance to alkaline hydrolysis with KOH
in methanolic phase. U1 –U8 , unknown glycerolipids 1–8; (+), (++), (+++), qualitative
detection scale; nd, not detected. In the case of U3 –U7 , occurrence in HFF cells was supposed
from co-migrating spots in Toxoplasma samples.
Table 2 gives the list of 26 Toxoplasma gene candidates for the
major steps in membrane lipid production, using either acyl-ACP
(acyl-carrier protein) or acyl-CoA as substrates. In this census,
gene candidates for enzymes involved in glycerolipid synthesis
are likely to occur in the apicoplast, endoplasmic reticulum and
mitochondria. In the apicoplast, first transfer of an acyl to a threecarbon backbone would occur due to a glycerol-3-phosphate acyltransferase candidate, using acyl-ACP as a substrate. Subsequent
production of PA may occur because of a putative 2-lysophosphatidate acyltransferase.
No gene candidates for chloroplast-like galactolipid syntheses
could be identified. In contrast, two sequences possibly encoding
enzymes of the PG synthesis pathway could be predicted as
apicoplast-targeted (Table 2). In the endoplasmic reticulum,
transfer of an acyl to a three-carbon backbone may not involve
a glycerol-3-phosphate acyltransferase as it occurs in plants. An
alternative pathway, known in animal endoplasmic reticulum,
utilizes glycerol-3-phosphate or dihydroxyacetone-3-phosphate
as a substrate for the three-carbon backbone. Using the yeast dual
dihydroxyacetone-3-phosphate/glycerol-3-phosphate acyltransferase as a probe, we could indeed identify, in the Toxoplasma genome annotation release [43–45], a gene candidate predicted to be
targeted to the endoplasmic reticulum. Genes possibly encoding
important steps of endoplasmic reticulum phospholipids (PC, PE,
PI, PS and PG) and ceramide metabolism were also predicted
(Table 2).
Table 2 shows gene candidates for acyl-lipid catabolism. In
this latter section of the Table, the few genes characterized might
occur in various cell compartments, as judged from the automated
target prediction. Thus, in addition to the inventory of possible
components of the membrane lipid synthetic machineries, we
could record some clues about lipid degradation enzymes that
may be responsible for diversion of some host cell lipids.
Toxoplasma tachyzoites
Lipid
Extracellular
Intracellular
Human HFF cells
+++
+++
+++
+
++
+++
++
+
+
nd
+++
+++
+++
++
++
+
+
++
Membrane glycerolipids
Ubiquitous phospholipids
PC
PE
PS
PI
PG
Mitochondrial phospholipids
DPG
Chloroplast-like glycolipids
MGDG
DGDG
SQDG
Uncharacterized glycerolipids
U1
U2
U3 (PA?)
U4
U5
U6
U7
U8
nd
+
nd
nd
nd
nd
nd
nd
nd
++
++
++
+
+
+
++
nd
nd
+
+
nd
nd
nd
+
+
nd
nd
+
+
+
+
+
nd
Membrane sphingolipids
GlcCer
LacCer
TriHexCer
Globoside
+
+
+
+
+
nd
nd
nd
++
+
+
+
the uncharacterized glycerolipids listed earlier [U3 (PA?), U2 and
U7 ] appeared to be also generated from host cell compounds
(Figure 4B). In the list of minor uncharacterized glycerolipids,
one could notice U8 (Figure 4B), which was detected neither in
extracellularly labelled tachyzoites nor in HFF cells. U8 is very
likely generated from modified host cell compounds and may be
specific to the intracellular parasitic life stage. In contrast, the
major U1 glycerolipid synthesized by extracellular tachyzoites
(Figure 2A) as well as the chloroplast-like DGDG were not synthesized from host cell precursors. U1 and DGDG might not be
synthesizable outside the apicoplast-dependent de novo biosynthetic pathways. Interestingly, intracellular tachyzoites exhibited a
high labelling of the mitochondrial cardiolipid (DPG; Figure 4B).
DPG is generated from PG, which could not be detected in
intracellular parasites; it is therefore likely to be imported directly
from the host cell.
Table 1 summarizes the lipid profiles obtained in the present
study. The use of host cell components for parasite lipid synthesis
is apparently a very complex process, either following routes of
direct import of lipids (DPG?), of close intermediates for polar
lipid syntheses [PA (U3 ?), NEFAs], or of pools of unrelated imported molecules that are largely degraded before serving as
building blocks for lipid production. Furthermore, the lipid metabolism of the parasite shifts from an autonomous de novo process
to a parasitic recycling of host cell material upon invasion.
However, at least some of the Toxoplasma-specific glycerolipids
(U1 , DGDG) may not be produced from host cell component
scavenging and may require functional apicoplast FA synthesis.
c 2006 Biochemical Society
DISCUSSION
In the present study, metabolic labelling profiles of membrane
lipids of Toxoplasma tachyzoites were analysed (Figure 1). Our
results highlighted for the first time the production of numerous
glycero- and sphingo-lipid classes in extracellular tachyzoites
(Figure 2, Table 1). More importantly, syntheses of all these lipids
were affected by haloxyfop, demonstrating that their de novo
syntheses necessarily required a functional apicoplast FAS II
pathway.
The major ubiquitous phospholipids (PC, PE, PS, PI and PG)
were abundantly synthesized under all conditions, consistent
with the massive need for such components in most membranes.
Mitochondrial membrane biogenesis seems also to occur during
the extracellular life stage, as DPG synthesis was also detected.
Part of the labelled PG might serve as an immediate precursor
for DPG synthesis. Interestingly, although the chloroplast-like
galactolipids MGDG and DGDG had been previously measured
in suspensions of partly lysed Toxoplasma cells, no synthesis of
MGDG could be observed under any of our experimental labelling
conditions. In contrast, we detected production of DGDG (Figure 2A). The de novo synthesis of DGDG seems therefore likely
to be the result of a channelled process, with no accumulation
of the MGDG intermediate. A similar process has been shown
to occur in Euglena gracilis whose plastid also finds its origin
from a secondary endosymbiosis. The E. gracilis chloroplastic
MGDG is so rapidly transformed into DGDG that its synthesis is
nearly not detectable [52]. In plants, plastids are also characterized
Fatty acid synthesis in the apicoplast
Table 2
203
Gene candidates for acyl-lipid metabolism in T. gondii
EC number: enzyme nomenclature following recommendation of the International Union of Biochemistry and Molecular Biology. Genes whose function is based on sequence similarity are in italic
characters; genes whose function is based on experimental data are in normal characters. Absence of a gene candidate is indicated by (nc), except in case of absence of experimental evidence for
the gene occurrence (no evidence). Plants having no known dual dihydroxyacetone-phosphate/glycerol-3-phosphate acyltransferase, the corresponding sequence from yeast (Uniprot accession no.
GPT1 YEAST P32784) was used as a probe. In the case of the plastidial CDP-DAG synthetase gene candidate, prediction of an Sp (*) could be assessed when computed with the second methionine
as a start. Ctp, chloroplast-like transit peptide; Mtp, mitochondrial transit peptide; Sa, signal anchor sequence; Sp, signal peptide.
Gene candidates
Acyl-ACP- or acyl-CoA-dependent lipid metabolism
EC number
A. thaliana
T. gondii
Target prediction
Synthesis of membrane lipids in plastids (PA, MGDG, DGDG, SQDG and PG)
using acyl-ACP
Glycerol-phosphate acyltransferase
2-Lysophosphatidate acyltransferase (PA synthase)
Plastidial phosphatidic acid phosphatase
MGDG synthase
2.3.1.15
2.3.1.51
3.1.3.4
2.4.1.46
At1g32200
At4g30580
At2g01180
At2g11810, At4g31780,
At5g20410
At3g11670, At4g00550
nc
At5g01220
At2g45150 , At3g60620 ,
At4g26770
At2g39290
nc
TgTigrScan 3735
TgTigrScan 0603
nc
nc
Sp Ctp
Sp Ctp
–
–
nc
nc
No evidence
TgTigrScan 4999
–
–
–
Sp Ctp*
TgTigrScan 4540
nc
Sp Ctp
–
At5g06090 , At3g11430
No evidence
nc
TgTwinScan 6545
–
Sp
No evidence
TgTwinScan 3802
Sp
At1g80950 , At3g18850 ,
At1g75020
At1g15080
nc
At1g68000, At4g38570
At4g25970, At5g57190
At1g15110
At3g25585
At1g62430 , At4g22340
At3g55030
nc
At4g36480 , At3g48780 ,
At5g23670
At3g25540 , At3g19260 ,
At1g13580
nc
–
DGDG synthase
Galactolipid:galactolipid galactosyltransferase
Sulpholipid synthase (SQDG synthase)
CDP-DAG synthetase
PG-phosphate synthase
PG-phosphate phosphatase (PG synthase)
Synthesis of membrane lipids in endomembrane systems (PA, APC, PE, PS, PI, PG and Cer)
using acyl-Co
Plant-like endomembrane glycerol-phosphate acyltransferase
Dual dihydroxyacetone-phosphate/glycerol-phosphate
acyltransferase (none in plants; in yeast: GPT1 YEAST)
Peroxisomal dihydroxyacetone-phosphate; ether lipid
syntheses (none in plants; in human: GNPAT-HUMAN)
2-Lysophosphatidate acyltransferase (PA synthase)
Phosphatidate phosphatase
Phosphatidylserine synthase (PS synthase)
Phosphatidylinositol synthase (PI synthase)
Phosphatidylserine decarboxylase
Ethanolamine/serine base-exchange enzyme
DAG cholinephosphotransferase (PC synthase)
CDP-DAG synthetase
PG-phosphate synthase
PG-phosphate phosphatase (PG synthase)
Serine palmitoyltransferase (LCB1-3)
Ceramide synthase (sphingosine N -acyltransferase)
Synthesis of membrane lipids in mitochondria (PA, PG and DPG) using acyl-CoA
Glycerol-phosphate acyltransferase
2-Lysophosphatidate acyltransferase
Phosphatidate phosphatase
PG-phosphate synthase
PG-phosphate phosphatase (PG synthase)
Glycerol-3-phosphate dehydrogenase
Cardiolipid synthase (DPG synthase)
Phosphatidylserine decarboxylase
2.4.1.184
2.7.7.41
2.7.8.5
2.3.1.15
2.3.1.51
3.1.3.4
2.7.8.8
2.7.8.11
4.1.1.65
2.7.8.2
2.7.7.41
2.7.8.5
2.3.1.50
2.3.1.24
TgTigrScan
nc
TgTigrScan
TgTigrScan
TgTigrScan
TgTigrScan
TgTigrScan
nc
nc
TgTigrScan
1948
1389
Sa
–
Sa
Sp
Sp
Sp
?
–
–
Sp
TgTigrScan 2444
Sp
nc
TgTigrScan 8310
nc
nc
nc
TgTigrScan 1884
nc
TgTigrScan 0325
–
Mtp
–
–
–
Mtp
–
Mtp
5902
0325
7983
0833
1888
4.1.1.65
At1g02390 , At1g06520
At4g30580
At3g02600
At4g04870
nc
At3g10370
nc
At4g16700
Synthesis and storage of triacylglycerols using acyl-CoA
Acyl-CoA: DAG acyltransferase
2.3.1.20
At2g19450
TgTigrScan 5720
?
Hydrolysis of acylglycerols and acyl-hydrolases
Acyl-CoA oxidase
1.3.3.6
At1g06290, At1g06310,
At2g35690 , At4g16760,
At5g65110
At3g15290
At1g04710 , At2g33150,
At5g48880
At1g52700 , At3g15650 ,
At4g22300 , At5g20060
At1g33270 , At3g57140 ,
At5g04040
At1g31480
At2g30720 , At5g48370
TgTigrScan 7878
Sa
TgTigrScan
TgTigrScan
TgTigrScan
TgTigrScan
3778
7504 ,
8000
5386
?
Mtp
Sp
?
TgTigrScan
TgTigrScan
TgTigrScan
TgTigrScan
2912 ,
4606
5764
1278
?
Sa
?
?
2.3.1.15
2.3.1.51
3.1.3.4
2.7.8.5
1.1.99.5
3-Hydroxyacyl-CoA dehydrogenase
Ketoacyl-CoA thiolase
1.1.1.35
2.3.1.16
Lysophospholipase
3.1.1.5
Carboxylic ester hydrolases (patatin-like)
3.1.1.*
Phospholipase A1
Acyl-CoA thioesterase
3.1.1.32
3.1.2.20
c 2006 Biochemical Society
204
C. Bisanz and others
by their content of SQDG (sulphoquinovosyldiacylglycerol), a
sulphonated glycolipid. We could never detect any synthesis
of SQDG, either in the present metabolic labelling study or in
independent attempts to assay an SQDG synthesis in partly lysed
parasites (results not shown). Consistently, no gene candidate
involved in SQDG synthesis could be inventoried (Table 2).
If the above results show that T. gondii is capable of synthesizing
all lipids required for its viability, including parasite-specific
glycerolipids, the profile of lipids synthesized by intracellular
tachyzoites using 14 C-labelled material from HFF host cells is less
complex (Figure 4B). The parasite diverts host cell precursors to
contribute to the syntheses of major phospholipids, mostly PC,
PE, PS, PI and the mitochondrial DPG, as well as some of the
unknown lipids (Table 1), suggesting that both de novo production
and recycling of building blocks diverted from host cells are
likely to coexist. This lipid acquisition would be facilitated by
the intimate and specific apposition of the membrane of the PV
with the host cell lipid biosynthesis apparatus, i.e. the endoplasmic
reticulum and the mitochondria [53].
Therefore most of the lipid metabolism would be a quantitative
shift from an autonomous to a partly dependent process. This
conclusion raises two important questions. First, why is haloxyfop
lethal, if most of the parasite lipids could be diverted from host
components? Part of the answer may lie in the characterization
and understanding of the biological roles of U1 and DGDG
glycerolipids that are specific to the parasite and strictly dependent
on the apicoplast FAS II pathway. Alternatively, apicoplast FAS
II may produce unique and vital FA molecular species, used for
dedicated purposes other than glycero- or sphingo-lipid manufacturing. Secondly, why would Toxoplasma be dependent on host
cell lipids to divide, when it is capable of synthesizing most of its
lipids autonomously? Obviously, processes other than membrane
biogenesis may be involved; nevertheless part of the answer may
also lie in the biological function of U8 , a glycerolipid that we
only detected after invasion and incorporation of host cell labelled
carbons (Figure 3B, Table 1).
In eukaryotic cells, redundant and branched pathways of production and transformation of glycerolipids occur in various membrane compartments. A given class of lipids can be scattered in
diverse pools and metabolized owing to distinct compartmentalized pathways. To that extent, no detailed conclusion on the
subcellular metabolism of lipids can be driven from the global
metabolic profile analyses presented here. However, we can
deduce an important hypothesis based on general considerations
regarding glycerolipid metabolism, particularly when addressing
the question of apicoplast-synthesized versus host cell-derived
FA sources. FAs are thiol-esterified either to ACP or to CoA. In
plant cells, acyl-ACPs are primarily generated in the chloroplast
owing to the FAS II pathway. They can be transported outside
the plastid after exchange with CoA, supplying the cytosol with
acyl-CoA. In most glycerolipid synthesis sites, the transfer of acyl
to a three-carbon backbone implies a glycerol-3-phosphate
substrate. It occurs either in the plastid inner membrane, using
stromal acyl-ACP, or in the endoplasmic reticulum and mitochondria membranes, utilizing acyl-CoA. PA produced can
be consequently hydrolysed into DAG (diacylglycerol) due to
a phosphatidate phosphatase. These two compounds are key
intermediates for glycerolipid syntheses in the plant plastid
membranes (for MGDG, DGDG and PG) [54], the endoplasmic
reticulum (for PC, PS, PE, PI and PG) and the mitochondrial membranes (for PG and DPG). Occurrence of glycerolipids outside
their biosynthetic sites further requires vesicular and non-vesicular trafficking systems. If the lipid metabolism of Toxoplasma
was indeed plant-like, one would deduce from our results that
apicoplast-synthesized FAs should be primarily coupled with
c 2006 Biochemical Society
ACP and be either directly available for apicoplast glycerolipid
syntheses or transported to other sites after exchange of ACP
by CoA. Alternatively, FAs imported from the host cell might
be coupled with CoA, producing acyl-CoA and be available
for glycerolipid syntheses in endomembranes and mitochondria.
The lack of U1 and DGDG synthesis in intracellular parasites
might indicate an incapacity for generating acyl-ACP from host
cell material. Thus the acyl-ACP → acyl-CoA route might be
non-reversible, a phenomenon that would explain the necessary
conservation of the apicoplast FAS II pathway during evolution.
The complex profile of lipids that the parasite can synthesize
from apicoplast acyl-ACP suggests that Toxoplasma contains
the entire corresponding enzymatic factories. Table 2 gives an
inventory of gene candidates from the Toxoplasma genome that
would contribute to de novo glycerophospholipid syntheses. No
complete apicoplast, endoplasmic reticulum or mitochondrial
pathway could be recovered, but gene candidates for most important steps were predicted. The accuracy of this list probed in the
early draft genomic annotation [43–45] will undoubtedly benefit
from the advances of the collaborative annotation project in the
near future. Some sequences may be confirmed, others rejected;
however, important trends may be deduced. In this enquiry, four
gene candidates might be targeted to the apicoplast, further supporting that, like plant plastids, this organelle is possibly an acylACP-dependent glycerolipid factory. Transfer from acyl-ACP
probably involves a glycerol-3-phosphate acyltransferase, as in
plant plastids. Whereas in plants a glycerol-3-phosphate acyltransferase isoenzyme is responsible for the first transfer of an acyl
from acyl-CoA in the endoplasmic reticulum, we could not
identify such a gene in Toxoplasma. In animal cells and yeast,
the substrate for the three-carbon backbone can alternatively
be dihydroxyacetone-3-phosphate. In the present census, we
could identify a gene candidate corresponding to an endoplasmic
reticulum bifunctional glycerol-3-phosphate/dihydroxyacetone3-phosphate acyltransferase (Table 2). This composite metabolic
picture would reflect the inheritance of an acyl-ACP/glycerol-3phosphate glycerolipid synthetic pathway from the ancestral alga
and of an acyl-CoA/glycerol-3-phosphate/dihydroxyacteone-3phosphate pathway from the ancestral protozoa. Since the
apicoplast is connected peripherally to the endomembrane system,
the four membranes that surround the organelle may function as
‘centrifugally specialized’ glycerolipid machinery, with the acylACP/glycerol-3-phosphate pathway in innermost membranes
and the acyl-CoA/dihydroxyacetone-3-phosphate/glycerol-3phosphate pathway in outermost membranes.
We thank Karine Musset for skilled technical support, especially for host cell and parasite
propagation, Nahid Azzouz (Institut für Virologie, Philipps-Universität, Marburg, Germany)
and Jacques Joyard (Laboratoire de Physiologie Cellulaire Végétale, UMR 5168, Grenoble,
France) for helpful advice on metabolic labelling during the initial phases of this work, and
Jean Gagnon (Lab. Transmission and Pathogenesis of Prion Diseases, FRE 2685, Grenoble,
France) for a critical reading of this paper. This work was funded by the CNRS through
the programme ‘Microbiologie Fondamentale’, by the ANVAR grant no. A0106220V/AT
assigned to E. M. and a grant ‘Region Rhône-Alpes’ assigned to M.-F. C.-D. Genomic data
were obtained from http://ToxoDB.org.Genomic provided by The Institute for Genomic
Research (supported by NIH grant no. AI05093) and by the Sanger Center (Wellcome
Trust).
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Received 14 April 2005/23 September 2005; accepted 24 October 2005
Published as BJ Immediate Publication 24 October 2005, doi:10.1042/BJ20050609
c 2006 Biochemical Society

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