Subcellular localization and dynamics of a
digalactolipid-like epitope in Toxoplasma gondii
Cyrille Botte ´,*,†Nadia Saı ¨dani,*,§Ricardo Mondragon,**Mo ´nica Mondrago ´n,**Giorgis Isaac,††
Ernest Mui,§§Rima McLeod,§§Jean-Franc ¸ois Dubremetz,§Henri Vial,§Ruth Welti,††
Marie-France Cesbron-Delauw,†Corinne Mercier,1,†and Eric Mare ´chal1,*
Unite ´ Mixte de Recherche 5168,* Centre National de la Recherche Scientifique-Commissariat a ` l’Energie
Atomique-Institut National de la Recherche Agronomique-Universite ´ Joseph Fourier, Institut de Recherches
en Technologies et Sciences pour le Vivant, 38058 Grenoble, France; Unite ´ Mixte de Recherche 5163,†
Centre National de la Recherche Scientifique-Universite ´ Joseph Fourier, Institut Jean Roget, Campus Sante ´,
38042 Grenoble, France; Unite ´ Mixte de Recherche 5235,§Centre National de la Recherche
Scientifique-Institut National de la Sante ´ et de la Recherche Me ´dicale-Universite ´ Montpellier II, 34095
Montpellier, France; Departamento de Bioquı ´mica,** Centro de Investigacio ´n y Estudios Avanzados del
Instituto Polite ´cnico Nacional, Avenida Instituto Polite ´cnico Nacional 2508, Col. San Pedro Zacatenco,
Distrito Federal, Me ´xico; Division of Biology,††Kansas State University, Kansas Lipidomics Research Center,
Manhattan, KS 66506-4901; and Department of Ophthalmology and Visual Sciences,§§Pediatrics (Infectious
Diseases), Pathology, and Committees on Genetics, Molecular Medicine, and Immunology, University of
Chicago, Chicago, IL 60637
terized by unique extracellular and intracellular membrane
compartments. The lipid composition of subcellular mem-
branes has not been determined, limiting our understanding
cesses involved in pathogenesis. In addition to a mitochon-
drion, Toxoplasma contains a plastid called the apicoplast.
The occurrence of a plastid raised the question of the pres-
ence of chloroplast galactolipids. Using three independent
rabbit and rat antibodies against digalactosyldiacylglycerol
(DGDG) from plant chloroplasts, we detected a class of
Toxoplasma lipids harboring a digalactolipid-like epitope
(DGLE). Immunolabeling characterization supports the
notion that the DGLE polar head is similar to that of
DGDG. Mass spectrometry analyses indicated that dihexosyl
lipids having various hydrophobic moieties (ceramide,
diacylglycerol, and acylalkylglycerol) might react with anti-
DGDG, but we cannot exclude the possibility that more
complex dihexosyl-terminated lipids might also be immuno-
labeled. DGLE localization was analyzed by immunofluo-
rescence and immunoelectron microscopy and confirmed
by subcellular fractionation. No immunolabeling of the
apicoplast could be observed. DGLE was scattered in pel-
licle membrane domains in extracellular tachyzoites and
was relocalized to the anterior tip of the cell upon invasion
in an actin-dependent manner, providing insights on a
possible role in pathogenetic processes.
tected in other Apicomplexa (i.e., Neospora, Plasmodium,
Babesia, and Cryptosporidium).—Botte ´, C., N. Saı ¨dani, R.
Mondragon, M. Mondrago ´n, G. Isaac, E. Mui, R. McLeod,
J-F. Dubremetz, H. Vial, R. Welti, M-F. Cesbron-Delauw,
Toxoplasma gondii is a unicellular parasite charac-
DGLE was de-
C. Mercier, and E. Mare ´chal. Subcellular localization and
dynamics of a digalactolipid-like epitope in Toxoplasma
gondii. J. Lipid Res. 2008. 49: 746–762.
Supplementary key words
diacylglcycerol & inner membrane complex & membrane domains
Apicomplexa & galactolipids & digalactosyl-
Toxoplasma gondii is the unicellular causative agent
of toxoplasmosis and one of the parasites of the large
Apicomplexa phylum, which includes numerous obli-
gate parasites of both human and veterinary importance
(e.g., Plasmodium, Cryptosporidium, Neospora, etc.). Like all
Apicomplexa, Toxoplasma is characterized by unique
extracellular and intracellular membrane compartmental-
ization. Toxoplasma resides within the host cell inside a
nonfusogenic parasitophorous vacuole (1–5). Its plasma
membrane is lined by an inner membrane complex formed
by two closely apposed membranes. Inside Toxoplasma cells,
the endomembrane system (endoplasmic reticulum, nu-
clear envelope, Golgi network, and plasma membrane) is
interconnected with specific apical secretory compart-
ments (i.e., micronemes, rhoptries, and dense granules).
These three compartments are sequentially involved in the
recognition of and attachment to the host cell (6) and the
formation and maturation of the parasitophorous vacuole
(4, 7). In addition to a mitochondrion, the envelope of
Manuscript received 19 October 2007 and in revised form 4 January 2008.
Published, JLR Papers in Press, January 8, 2008.
1To whom correspondence should be addressed.
e-mail: firstname.lastname@example.org (C.M.);
Copyright D2008 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
746Journal of Lipid Research
Volume 49, 2008
by guest, on June 1, 2013
which has membranes that are disconnected from the
endomembrane system, a second semiautonomous or-
ganelle has been described in Toxoplasma (i.e., a non-
photosynthetic plastid called the apicoplast) (8–10). The
apicoplast is surrounded by four membranes and was ac-
quired by a secondary endosymbiosis of a red alga (8–11).
The outermost membranes of the apicoplast are thought
to be reminiscent of the alga plasma membrane and the
endosymbiotic phagosome, consistent with their connec-
tion with the trafficking vesicular system (12). The two
innermost membranes are believed to derive from the
envelope membranes of the ancestral algal chloroplast,
although no evidence could be provided that the con-
stituents of these membranes were similar to those of the
plant chloroplast envelope. Global analysis of the major
lipid classes of Toxoplasma was achieved recently using
mass spectrometry lipidomic profiling(13). The lipid com-
position of each membrane compartment has not been
determined yet, limiting our understanding of the mecha-
ing of membrane lipids in Toxoplasma, a series of processes
that are critical to understanding pathogenesis.
Intense remodeling of membrane compartments is ob-
served during the Toxoplasma life cycle. The biogenesis of
the parasitophorous vacuole and cell division require large
amounts of polar lipids. Data on membrane lipid synthesis
are fragmentary, but they highlight the fact that Toxoplasma
is an auxotroph for sterols (14) and that the production
of acyl lipids relies on orchestrated de novo synthesis and
the diversion of precursors from the host cell. Three major
and possibly redundant fatty acid synthetic machineries
can operate for de novo synthesis: an apicoplast fatty acid
synthase of type II (FAS II) (10, 15–21), a cytosolic fatty
acid synthase of type I (FAS I) (16), and cytosolic fatty acyl
elongases (FAEs) (22, 23). Based on metabolic labeling
experiments, Bisanz et al. (24) showed that in free stages,
de novo fatty acid synthesis was a source for the acyl moiety
of Toxoplasma glycerolipids. Because acyl-lipid labeling is
abolished by haloxyfop [an inhibitor of plastid acetyl-CoA
carboxylases (16)], Bisanz et al. (24) concluded that an
active FAS II was essential for the bulk of the acyl-lipid
synthesis. Combining conditional mutant analyses and
metabolic labeling, Mazumdar et al. (23) showed that FAS
II was indeed critical for the biogenesis of the apicoplast
itself, and subsequently for the parasite survival, but was
unlikely to be the source of acyls for the bulk of acyl lipids.
Rather, most Toxoplasma glycerolipids appear to be
produced using acyls generated by FAS I and/or FAEs,
based on thiolactomycin resistance and cerulenin sensitiv-
ity (23). Together, these analyses highlight the importance
1) of Toxoplasma FAS and FAEs for bulk acyl-lipid syntheses
in free stages and 2) of FAS II activity for apicoplast bio-
genesis. Upon invasion, in spite of its autonomous capacity
to synthesize acyl lipids, Toxoplasma massively scavenges host
cell lipid precursors for its membrane biogenesis (24–26).
The presence of the apicoplast in Toxoplasma suggested
that glycerolipids that are unique to alga and plant plastids
might be synthesized by the parasite as well. In particular,
monogalactosyldiacylglycerol (MGDG) and digalactosyldi-
acylglycerol (DGDG), which constitute .70% of the mem-
brane lipids from chloroplasts and cyanobacteria (27–30),
were sought. The synthesis of three classes of galactolipids
has been detected in both Plasmodium and Toxoplasma cell
suspensions after metabolic labeling with radiolabeled
UDP-galactose (30). Identification of these low-abundance
lipids was attempted by comigration in thin-layer chroma-
tography with standard galactolipids from mammals and
plants, hydrolysis of polar heads by galactosidases, and
hydrolysis of acyl esters by alkaline treatments. One class
exhibited the chromatographic behavior of monogalacto-
sylceramide; the two others coincided with plant MGDG
and DGDG (30). After metabolic labeling of acyls with
radiolabeled acetate, Bisanz et al. (24) confirmed the syn-
thesis of a galactolipid comigrating with DGDG. Toxoplasma
lipids exhibiting the analytical and biochemical properties
of plant MGDG and DGDG raise the questions of their
precise structures, biosynthesis, abundance, localization,
and possible roles.
The analysis of minor lipids of Toxoplasma is a technical
challenge, because of the difficulty of producing sufficient
amounts of biological material and the sensitivity of ex-
isting techniques. Polyclonal antibodies have been intro-
duced as a new tool to probe the subcellular localization
and trafficking of DGDG in plants (31). In this study,
we used anti-DGDG antibodies obtained by three inde-
pendent immunizations of two rabbits and one rat with
homogeneously pure chloroplast DGDG for the immuno-
detection of a class of lipids harboring a digalactolipid-like
epitope (DGLE) by immunofluorescence (IF) and immu-
noelectron microscopy (IEM) throughout the life cycle of
Toxoplasma. Cell membrane fractionation and mass spec-
trometry were used to attempt to characterize DGLE struc-
ture. The occurrence of DGLE in other apicomplexans
was also investigated by immunostaining approaches.
MATERIALS AND METHODS
Lipids were either purified from plant material or purchased.
The purity of lipids, which is critical for this study, was ana-
lyzed carefully by two-dimensional thin-layer chromatography
(2D-TLC). MGDG, DGDG, trigalactosyldiacylglycerol (TriGDG),
and sulfolipid (SL) were purified from spinach leaf chlo-
roplasts. Briefly, chloroplast lipids were extracted (see below)
and a first series of separations by 2D-TLC (see below) allowed
the separation of each lipid class; if required, a second series
of 2D-TLC allowed the purification to homogeneity of each
lipid class. Diacylglycerol (DAG), phosphatidylethanolamine (PE),
phosphatidylcholine (PC), phosphatidylglycerol (PG), mono-
galactosylcerebroside (MGCB), lactocerebroside (LCB), and
sphingomyelin (SM) were purchased from Sigma and checked
Rabbit and rat anti-DGDG antibodies
Two rabbit polyclonal sera were raised against DGDG (anti-
DGDG) by immunization of New Zealand White rabbits (Charles
River Laboratories) with 2.5 mg of homogeneously pure DGDG
extracted from spinach chloroplast membranes, as described
Digalactolipid-like epitope in Apicomplexa747
by guest, on June 1, 2013
(30). DGDG used for immunization was purified by 2D-TLC.
Briefly, the immunization procedure consisted of a first sub-
cutaneous injection at day 1 (0.35 mg of DGDG in Freund’s
adjuvant), a second and third series of subcutaneous and
intramuscular injections at days 10 and 21 (0.35 mg of DGDG
in Freund’s adjuvant for both subcutaneous and intramuscular
injections), and a fourth and fifth series of injections at days
36 and 50 (0.35 mg of DGDG in Freund’s adjuvant for sub-
cutaneous injections and 0.35 mg of DGDG without adjuvant
for intramuscular injections) before serum collection at day 57.
A rat polyclonal anti-DGDG serum was obtained by immuniza-
tion of a Lewis rat (Charles River Laboratories) with 2.5 mg of
Photosynthetic Arabidopsis thaliana cell suspension was cul-
tured and processed for the IF detection of DGDG in chloroplast
membranes as described (31). Chloroplasts and chloroplast
envelope membranes from spinach leaves were purified as
Culture of Toxoplasma gondii, Neospora caninum,
Cryptosporidium parvum, and Plasmodium falciparum
Toxoplasma gondii tachyzoites [RH; American Type Culture
caninum tachyzoites were propagated in human foreskin fibro-
blasts (HFFs; ATCC CRL-1635). For pellicle enrichment experi-
ments, parasites were amplified in human cervix adenocarcinoma
epithelial HeLa cells (ATCC CCL-2). Human cells were grown in
DMEM supplemented with 100 U/ml penicillin, 100 mg/ml
streptomycin, 2 mM glutamine, and 10% FBS (Gibco). Extracel-
lular parasites were forced through a 26.5 gauge needle to break
host cells and were purified by filtration through a 3 mm poly-
carbonate membrane before use. Cryptosporidium parvum oocysts
(bovine genotype 2) were purified from feces obtained from
calves experimentally infected with an isolate maintained at the
Institut National de la Recherche Agronomique Laboratory of
Avian Pathology (Nouzilly, France). Feces were layered on a dis-
continuous sucrose density gradient, and purified oocysts were
bleached, counted, and permitted to excyst in a 1.5% taurocho-
lic acid solution in BHK 21 medium (Gibco) for 90 min in a
37jC humidified, 5%CO2atmosphere. Parasite suspensions were
purified through a 5 mm cellulose acetate filter (Sartorius).
Twenty-four hours before infection, confluent human ileocecal
adenocarcinoma cells (HCT-8; ATCC CCL 244) maintained
in RPMI 1640 medium (Gibco) supplemented with 10% FBS,
100 U/ml penicillin, and 100 mg/ml streptomycin were trypsinized
slides; Nunc). A total of 1.25 3 105parasites were allowed to in-
vade each well containing monolayer at 80–85% confluence for
2 h at 37jC. Parasites that did not enter cells were removed; fresh
medium (RPMI 1640 containing 35 mg/l ascorbic acid, 25 mM
glucose, 0.1 IU/ml insulin, 15 mM HEPES, 1 mg/ml strepto-
and parasites were allowed to multiply for 48 h. Plasmodium
falciparum strain 3D7-infected erythrocytes were maintained at
37jC as described (33). 3D7 P. falciparum cultures were enriched
in gametocytes as described (34).
Treatment of T. gondii with cytoskeleton-specific drugs
Confluent HFF cells grown on coverslips were infected with
RH tachyzoites for 24 h and treated with the dinitroaniline her-
bicide oryzalin (0.5, 1, or 2.5 mM) for 24 h (35), cytochalasin D
(1 or 5 mM) for 30 min (36), or butanedione monoxime (20, 30,
or 50 mM) for 1 h (36) before IF analysis.
For most experiments, purified extracellular Toxoplasma cells
were allowed to settle for 10 min on polylysine-coated coverslips.
Suspensions of purified tachyzoites were processed for IF, with or
without fixation and permeabilization, as mentioned in the text
and figures. To visualize the plasma membrane and the inner
membrane complex separately, extracellular tachyzoites were
incubated in 5% glycerol in PBS for 30 min at 37jC on an orbital
wheel, centrifuged at 2,000 rpm for 10 min, and suspended in
PBS before being deposited on coverslips. For intracellular ob-
servations, HFF cells were grown to confluence on glass coverslips
deposited on four-well plates and infected with the different
strains of parasites. Toxoplasma cells as well as infected cells were
fixed for 20 min in 4% paraformaldehyde in PBS, permeabilized
with 0.1% Triton X-100 (v/v) in PBS, and blocked using 10%
FBS in PBS. For P. falciparum 3D7 analyses, infected erythrocytes
were fixed in 4% paraformaldehyde in PBS and immobilized
on polylysine-coated slides. Fixed parasites were briefly perme-
abilized in 0.1% Triton X-100 in PBS, and unspecific binding sites
were blocked using 10% FBS in PBS. Parasites and infected cells
were stained with the following primary antibodies: polyclonal
anti-DGDG sera (1:25 or 1:50) (30), polyclonal rat anti-DGDG
serum (1:25), polyclonal rabbit anti-IMC1 serum (1:500; a kind
gift from C. Beckers, University of North Carolina) (37), poly-
clonal rabbit anti-GRA6 serum (1:500; a kind gift from L. D.
Sibley, Washington University School of Medicine) (38), mono-
clonal TG19.179 anti-GRA2 antibody (1:500) (39), monoclonal
TG054 anti-SAG-1 antibody (1:500) (40), monoclonal
T8.4A12.1C3 anti-SRS9 antibody (previously called P36 or BSR4)
(41), and polyclonal rat anti-Cryptosporidium serum (42). All anti-
bodies were diluted in 1%FBSin PBS and detectedusing BODIPY
or Texas Red-conjugated goat anti-mouse, anti-rabbit, or anti-rat
IgG (H1L) antibodies (1:500) (Molecular Probes).
DNA was labeled with 0.5 mg/ml Hoechst (Molecular Probes)
for 5–20 min. Coverslips were mounted with the ProLong Antifade
Kit (Molecular Probes). Fluorescent images were acquired with
an Axiocam MRm (Zeiss) on an inverted Axioplan 2 microscope
(Zeiss), and images were acquired with the Axiovision 3.1 soft-
ware (Zeiss). For P. falciparum analyses, parasites containing a
single nucleus and lacking the digestive vacuole were considered
as ring-stage parasites, those containing a single nucleus and a
digestive vacuole were considered as trophozoite-stage parasites,
and those containing two nuclei or more and a digestive vacuole
were considered as schizont-stage parasites. Anti-DGDG antibod-
ies were incubated at a 1:25 dilution in 10% FBS in PBS during
1 h, and secondary antibodies (either Alexa Fluor 488 or Alexa
Fluor 592 goat anti-rabbit) were incubated using a 1:1,000 dilu-
tion in 10% FBS in PBS. Nuclei were labeled using Hoechst 33258
by a 5 min incubation at a 1:10,000 dilution in PBS. Detection of
DGDG in Arabidopsis cells was carried out as described (31) with
anti-DGDG at a 1:25 dilution. The fluorescence of chlorophyll
(excitation, 543 nm) was collected at 652 nm. Coverslips were
mounted with Immu-Mount (Thermo Electron Corp.). Fluores-
cent images were acquired with a MicroMax 1300 Y/HS charge-
coupled device camera (Princeton Instruments) under the
control of the Metavue imaging system (Universal Imaging Corp.)
on an upright Leica DMRA2 microscope.
Immunolocalization of DGLE was achieved on extracellular
tachyzoites as described (43). Human larynx carcinoma epithe-
lial cells (Hep-2; ATCC-CCL 23) maintained in DMEM (Gibco),
748Journal of Lipid Research
Volume 49, 2008
by guest, on June 1, 2013
supplemented with 10% FBS (Equitech-Bio) under a 5% CO2
atmosphere at 37jC, were infected by Toxoplasma (infection ratio
of 10 parasites to 1 host cell) tachyzoites for 24 h. Infected Hep-2
cells and isolated tachyzoites (5–7 3 107/ml) were washed three
times with PBS and fixed in 4% paraformaldehyde containing
0.1% glutaraldehyde in serum-free PBS for 1 h at room temper-
ature. Washed parasites were gradually dehydrated in ethanol
and embedded in LR White resin (London Resin Co.), which was
polymerized overnight, under ultraviolet light, at 4jC. Thin sec-
tions were mounted on Formvar-covered nickel grids. Immuno-
labeling was carried out at room temperature by flotation of the
mounted sections on drops of each solution; to minimize non-
specific labeling, grids were incubated with PBS containing 1%
skim milk and 0.05% Tween-20 (PBS-MT) for 30 min, and sec-
tions were incubated with the anti-DGDG rabbit polyclonal
serum (dilution, 1:5 in PBS-MT) for 1 h at room temperature
and overnight at 4jC. Grids were thoroughly washed with PBS-
T (PBS 1 0.05% Tween-20) and then incubated for 2 h at
room temperature, with the corresponding secondary antibody
(goat anti-rabbit polyclonal antibody) coupled to 10 nm gold
particles (Axell) (dilution, 1:40 in PBS-T). Incubation with each
antibody solution was performed in a humid chamber with
intervening washes. After thorough washing in PBS and dis-
tilled water, sections were contrasted with 2% uranyl acetate and
a satured solution of lead citrate and then examined with a
transmission electron microscope (JEOL 2000 EX). As negative
controls, sections were incubated with normal rabbit serum
diluted in PBS-MT and then with the secondary antibody cou-
pled to gold particles. As a positive control, sections were incu-
bated with a rabbit polyclonal serum against a whole extract of
Toxoplasma tachyzoites and then revealed with the secondary
antibody coupled to gold particles. Immunolocalization of DGLE
in tachyzoite cytoskeletal preparations was carried out as de-
Purification and analysis of Toxoplasma pellicle
Pellicle (inner membrane complex and plasma membrane)
was purified from extracellular Toxoplasma parasites by sucrose
gradient centrifugation and high-salt glycerol treatment as de-
Purification and analysis of Toxoplasma
Detergent-resistant membranes (DRMs) were isolated from
Toxoplasma tachyzoites according to the procedure described
Glycerolipid extraction, separation, and quantification
Lipids were extracted from T. gondii tachyzoites (2 3 109cells)
and from purified membrane fractions as described (49). Quan-
tification of Toxoplasma glycerolipids was performed after
methanolysis. Briefly, known amounts of C21:0 fatty acid were
added to lipid extracts, then acyl esters were transesterified to
the methyl esters and FAs were methylated using 3 ml of
2.5% H2SO4in methanol for 1 h at 100jC. The reaction was
stopped by the addition of 3 ml of water and 3 ml of hexane.
The hexane phase was analyzed by gas-liquid chromatography
(Perkin-Elmer) on a BPX70 (SGE) column. Retention times
and peak intensities of fatty acid methyl esters were compared
with those of standards. The obtained amount of fatty acids
was used to calculate the initial glycerolipid content. Quantified
lipids were dried under argon and frozen at 220jC for subse-
Sterol extraction and quantification
Sterols (including cholesterol and steryl esters) were extracted
and quantified by a resofurin-based fluorometric assay (Calbiochem)
according to the manufacturer’s instructions.
Acyl-lipid mass spectrometry analysis
Mass spectrometry analyses of Toxoplasma pellicle lipid extracts
were carried out by injecting 2–20 nmol of total polar lipids per
milliliter. Analyses were performed as described previously (13).
The sample in chloroform-methanol-300 mM aqueous ammo-
nium acetate (300:665:35) was infused into an Applied Biosys-
tems Q-TRAP with an Advion Triversa microchip electrospray
system at 0.11 ml/min (ionization voltage was set to 1.8 kV and
gas pressure to 0.1 p.s.i.). Polar lipids from pellicle membranes
were determined and quantified based on known amounts of
internal standards for each lipid class: phosphatidic acid (PA),
LysoPC, PC, ePC [alk(en)yl-acyl phosphocholine], LysoPE, PE,
ePE [alk(en)yl-acyl phosphoethanolamine], phosphatidylinosi-
tol, phosphatidylserine (PS), ePS [alk(en)yl-acyl phosphoserine],
MGDG, DGDG, Cer, EthCer (ceramide phosphoethanolamine),
SM (also termed ChoCer or choline ceramide in this paper), and
MHexCer and DHexCer. Amounts of lipids with masses coin-
ciding with those of MGDG, DGDG, and PA include weak peaks
close to the noise/contamination baseline. To identify potential
glycolipids, scans to detect ions producing a neutral loss of 179
(i.e., loss of ammoniated hexose minus water or [C6H13O5N]) or
ions corresponding to neutral loss of 341 (i.e., loss of ammo-
niated dihexose minus water or [C12H23O10N]) were performed.
The final profile of acyl lipids, calculated in mol%, was normal-
ized by taking into account the sterol content quantified in the
Lipid nitrocellulose dot-blotting and immunolabeling with
MGDG, DGDG, TriGDG, PE, PC, SL, DAG, PG, SM, MGCB,
LCB, and total lipid extracts from spinach chloroplast envelope,
HFF cells, and Toxoplasma were solubilized in butanol and spotted
onto a nitrocellulose membrane. Membranes were saturated for
1 h in TBS (10 mM Tris, pH 7.5, and 9 g/l NaCl) complemented
with 1% (w/v) nonfat dry milk, incubated with rabbit anti-DGDG
serum (1:100), washed, and developed with a goat serum anti-
(Sigma). For competition assays, anti-DGDG serum [100 ml, 1:100
in 1% (w/v) nonfat dry milk in TBS] was preincubated for 16 h at
4jC with 100 mg of DGDG purified from spinach. Peroxidase
activity was revealed in 100 mM Tris-HCl, pH 8.5, 12.5 mM luminol
(3-aminophalhydrazine), and 0.2 mM coumaric acid in the pres-
ence of H2O2. Autoradiography was performed with Hyperfilm
Proteins of whole cell extracts or obtained after pellicle frac-
tionation were quantified (50) and separated by SDS-PAGE. After
electrophoresis, proteins were stained in isopropanol-acetic acid
(3:1, v/v) containing 0.25% (w/v) Coomassie Brilliant Blue
(R-250; Sigma) or electrophoretically transferred to nitrocellu-
lose membranes for immunoblotting. Membranes were blocked
for 1 h with saturation solution (5% powdered milk, 5% goat
serum, 0.05% Tween-20, and 0.05% Triton X-114 in PBS) and
incubated for 1 h with primary antibodies: rabbit anti-DGDG
serum (1:100), rabbit anti-IMC1 (1:5,000), or monoclonal TG
17.054 anti-SAG1 (1:5,000). After incubation with peroxidase-
conjugated goat secondary antibodies (Jackson ImmunoResearch
Laboratories), immunolabeled polypeptides were detected using
the Supersignal ECL system (Pierce Chemical).
Digalactolipid-like epitope in Apicomplexa749
by guest, on June 1, 2013
Specific antibodies raised against spinach chloroplast
DGDG react with a class of Toxoplasma lipids harboring
Metabolic labeling experiments with radiolabeled UDP-
galactose and acetate have demonstrated the synthesis of
a lipid class comigrating with DGDG in Toxoplasma (24,
30). In the present study, we attempted to characterize
this digalactolipid-like lipid and to provide additional in-
formation on its subcellular localization. We made use of
rabbit polyclonal antibodies raised against DGDG homo-
geneously purified from spinach chloroplasts (30, 31).
Anti-lipid antibodies are usually more difficult to manip-
ulate than anti-protein antibodies (mainly because of the
very different molecular size and hydrophobicity of lipids
vs. proteins); they must be used at higher concentrations
(dilution 1:100 in nitrocellulose immunostaining assays
and 1:25 in IF experiments), require an accurate exposure
of the lipid epitope (regions where the lipid is sufficiently
concentrated), and cannot be easily purified without an
existing method to accurately graft the lipid epitope on a
purification matrix. For these reasons, we checked the
validity of the experiments presented here with three in-
dependent rabbit and rat anti-DGDG sera, controlled the
results with those obtained with preimmune serum, and
carried out competitive labeling with homogeneously pure
DGDG. In this study, the class of Toxoplasma lipids harbor-
ing a DGLE was named DGLE.
Figure 1 shows the results obtained with one of the
rabbit anti-DGDG sera. Purified DGDG (10 mg) and total
lipids from the spinach chloroplast envelope (200 mg)
spotted on nitrocellulose membranes were used as positive
controls for the immunostaining of lipids dot-blotted on
nitrocellulose membranes (Fig. 1A). No signal was de-
tected with DAG, phospholipids (PG, PC, PE), SM,
monogalactolipids or trigalactolipids (MGDG, TriGDG),
sulfoquinovosyldiacylglycerol (SL), MGCBs, or LCBs
(Fig. 1A). Other than LCB, MGCB, and SM, all of these
lipids share a DAG hydrophobic moiety with DGDG.
Therefore, this immunoreactivity profile shows that the
rabbit anti-DGDG antibodies react with the digalactolipid
polar head. The rabbit polyclonal anti-DGDG antibodies
consistently reacted with lipids extracted from the spinach
chloroplast envelope and did not react with HFF lipids
(200 mg) (Fig. 1A). As a negative control, preimmune
serum failed to react with purified DGDG (10 mg), spinach
chloroplast envelope lipids (200 mg), or Toxoplasma total
lipids (200 mg) (Fig. 1B). Consistent with a previous report
by Jouhet et al. (31), Fig. 1C shows that the rabbit anti-
DGDG serum allows the specific detection of chloroplast
membranes of permeabilized Arabidopsis cells (DGDG is
not extracted from membranes in IF permeabilization
treatments). The rabbit anti-DGDG serum did not show
any significant cross-reactivity with total protein extracts
from Toxoplasma or HFF proteins (Fig. 1D).
Figure 1E, F shows the reactivity of the rabbit anti-
DGDG serum with Toxoplasma lipids. Preincubation of
anti-DGDG with purified DGDG abolished the reactivity
on purified DGDG and on Toxoplasma total lipid extract
(Fig. 1E). These results showed that DGDG saturated the
anti-DGDG antibodies of the rabbit serum and competed
efficiently with the Toxoplasma lipids, highlighting the
structural similarity between DGDG and DGLE polar heads.
Thus, we reasonably supposed that DGDG and DGLE were
immunostained with a similar intensity and that a dose-
dependent immunoreactivity of the anti-DGDG rabbit
serum with increasing amounts of spotted DGDG (Fig. 1F)
allowed the relative quantification of DGLE in various
Toxoplasma and HFF samples. Therefore, the DGLE pro-
portion in Toxoplasma total lipids was estimated at
?0.25 mol% (Fig. 1F) and represents a minor lipid class.
A second rabbit and a rat were independently immu-
nized with homogeneously pure DGDG according to the
same procedure. The two additional sera obtained after
immunization with DGDG reacted similarly with Toxo-
plasma DGLE (data not shown). Collectively, these results
support the notion that both rabbit and rat polyclonal
antibodies, raised against plant chloroplast DGDG, immu-
noreact with a class of Toxoplasma lipids whose polar heads
share structural features with that of DGDG [i.e., termi-
nated by a dihexosyl group structurally close to a-galactosyl
(1Y6)b-galactose]. Anti-DGDG sera have been used to
investigate the subcellular localization of DGLE by IF and
IEM experiments. In the experiments described below,
results obtained with the first rabbit anti-DGDG serum are
presented, except as indicated.
In extracellular Toxoplasma parasites, DGLE is localized
in membrane domains, at the surface of the plasma
membrane, and in the inner membrane complex
To localize DGLE in extracellular parasites, freshly lysed
tachyzoites were labeled with the rabbit anti-DGDG serum
without any fixation and permeabilization and then ob-
served by epifluorescence. In parallel, parasites were fixed,
permeabilized with Triton X-100, and labeled with anti-
DGDG (Fig. 2A). IF images were captured and signal in-
tensity was enhanced using Axiovision 3.1 software (Zeiss).
Preimmune serum did not allow any labeling (Fig. 2A,
left). The absence of labeling with anti-IMC1, an antibody
raised against an inner membrane protein (IMC-1) (37),
was used as a control for plasma membrane integrity
(Fig. 2A, right). DGLE was localized with a dotted pattern
at the parasite periphery, independently of the perme-
abilization step (Fig. 2A, center). A series of images fo-
cused at the top, medial, and bottom sections of the same
parasite showed that the dotted patterns of both non-
permeabilized and permeabilized parasites were scattered
around the parasitic surface (Fig. 2B) and that no intense
labeling could be detected in central compartments of
To refine the localization of DGLE at the cell periphery,
extracellular tachyzoites were treated with 1% glycerol to
allow local swelling and osmotic separation of the plasma
membrane from the inner membrane complex. Under
these conditions, the plasma membrane forms small
blebs at the cell surface. Figure 2C shows that the rabbit
anti-DGDG serum labeled both the plasma membrane
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Fig. 1. Immunoblot analyses of lipid standards and T. gondii lipid extracts using rabbit anti-
digalactosyldiacylglycerol (DGDG) polyclonal antibodies. A: Immunoreactivity of the rabbit anti-DGDG
serum toward lipid standards (10 mg) [i.e., diacylglycerol (DAG), DGDG, lactocerebroside (LCB), mono-
galactosylcerebroside (MGCB), monogalactosyldiacylglycerol (MGDG), trigalactosyldiacylglycerol
(TriGDG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG),
sulfolipid (SL), and sphingomyelin (SM)] and total lipids purified from spinach chloroplast envelope
(25 mg) and human foreskin fibroblast (HFF) cells (200 mg). To assess the specificity of the anti-DGDG
serum, purified DGDG and total lipids from the spinach chloroplast envelope were used as positive controls.
No signal was detected with spotted DAG, phospholipids (PG, PC, and PE), sphingolipid (MGCB, LCB, and
SM), or simple glycoglycerolipids such as galactolipids (MGDG and TriGDG) or sulfoquinovosyldiacylglyc-
erol (SL). Therefore, this immunostaining profile shows that the anti-DGDG antibodies react with the lipid
polar head. B: Immunoreactivity of the preimmune serum against purified DGDG (10 mg), total spinach
chloroplast envelope (200 mg), or total T. gondii lipids (200 mg). C: Immunofluorescence (IF) immuno-
staining of chloroplasts, in permeabilized Arabidopsis cells, with the rabbit anti-DGDG serum. D: SDS-PAGE
of protein extracts from Toxoplasma parasites (Tg) and from HFF host cells (HC) revealed either by
Coomassie blue staining (Coomassie) or by immunoblot analysis with the rabbit anti-DGDG serum. E:
Immunoreactivity of the rabbit anti-DGDG serum, preincubated for 12 h with purified DGDG, with purified
DGDG (0.25 mg) or total T. gondii lipids (200 mg) (lower lane) and immunoreactivity of the untreated rab-
bit serum anti-DGDG with the same lipids (upper lane). This result highlights the very high structural
similarity between the DGDG epitopes and the digalactolipid-like epitope (DGLE). F: Dose-dependent
reactivity of the rabbit serum anti-DGDG to increasing quantities of purified spinach DGDG (1, 5, or 10 mg)
spotted ona nitrocellulose membrane. Supposing that DGDG and DGLEwere immunostained witha similar
intensity, the dose-dependent immunoreactivity of the anti-DGDG rabbit serum allowed a relative quan-
tification of DGLE in various Toxoplasma samples. Total lipid extracts from T. gondii (200 mg) as well as
pellicle (200 mg) and raft (200 mg) lipid extracts were spotted onto nitrocellulose membranes and probed
with the rabbit anti-DGDG serum. DRM, detergent-resistant membrane.
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Fig. 2. Immunolocalization of DGLE in extracellular T. gondii tachyzoites. A: IF analysis of DGLE in
extracellular parasites. Freshly lysed tachyzoites were labeled with rabbit preimmune (left) or anti-DGDG
(center and right) serum without any fixation and permeabilization (no perm.) and then observed by
epifluorescence. In parallel, parasites were fixed, permeabilized with Triton X-100, and then labeled with
anti-DGDG (Triton X-100). The absence of labeling with anti-IMC-1 was used as a control for the absence
of cell permeabilization. B: Three-dimensional distribution of DGLE in extracellular T. gondii. Immuno-
fluorescence was analyzed by adjustment of imaging focus at the top (1), medial (2), or bottom (3) sections
of the same parasite. DGLE is detected as microdomains at the parasite periphery. C: IF analysis of DGLE in
the plasma membrane and inner membrane complex after pellicle physical membrane separation. Parasites
were treated with 1% glycerol for 1 h, allowing physical separation of the plasma membrane (arrowheads)
from the inner membrane complex (arrows) and producing plasma membrane blebs (circles). Treated
parasites were colabeled with both monoclonal anti-SAG1 antibody (red) and rabbit anti-DGDG serum
(green) or with both rabbit anti-IMC1 (purple) and rat anti-DGDG (green) sera. D: Immunogold labeling of
extracellular parasites with rabbit anti-DGDG serum. 1, Labeling at the level of the pellicle (circle). 2, Dotted
labeling at the surface of the plasma membrane and at the level of the inner membrane complex
(arrowheads). Bars 5 500 nm.
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(colabeling with antibodies raised against the surface pro-
tein SAG1) and the inner membrane complex (colabeling
with antibodies raised against IMC-1). When freshly iso-
lated parasites were immunogold-labeled with the rabbit
anti-DGDG serum, DGLE was consistently detected at the
cell periphery (Fig. 2D, panel 1) and exposed at the plasma
membrane surface or within the inner membrane complex
(Fig. 2D, panel 2).
To assess the localization of DGLE in the pellicle,
extracellular parasites were fractionated on sucrose gra-
dients (45) to obtain pellicle-enriched fractions (Fig. 3A).
Proteins from these fractions were analyzed by Western
blot using compartment markers: clear enrichments in
both SAG1 and IMC1 were observed in the pellicle fraction
(Fig. 3A). When lipids were extracted, spotted onto nitro-
cellulose, and probed with the rabbit anti-DGDG serum,
DGLE was immunodetected (Fig. 3A). DGLE represented
?0.5% of the total lipid content of the pellicle fractions
Attempt to assess the structure of DGLE by mass
spectrometry lipidomic analysis of the pellicle membranes
purified from extracellular Toxoplasma
We analyzed the lipid profile of the pellicle-enriched
fraction by mass spectrometry and attempted to identify
DGLE candidate(s) in the minor peaks corresponding to
accurate glycolipidic structures (Fig. 3B). The amounts of
the main phospholipids, and Toxoplasma-specific enrich-
ment in phosphoethanolamine ceramide, confirmed pre-
vious analyses performed on whole parasites (13, 24). Based
on immunoblot analyses, the proportion of lipids react-
ing with the antisera was estimated to be in the range of
1.2–6 nmol/mg total glycerolipids. Immunolabeling of
DGLE is consistent with 1) the occurrence of DGDG but
also with 2) digalactolipids having an alternative hydro-
phobic moiety (i.e., acylalkylglycerol or ceramide; Fig. 3B)
and 3) dihexosyl lipids that might be cross-detected by
the antibody but harboring different sugars, such as Glc,
GalNAc, or GlcNAc. Furthermore, we cannot exclude the
possibility that antibodies react with 4) more complex
glycolipids terminated by a dihexosyl group. Here, we
particularly examined nonsubstituted monohexosyl and
dihexosyl lipids. Scans for neutral loss of 179 [i.e., loss of
ammoniated hexose minus water (C6H13O5N)] or neutral
loss of 341 [i.e., loss of ammoniated dihexose minus water
(C12H23O10N)] produced a few peaks close to the detec-
tion thresholds in the MGDG/MGAAG (700–900) or
DGDG/DGAAG (890–1,050) mass ranges, respectively. In
the neutral loss 179 scan, consistent with the loss of one
hexosyl residue, minor peaks were detected at m/z 732,
734, 760, 764, 776, 792, and 804. In the neutral loss
341 scan, consistent with the loss of two hexosyl residues,
minor peaks were detected at m/z 894, 896, 908, 910,
922, 936, 954, and 966. These weak peaks might be attrib-
utable to the occurrence of hexosyl diacyl lipids in the
sample in the low picomole range, consistent with the
quantity detected in the immunostaining dot-blot ex-
periments. Some of the weak peaks, including those at
m/z 894, 896, and 922, are distinct from the standard
galactolipids and could correspond, as [M 1 NH4]1, to
In contrast with glycosylated glycerolipids, the mono-
hexosylceramides and dihexosylceramides were detected
in higher quantity (Fig. 3B). Among dihexosylceramides,
digalactosylceramide could contribute to the binding of
anti-DGDG antibodies, particularly if the digalactosyl
conformation resembles that of the DGDG polar head
Although the low abundance of DGLE is a technical
limitation for global characterization, in the range of
low-level contamination in the mass spectrometer, so that
no conclusive result could be drawn regarding the struc-
ture of the DGLE hydrophobic moiety, mass spectrometry
lipidomic analyses indicate the clear presence of dihexosyl
lipids with a ceramide moiety and allow for the possibility
of other hydrophobic moieties (DAG, acylalkylglycerol) at
low levels. Any of these structures may represent the DGLE
that reacts with the anti-DGDG antibodies.
During host cell invasion and endodyogenic
multiplication, DGLE relocates to the anterior tip of
the Toxoplasma cells
Localization of DGLE was analyzed during intracellu-
lar parasite development. Figure 4A shows that the pre-
immune serum did not react with human or Toxoplasma
cells. Because the parasite population was not synchro-
nized at the time of host cell invasion, different stages of
parasite development were observed on the same slide at
24 h after infection. Figure 4B summarizes these observa-
tions. At the time of host cell invasion, when the parasite
squeezes through the moving junction (51), a relocaliza-
tion of DGLE was observed. At the posterior end of the
parasite (Fig. 4B, panel 1), which was still outside the host
cell, a dotted labeling was observed, reminiscent of that
observed at the surface of extracellular parasites (Fig. 2).
By contrast, concentration of DGLE was observed in the
apical part of the parasite, which was already inside the
intracellular forming vacuole. Early after vacuole forma-
tion, Toxoplasma is known to accumulate at its posterior
end the dense granule protein GRA2 (4). Colabeling of re-
cently invaded parasites with anti-DGDG rabbit antibodies,
a monoclonal anti-GRA2, and Hoechst reagent showed
that DGLE was concentrated opposite to GRA2 (i.e., at the
anterior part of the parasite) (Fig. 4C). Together, Fig. 4B,
C show that after invasion and the formation of the
parasitophorous vacuole, DGLE relocates rapidly to the
anterior tip of the parasite.
Duringthefirstdivisionoftheparasite (Fig.4B, panel2),
DGLE remained concentrated as a gradient at the anterior
of the mother cell and was also detected in duplicated
structuresattheapex of thedaughter cells.Colabeling with
anti-DGDG rat serum and monoclonal anti-IMC1 (Fig. 4D)
showed that DGLE did not fully overlap with IMC1, the
localization of which follows the complete inner mem-
brane complex of daughter cells (Fig. 4D, arrowheads).
Therefore, anti-DGDG/anti-IMC1 colabeling highlights a
gradient of DGLE at the anterior part of the inner mem-
brane complex. DGLE participation in apical pole devel-
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Fig. 3. Lipid analyses of pellicle fractions purified from extracellular T. gondii tachyzoites. A: Purification of pellicle membranes. Pellicle
membranous structures were fractionated from extracellular T. gondii tachyzoites (scheme at left) after isopycnic centrifugation on a
saccharose gradient (center). Enrichment in pellicle-specific proteins (Pellicle) was assessed by immunoblot analysis using plasma mem-
brane and inner membrane complex markers (i.e., monoclonal anti-SAG1 and rabbit anti-IMC1 serum). Loaded samples (Total extract and
Pellicle) correspond to the same initial amount of unfractionated and fractionated parasites (108parasites). Lipids were extracted and
200 mg was analyzed by immunoblot with rabbit anti-DGDG serum. B: Lipid profile of Toxoplasma pellicle membranes. Polar lipids from
pellicle membranes were analyzed by mass spectrometry and quantified based on known amounts of internal standards for each lipid class.
Sterols (including cholesterol and steryl esters) were quantified by a resofurin-based fluorometric assay. The histogram gives average values
of two series of quantifications. Glycosylated lipids were analyzed based on the presence of one or two hexosyl residues on the polar
head, consistent with the structure of one or two galactosyl residues. Lipids that might terminate by an a-galactosyl(1Y6)galactose and
be detected by anti-DGDG antibodies are labeled with asterisks. Amounts of MHexDG/MHexAAG, DHexDG/DHexAAG, and PA in-
clude weak peaks corresponding to structures that were not verifiable as belonging to these classes. PA, phosphatidic acid; LysoPC,
lyso-phosphatidylcholine; ePC, ether-linked PC [i.e., alk(en)yl, acyl PC]; LysoPE, lyso-phosphatidylethanolamine; ePE, ether-linked PE
[i.e., alk(en)yl, acyl PE]; PI, phosphatidylinositol; PS, phosphatidylserine; ePS, ether-linked PS [i.e., alk(en)yl, acyl PS]; MHexDG,
monohexosyldiacylglycerol (including MGDG); MHexAAG, monohexosyl-alk(en)yl-acyl glycerol (including MGAAG); DHexDG,
dihexosyldiacylglycerol (including DGDG); DHexAAG, dihexosyl-alk(en)yl-acyl glycerol (including DGAAG); Cer, ceramide; EthCer, phos-
phoethanolamine ceramide; ChoCer; phosphocholine ceramide or SM; MHexCer, monohexosylceramide (including galactosylceramide);
DHexCer, dihexosylceramide (including digalactosylceramide).
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Fig. 4. Immunolocalization of DGLE in intracellular parasites. A: Immunostaining of intracellular parasites
with preimmune serum. B: Sequence of intracellular parasites labeled with rabbit anti-DGDG. 1, Arrows
indicate the moving junction. 2, 3, Arrowheads indicate the anterior tip of daughter parasites forming within
the mother cell. 5, The arrow shows the intravacuolar residual body. C: Parasites recently invaded (,1 h) and
colabeled with rabbit serum anti-DGDG, monoclonal anti-GRA2, and Hoechst reagent. ap, anterior part of
the intracellular parasite; d, posterior GRA2 dot. The arrowhead indicates the apicoplast nucleic content.
D: Colabeling of dividing intracellular parasites with both rat anti-DGDG serum and rabbit anti-IMC1 serum.
Arrowheads indicate the anterior part of parasites, including those of daughter cells. E: Colabeling of
dividing parasites with rabbit anti-DGDG serum and Hoechst reagent. Arrowheads indicate localizations
where DGLE is concentrated in dividing parasites.
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opment was further supported by superimposition of the
divided nucleus and the anti-DGDG labeling shaping the
future apical inner membrane complex of daughter para-
sites (Fig. 4E). During the subsequent rounds of division
(Fig. 4B, panels 3–5), DGLE localization was similar, at the
anterior tips of the parasites and of their dividing daughter
by a vacuolar structure known as the residual body, which
contains remnants of the mother parasites (52). Within the
vacuolar compartment, DGLE was detected at the level of
this residual body but was not detected in any other vacu-
olar membrane, as shown by IF (Fig. 4B, panel 5).
DGLE localization in membrane domains of the
parasite pellicle is affected by the depolymerization of
Because DGLE was localized in patches at the parasite
periphery of extracellular parasites and at the anterior
tip of intracellular parasites, where it remained during
endodyogeny, we addressed the question of a possible
clustering of DGLE in DRMs (or rafts) and the potential
relation with the parasite cytoskeleton.
DRMs were isolated from extracellular parasites, accord-
ing to the protocol recently reported by Azzouz et al. (48).
Sterol and steryl ester quantification demonstrated a con-
sistent enrichment in the DRM fraction (11.35 mol% of
the membrane lipids) compared with the pellicle fraction
(3.3 mol%). When lipid extract from the DRM fraction was
spotted onto nitrocellulose and incubated with the rabbit
anti-DGDG serum, DGLE was detected: the DGLE content
of the DRM fraction was estimated at ?0.1%, based on the
intensity of the immunostaining with anti-DGDG anti-
bodies (Fig. 1F). Because this proportion is lower than that
of pellicle lipids (i.e., 0.5%) (Fig. 1F), this analysis shows
that the DGLE membrane domains visualized by imaging
experiments do not coincide strictly with sterol-rich DRMs.
To characterize the possible relation of DGLE mem-
brane domains with the cytoskeleton, various treatments
with cytoskeleton-destabilizing drugs, either depolymeriz-
ing microtubules (oryzalin) or actin (cytochalasin D) or af-
fecting myosin A (butanedione monoxime), were carried
out before DGLE visualization (Fig. 5A, B). Oryzalin or
butanedione monoxime treatment did not change the
distribution of DGLE at the anterior part of the parasite
(Fig. 5A, left and central panels). By contrast, treatment of
infected cells with cytochalasin D resulted in the redistri-
bution of DGLE to the periphery of the cell (Fig. 5A, right
panel). These results suggest that DGLE might be directly
or indirectly linked to parasite polymerized actin, which is
atypically localized between the plasma membrane and the
inner membrane complex in Apicomplexa (53), and that
DGLE does not interact with microtubules or myosin A.
Actin filaments are particularly difficult to observe in
apicomplexan parasites (53). Nevertheless, filamentous
actin was recently detected as short filaments located be-
tween the subpellicular network and the plasma mem-
brane of Toxoplasma (44). When preparations of parasite
subpellicular network, known to preserve both micro-
tubules and filamentous actin, were incubated with the
rabbit anti-DGDG serum, labeling was observed at the
extreme tip of the conoid (Fig. 5C, panels 1, 2), along the
subpellicular microtubules (Fig. 5C, panel 3), and along
the whole cell surface (Fig. 5C, panels 4, 5). These obser-
vations further support a possible association of DGLE
with parasite filamentous actin.
Search for DGLE in other apicomplexan parasites
We examined the possible occurrence of DGLE in other
apicomplexan parasites (Fig. 6). A distribution pattern
similar to that observed in Toxoplasma was detected in the
closely related apicomplexan parasite N. caninum (Fig. 6A).
DGLE was also detected in C. parvum, an Apicomplexa
withoutplastid(Fig. 6B).InP.falciparum, themain malarial
parasite, DGLE was detected, with a subcellular localiza-
tion that appeared to be remodeled along parasitic stages
in the human host (Fig. 6C). In ring stages, DGLE showed
a crescent-like distribution as marginal or peripheral dots
(Fig. 6C, panels 1, 2). Trophozoite-stage parasites (Fig. 6C,
panel 3) exhibited DGLE domains at the cell periphery. A
localization of the DGLE at the level of the developing
merozoite membranes was then observed in the schizont-
stage parasites (Fig. 6C, panels 4, 5). Eventually, an inner
membrane complex-like distribution was observed in the
gametocyte stage, with a regular network of superficial dots
(Fig. 6C, panel 6). We further detected DGLE in Babesia
divergens (data not shown). We did not detect DGLE in
Trypanosoma brucei, a nonapicomplexan unicellular para-
site (data not shown). Together, these results show that
the anti-DGDG serum labels a lipid found in numerous
parasites of the Apicomplexa phylum. In all cases, DGLE
appears to be clustered in membrane domains at the
cell periphery and to be mobilized at the anterior tip
The presence of a plastid in Toxoplasma suggested that
MGDG and DGDG, the main chloroplast and cyano-
bacteria lipids (27–29), might also be important constitu-
ents of the apicoplast membranes. In plants and algae,
MGDG and DGDG are synthesized within plastid envelope
membranes (29, 31, 54–58). In plants, MGDG is produced
by the galactosylation of DAG, whereas it is generated by a
two-step glucosylation and epimerization in cyanobacteria
(59). In both plants and cyanobacteria, DGDG is formed
by the addition of a galactosyl residue to MGDG (29, 54).
Upon phosphate deprivation, DGDG is exported outside
plastids to the plasma membrane (60) and the mitochon-
dria (31). No MGDG synthase or DGDG synthase homolo-
gous sequences could be detected in the genome of some
plastid-containing organisms, such as Euglena, in which
MGDG and DGDG are well-established constituents (61),
suggesting that their synthesizing enzymes might have
strongly diverged during evolution. Searching both Toxo-
plasma and Plasmodium databases did not reveal any gene
candidate for MGDG synthesis, leaving the question of
the monogalactolipid-synthesizing enzymes unresolved. A
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Fig. 5. Analysis of the relation between DGLE pellicle membrane domains and the T. gondii cytoskeleton. A: Evolution of DGLE pellicle
membrane domains after chemical impairment of the T. gondii cytoskeleton of intracellular tachyzoites with oryzalin, butanedione
monoxime, and cytochalasin D. Infected cells were treated with oryzalin, a tubulin-destabilizing agent, butanedione monoxime, a myosin A
binding drug, or cytochalasin D, an actin-depolymerizing factor. After labeling with rabbit anti-DGDG serum, a relocalization of DGLE
similar to that observed in extracellular parasites was observed only under conditions in which actin filaments were disrupted. DGLE
membrane domains formed spontaneously after the cytochalasin D time course. B: Evolution of DGLE pellicle membrane domains after
treatment of the T. gondii cytoskeleton of extracellular tachyzoites with cytochalasin D. Extracellular parasites were treated with 5 mM
cytochalasin D for 30 min and labeled with rabbit anti-DGDG serum. No apparent change in DGLE localization was observed. C:
Immunogold labeling of the parasite subpellicular network with the rabbit anti-DGDG serum. Immunolocalization of DGLE in cytoskeletal
preparations was carried out as described (44). DGLE is concentrated mainly at the extreme tip of the conoid (1, circle) as well as along the
inner membranes, in closeproximity to subpellicular microtubules, which are preserved after detergent treatment (2–4, arrows and circles).
DGLE was also found concentrated at the posterior end (5, circle). Bars 5 200 nm.
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single gene in Toxoplasma (49.m00002; PF10_0316 in P.
falciparum), coding for a 624 amino acid protein, contains
a 387 amino acid segment with ?24% identity with cyano-
slr1508) (62), and also has strong homology with lipo-
polysaccharide glycosyltransferases involved in glycosyl-
phosphatidylinositol anchor synthesis. This gene might
encode an enzyme of broad specificity catalyzing the syn-
thesis of DGLE and should be functionally analyzed in
Here, we used specific polyclonal antibodies from rabbit
or rat, raised against plant DGDG, to investigate the oc-
currence and cell dynamics of a class of lipids harboring a
DGLE in Toxoplasma and other Apicomplexa. We did not
detect any reactivity of any of the anti-DGDG antibodies
with mammalian lipid extracts or cell membranes. Analysis
of the anti-DGDG specificity assessed by nitrocellulose im-
munostaining experiments (Fig. 1) shows that the anti-
bodies have no significant cross-reactivity with Toxoplasma
proteins and that they do not specifically bind to the
hydrophobic moiety of DGDG (i.e., the DAG structure).
Because preincubation of the antibodies with DGDG com-
petes with both chloroplastic and Toxoplasma lipids, a strong
structural similarity to the polar head of DGDG seems
Fig. 6. Localization of DGLE in N. caninum, C. parvum,
and P. falciparum. A: Immunolabeling of N. caninum
tachyzoites with rabbit anti-DGDG serum. A1, Recently
invaded tachyzoite. A2, Replicating pair of parasites. A3,
Vacuole containing four parasites. Arrowheads indicate
the newly formed parasites within the mother cell; the
arrow shows the intravacuolar residual body. B: Immuno-
labeling of C. parvum cells with both rabbit anti-DGDG
serum and rat anti-Cryptosporidium serum. B1, Extracellu-
lar sporozoites exhibiting an apical distribution of DGLE.
B2, B3,DGLE localization at the periphery of intracellular
sporozoites, with stronger presence at the apical pole. B4,
Distribution of DGLE at the membrane of the meront-
stage parasites. C: Distribution of DGLE during the asex-
ual cycle of P. falciparum. Cultures of P. falciparum-infected
red blood cells were fixed and permeabilized using Triton
X-100. Immunolabeling was performed using anti-DGDG
antibody, and nuclei were stained with Hoechst 33258.
Acquisitions were ordered according to the life stage of
the parasites. C1, C2, A crescent-like distribution of DGLE
as marginal dots was observed in the ring stages. C3, The
DGLE distribution is localized as peripheral dots close to
the membranes of the trophozoite-stage parasites. C4, C5,
Reorganization of DGLE to the developing merozoite
brane complex-like distribution as well as reticulated su-
perficial dots were observed in the gametocyte stage.
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required for the antibody binding. Based on these anal-
yses, DGLE detected in Toxoplasma lipid extracts is pos-
sibly a dihexosyl lipid whose polar head is close to an
a-galactosyl(1Y6)b-galactose and, based on previous
metabolic analyses (24, 30), whose hydrophobic moiety
contains at least one hydrolyzable acyl ester (30). We do
not exclude the possibility that other lipid structures, with
a more complex polar head terminated by a dihexosyl
group, mightalso bedetectedusinganti-DGDG antibodies.
Inconsistent with the initial idea sustaining the search
for chloroplast galactolipids in Apicomplexa, none of our
IF or IEM experiments allowed the detection of DGLE in
an internal structure of Toxoplasma that resembles the
tion, or access of antibodies to internal membranes might
not allow immunodetection in this organelle (Fig. 4C).
Galactolipids might be synthesized by apicoplast enzymes
but rapidly exported to other cell compartments, in a
similar manner to which DGDG is exported outside chlo-
roplasts in phosphate-deprived plants (31, 60). Alterna-
tively, evolution of the galactolipid synthetic machinery
or it may be completely independent on any chloroplast-
related process. In extracellular life stages of Toxoplasma,
DGLE was detected at the periphery of the cell by both IF
and IEM imaging, partly exposed at the surface of the
plasma membrane (Fig. 2A, B, D), but also in the inner
membrane complex (Fig. 2C, D). Localization in pellicle
membranes was confirmed by immunostaining of lipid
extracts of pellicle-enriched fractions (Fig. 3A). Therefore,
DGLE appears as a minor constituent of the pellicle of
tachyzoites, with a dotted or patched pattern that suggests
a concentration in membrane domains.
Upon invasion, DGLE relocates to the anterior part
of the cell (Fig. 4). Interestingly, we could apparently
reverse the relocation of DGLE by treating intracellular
Toxoplasma with cytochalasin D, an actin-depolymerizing
agent (Fig. 5A). This result suggests that DGLE reloca-
tion might be directly or indirectly determined by actin-
dependent processes and that DGLE might be a factor of
the invasion mechanism. In the current understanding
of invasion, conserved multiprotein machineries are in-
volved, including both the parasite acto-myosin motor
located between the parasite plasma membrane and the
inner membrane complex and the microneme secretory
apparatus (44, 52, 63). During invasion, adhesive trans-
membrane proteins (secreted from micronemes) bind to
the host cell. These adhesive proteins are linked to the
invasive motor via an interaction between their cyto-
plasmic tail and aldolase, which in turn interacts with ac-
tin linked to myosin A. The unconventional myosin A is
rigidly anchored to inner membrane complex proteins
(IMC-1 and IMC-3) through an interaction with protein
intermediates, which include the myosin A-interacting
protein (MTIP in Plasmodium and MLC1 in Toxoplasma)
and the gliding-associated proteins of 45 and 50 kDa
(GAP45 and GAP50). The cytosolic face of the inner
membrane complex is then associated directly with 22 sub-
pellicular microtubules, which maintain the parasite shape
and which are also involved in the cell-gliding motility (44,
52, 63). Therefore, DGLE that rapidly moves to the ante-
rior part of the cell during invasion might be associated
with some of these constituents, an hypothesis further
supported by IEM detection of DGLE in the vicinity of
cytoskeleton structures (Fig. 5C). Because the DGLEgradient
was not disorganized by oryzalin, a tubulin-destabilizing
agent, or butanedione monoxime, affecting myosin A
(Fig. 5A), our study suggests that during invasion DGLE
might be associated with actin and/or micronemal pro-
teins. Alternatively, Johnson et al. (64) recently showed
that GAP50 and the myosin complex are immobilized
within the inner membrane complex at the level of DRM
domains, which are enriched in sterols and have a higher
density than DRMs classically reported in eukaryotic cells.
DGLE might be a component of such inner membrane
Toxoplasma division is a binary process called endody-
ogeny during which a single chromosome replication is
followed by concurrent mitosis and parasite budding (65).
In our study, DGLE was detected within dividing cells at
the tip of the duplicated inner membrane complexes of
the daughter cells (Fig. 4). Because DGLE was also de-
tected at the tip of the mother cell and as part of the re-
sidual body, the DGLE of the daughter cells is likely newly
synthesized rather than recycled.
Attempts to determine the precise structure of DGLE by
mass spectrometry analyses of the pellicle membranes
(Fig. 3B) allowed us to inventory some minor lipids, par-
ticularly dihexosyl lipids with various hydrophobic moie-
ties (ceramides, DAG, acylalkylglycerol), that might
react with the anti-DGDG antibodies. Immunolabeling
of DGLE is consistent with the occurrence of DGDG, and/
or digalactolipids having an alternative hydrophobic
moiety (i.e., acylalkylglycerol or ceramide; Fig. 3B), and/
or dihexosyl lipids that might be cross-detected by the
antibody but harboring different sugars, such as Glc,
GalNAc, or GlcNAc. Furthermore, we cannot exclude the
possibility that antibodies react with more complex glyco-
lipids terminated by a dihexosyl group. Whereas dihexosyl
ceramides could be unambiguously assessed, peaks corre-
sponding to dihexosyl DAG or dihexosyl acylalkylglycerol
were close to the detection threshold, and no conclusive
result could be drawn regarding the unambiguous deter-
mination of the DGLE structure. Previous reports of the
metabolic labeling of a lipid comigrating with DGDG after
incubation with radiolabeled UDP-galactose (30) and
acetate (24) support the idea that the DGLE hydrophobic
moiety is DAG or acylalkylglycerol. Because numerous
glycolipids generated by the same enzyme can share an
identical polar head but harbor different hydrophobic
moieties (e.g., MGCB and MGDG generated by galacto-
cerebroside synthase based on the supplied substrate,
ceramide or DAG, respectively), the possibility that the
detected DGLE corresponds to glycosyl glycerolipids or
glycosyl ceramides or to both classes cannot be rigorously
excluded. Recently, the parasitic trematode Fasciola
hepatica was shown to exhibit mammalian-type glycolipids,
including a-galactosyl(1Y4)b-galactose- and a-galactosyl
Digalactolipid-like epitope in Apicomplexa 759
by guest, on June 1, 2013
(1Y3)b-galactose-terminating glycoceramides, as well as
ing glycoceramides that account for cestode serological
cross-reactivity (66). It is possible, therefore, that like
glycolipids of the neogala series [i.e., b-galactosyl(1Y6)b-
galactose], the a-galactosyl(1Y6)b-galactose-terminating
glycolipids might be related to the pathogenic process.
We also attempted to refine the distribution of DGLE
in membrane domains by analyzing the lipid content of
DRMs. In a recent report, DRMs isolated from the pellicle
of extracellular parasites were shown to be enriched in
cholesterol, GM1 ganglioside, SM, phospholipids, and still
unidentified lipids. They were also shown to be associated
with proteins involved in invasion and motility, suggesting
their localization at the inner membrane complex (53).
Here, DRM fractions purified according to the same pro-
cedure were enriched in sterols and contained DGLE,
although no specific enrichment in DGLE could be
measured (Fig. 1F). The galactosyl head groups of lipids
are known to interact and promote the organization of
stacked domains (67). Therefore, DGLE might sponta-
neously form membrane domains, possibly associated with
DRMs, with some specific cellular functions including
protein anchoring. Thus, the actin-dependent migration
of DGLE to the anterior part of the parasite, occurring
during early infection processes, might be part of a mecha-
nism recruiting other components to the apex of the cell.
DGLE was detected in important apicomplexan para-
sites (Toxoplasma, Neospora, Plasmodium, Babesia, and
Cryptosporidium) (Fig. 6). In all cases, DGLE was localized
at the periphery of the cell, either as pellicle membrane
domains or as an apical gradient. This study shows that
DGLE is found broadly in the Apicomplexa phylum, in
both apicoplast-containing (Toxoplasma, Neospora, Plasmo-
dium, and Babesia) and apicoplast-free (Cryptosporidium)
species. If DGLE synthesis derived from an ancestral chlo-
roplastic DGDG synthetic machinery, this result suggests
that the corresponding enzymes are no longer localized
within a plastid, at least in Cryptosporidium.
After this descriptive work, future studies include the
precise characterization of the hydrophobic moiety of
DGLE, requiring very large-scale cultures of the parasites,
lipid class purification, and analysis. Although this work
was motivated by the analysis of chloroplast galactolipids
in Apicomplexa, based on a series of experimental evi-
dence, technical limitations did not allow us to give any
definitive demonstration regarding the occurrence of
these precise lipidic structures in Apicomplexa. It becomes
clear that the analysis of apicoplast lipids requires a puri-
fication procedure for this organelle, respecting its mem-
brane integrity. The current understanding of galactolipid
synthesis in plastid-containing organisms is still incom-
plete, lacking some of the enzymes in important photo-
synthetic organisms that might be helpful to explore
Apicomplexa genomes. In spite of the apparently small
number of glycosyltransferases in Apicomplexa [as in-
ventoried in the CAZy database (68)], genes sharing some
similarity with cyanobacteria dgdA can be detected, and
efforts should focus on their analyses and on the search for
other glycolipid-synthesizing enzymes to functionally ad-
vance our understanding of simple glycolipid classes in the
context of Apicomplexa pathogeneses.
The authors are indebted to S. Gonza ´lez (Unidad de Microscopia
Electro ´nica, Centro de Investigacio ´n y de Estudios Avanzados
del Instituto Polite ´cnico Nacional, Me ´xico) for expert micros-
copy; to A. Zoppe ´ (Commissariat a ` l’Energie Atomique, Grenoble,
France), A. Sparks and M. Roth (Kansas State University) for
technical assistance; and to C. Bisanz (Institut Jean Roget,
Grenoble, France), C. Beckers (University of North Carolina),
M. A. Hakimi (Institut Jean Roget), S. Khaldi and G. Gargala
(Universite ´ de Me ´decine-Pharmacie, Rouen, France), A.
Grichine (Institut Albert Bonniot, Grenoble, France), J. Jouhet
(Commissariat a ` l’Energie Atomique), L. Lecordier (Universite ´
Libre de Bruxelles, Belgium), K. Musset (Institut Jean Roget),
L. D. Sibley (Washington University School of Medicine, St.
Louis, MO), D. Soldati (University of Geneva, Switzerland),
and G. Wards (University of Vermont, Burlington) for sharing
invaluable reagents (lipids, antibodies, and cell lines). The
authors thank M. A. Block and M. A. Hakimi for fruitful dis-
cussions. This work was funded by grants from Ose ´o-Innovation
(Grants A0106220V and A0502020V), the Agence Nationale
de la Recherche (Grant ANR 05EMPB01702), and the Conseil
Re ´gional Rho ˆne-Alpes, Cluster 9 (to E.M.); by the National
Insitutes of Health (Grants RO1 NIAID TMP 16945 01-20,
27530 01-20, 4328x 01-11) and the Research to Prevent Blind-
ness Foundation (to R.M.); and by fellowships from the French
Ministry of Research (to C.B.) and the Conseil Re ´gional Rho ˆne-
Alpes, Emergence (to N.S.). The Kansas Lipidomics Research
Center was supported by the National Science Foundation
(Grants EPS 0236913, MCB 0455318, and DBI 0521587), the
Kansas Technology Enterprise Corporation, K-IDeA Networks
of Biomedical Research Excellence of the National Institutes of
Health (Grant P20 RR-16475), and Kansas State University.
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