Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO 63110, USA
* Author for correspondence (e-mail: sibley{at}borcim.wustl.edu )
Accepted 17 May 2002
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Summary |
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Key words: Cholesterol, Endocytosis, Exocytosis, Fatty acid, Organelle association, Parasitophorous vacuole, Phospholipid, Phosphatidylcholine, Vesicular transport
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Introduction |
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The inability of T. gondii to grow in vitro in the absence of a
host cell suggests that, in addition to protection from extracellular immune
confrontations, its intracellular lifestyle provides the parasite with some
essential factor(s) unobtainable in the extracellular milieu. The isolation
from host cell endo/exocytic pathways negates the possibility that the
parasites require proteins or lipids whose trafficking is restricted to those
pathways and suggests that the needed host components are selectively
mobilized via non-vesicular transport routes. Candidate components include
nucleobases, amino acids, cytosolic proteins, and lipids. T. gondii
is in fact auxotrophic for both purines and tryptophan
(Pfefferkorn, 1984;
Schwartzman and Pfefferkorn,
1982
), and is afforded access to small molecules (<1400 Da) via
pores in the PVM (Schwab et al.,
1994
). In addition, low density lipoprotein (LDL)-complexed
cholesterol is integrated into intracellular parasite membranes by an
undefined mechanism (Coppens et al.,
2000
), indicating that T. gondii is competent to scavenge
this lipid from its host. Because rapid parasite replication must coincide
with significant biogenesis of parasite membranes and the concomitant
enlargement of the PVM, it is plausible that parasites are adept at diverting
and/or metabolizing host cell membrane lipids or lipid precursors. This
hypothesis was tested in the current study, which made use of fluorescent and
radioactive lipid probes in conjunction with fluorescence microscopy and thin
layer chromatography.
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Materials and Methods |
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Liposomes/lipids
Liposomes comprising NBD-cholesterol or BODIPY-phosphatidylcholine were
prepared by combining either fluorescent cholesterol with unconjugated
phosphatidylcholine (PtdCho) and phosphatidylglycerol or fluorescent PtdCho
with unconjugated cholesterol and phosphatidylglycerol to yield a final ratio
of 1 cholesterol:0.9 PtdCho:0.1 phosphatidylglycerol. Lipid solutions were
combined in round-bottom flasks and immediately dried under a stream of argon
with constant agitation with glass beads in a 40°C water bath. After
drying, the flask containing the lipid cake, phosphate-buffered saline (PBS),
and the glass beads was rapidly rotated in a water bath sonicator set at
40°C. Liposomes were collected and stored under argon at room temperature
in glass vials. Immediately prior to each use the liposomes were sonicated in
a water bath sonicator for 10 minutes at 37°C. Dried lipids and
non-liposome lipid solutions were stored dessicated at -20°C in single-use
aliquots until needed. The optimal dilution of each liposome and organic lipid
solution was determined empirically for each experimental procedure.
Cell culture
Tachyzoites of the RH strain of T. gondii were maintained by
serial passage in human foreskin fibroblasts (HFFs) as previously described
(Morisaki et al., 1995). HFFs
and intracellular parasites were routinely cultured in D10 media (Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum, 10 mM Hepes, 2 mM
L-glutamine, and 2 µg/ml gentamycin). Mutant 89.1 cells and parental CHO-K1
cells were cultured in H10 (Hams F12 media containing 10% fetal bovine serum,
10 mM Hepes, 2 mM L-glutamine, and 2 µg/ml gentamycin). In preparation for
experiments, host cells were grown to confluence in 12-well dishes or 100 mm
diameter dishes or to near confluence on glass coverslips.
Infection and labeling
HFFs
Host cells were washed with D10 in which lipid-containing serum was
substituted with delipidized bovine serum (DLS-D10). Rinsed cells were either
prelabeled with lipids before parasite infection or preinfected with parasites
before lipid labeling. In the first case, lipids diluted in DLS-D10 were added
to each well and the labeling performed for either 1 hour (for fluorescence
experiments) or 3 hours (for radioactive experiments) in a tissue culture
incubator. Following replacement of the lipid-containing solution with
DLS-D10, parasites suspended in DLS-D10 were added to each well at a
multiplicity of infection (MOI) of 20. Infection was performed for 3 hours
(for radioactive experiments) or as otherwise noted (for fluorescence
experiments) in a tissue culture incubator. In the second case, the order of
labeling/infection was simply reversed. [14C]Ethanolamine,
[14C]serine, [14C]choline, and
[14C]phosphatidic acid were all used at a final concentration of 2
µCi/ml, whereas [14C]acetic acid and [14C]butyric
acid were both used at 20 µCi/ml.
89.1s/CHOs
Monolayers were either mock-infected or infected at an MOI of 20 with
T. gondii. Approximately 6 hours before imminent infected cell lysis,
host cells were gently washed with H10 in which lipid-containing serum was
substituted with delipidized bovine serum (DLS-H10). DLS-H10 containing 2
µCi/ml [14C]choline was added to each well and labeling
performed for 30 minutes in a tissue culture incubator. The labeling solution
was then removed and the cells chased with label-free DLS-H10.
Extracellular parasites
Approximately 1x108 parasites were collected from a
lysing-out culture and purified by sequential passage through 20, 22 and 25 G
needles (to disrupt monolayer debris) and finally through a 3 µm pore
polycarbonate filter (Nuclepore, Whatman, Clifton, NJ). Purified parasites
were pelleted by centrifugation at 1200 g for 10 minutes,
resuspended in a small volume of DLS-D10, and incubated with lipid label for 3
hours at either 2°C or 37°C. Both phosphatidic acid and choline were
used at a final concentration of 2 µCi/ml, while acetic acid was used at 20
µCi/ml. Following labeling, the label was removed, the parasites were
washed in PBS and pelleted by centrifugation, and the pellet prepared for thin
layer chromatographic analysis as described below.
Fluorescence microscopic analysis of lipid recruitment
The subcellular distribution of fluorescent lipid analogs was examined in
uninfected hosts exposed to lipids following mock infection, hosts exposed to
lipids prior to parasite infection, and hosts exposed to lipids subsequent to
parasite infection. Infection and lipid labeling was carried out as described
above. The coverslips were then rinsed in ice-cold PBS+ (PBS
containing 1 mM each of CaCl2 and MgCl2). The coverslips
were mounted upside down on microscope slides in ice-cold PBS+,
sealed onto the slide with nail polish, placed on ice, and immediately imaged
live using a Zeiss LSM510 laser scanning confocal microscope. Fluorescence and
brightfield images were obtained using a 63x oil plan-apochromat
objective lens (NA 1.4, Zeiss) and He-Ne and Kr-Ar lasers. Each image depicts
a 0.4 µm-thick focal slice. Images were processed using Zeiss ImageBrowser
software and imported into Adobe PhotoShop for final arrangement.
Immunofluorescence microscopy of the T. gondii lipid
compartment
Host cells were inoculated with parasites at an MOI of 20 and replaced into
a tissue culture incubator. After 6 hours the uninvasive parasites were
removed and the media was replaced with DLS-D10 containing C4-BODIPY-C9. This
solution was removed 4 hours before host cell lysis and substituted with
lipid-free DLS-D10. The lysed-out parasites were collected, forced through 20
G and 25 G needles (to disrupt monolayer debris), and purified by passage
through a 3 µm pore filter. Parasites were resuspended in DLS-D10 and
either deposited upon poly-L-lysine-coated glass coverslips or added to
coverslips seeded with host cells. After a 1 hour incubation at 20°C, the
extracellular parasites on coverslips were rinsed in PBS and fixed with 4%
(w/v) paraformaldehyde dissolved in PBS+. After either 10 minutes
(for MIC4 and ROP2) or 30 minutes (for GRA2) the media and parasites in the
host-containing samples was removed and replaced with fresh DLS-D10 for either
1 hour or 6 hours. At the appropriate time, these samples were rinsed with PBS
and fixed. The autofluorescence in all samples was quenched by a 10 minute
incubation in 50 mM NH4Cl dissolved in PBS+. The samples
were permeabilized with 0.05% (w/v) saponin dissolved in PBS containing 0.2%
(v/v) fish skin gelatin (PBS/FSG/sap) for 10 minutes. The permeabilized
samples were rinsed and incubated in a humidified chamber for 1 hour at
37°C in PBS/FSG/sap in which one of the following antibodies was diluted
to a final IgG concentration of 1 µg/ml: pAb anti-ROP2, pAb WU1228
(anti-GRA2), or mAb 5B1 (anti-MIC4) ascites. The coverslips were rinsed
extensively in PBS/FSG/sap and stained with Cy5-conjugated anti-mouse or
anti-rabbit antibodies for 30 minutes at 37°C. Alternatively, coverslips
not incubated with primary antibodies were labeled with either the nucleic
acid stain TOPRO3 or the lipophilic dye Nile red for 30 minutes at 37°C.
Finally, all coverslips were rinsed, mounted in Vectashield, and confocal
images collected as described above.
Electron microscopy
Host cells were inoculated with parasites at an MOI of 20 and replaced into
a tissue culture incubator. After 6 hours, the media and uninvasive parasites
were replaced with DLS-D10 containing NBD-cholesterol liposomes. This solution
was removed 4 hours before host cell lysis and substituted with DLS-D10.
The lysed-out parasites were collected, forced through 20 G and 25 G needles,
and purified by passage through a 3 µm pore filter. Parasites were
resuspended in DLS-D10 and deposited onto thermanox coverslips seeded with
host cells. After 1 hour at 37°C, all coverslips were rinsed in PBS and
fixed for 1 hour at 4°C with chilled 4% (w/v) paraformaldehyde, 2% (w/v)
glutaraldehyde (both from Polysciences, Warrington, PA) dissolved in
PBS+. Coverslips were rinsed with 125 mM Pipes buffer (4x5
minutes). The samples were then incubated for 10 minutes in diaminobenzadine
(DAB) at a concentration of 1.5 mg/ml in 0.1 M Tris pH 7.6. The coverslips
were placed cell-side up in DAB solution on a slide and covered with a second,
larger coverslip resting on nail polish posts. This assembly was immediately
subjected to photoactivation: the light from the FITC filter cube of a Zeiss
Axioskop epifluorescence microscope was focused on the parasites/HFFs with the
10x dry objective until fluorescence was undetectable. Samples were
washed in 100 mM Pipes, pH 7 and fixed in a freshly prepared mixture of 1%
(w/v) glutaraldehyde and 1% (w/v) osmium tetroxide (Polysciences, Warrington,
PA) in 100 mM Pipes buffer at 4°C for 30 minutes. The samples were then
rinsed extensively in dH2O prior to en bloc staining with 1% (w/v)
aqueous uranyl acetate (Electron Microscopy Sciences, Ft. Washington, PA) for
3 hours at 4°C. Following several rinses in dH2O, samples were
dehydrated in a graded series of ethanol and embedded in Eponate-12 (Ted
Pella, Redding, CA). Sections of 70-80 nm were cut, stained with uranyl
acetate, and viewed on a JEOL 1200 EX transmission electron microscope.
Thin layer chromatographic analyses of lipid acquisition
Thin layer chromatography was performed on lipid-labeled extracellular
parasites, uninfected hosts, uninfected host elements potentially
contaminating purified parasite preparations, parasites exposed to lipid
subsequent to the establishment of a parasitophorous vacuole, and parasites
allowed to invade hosts previously exposed to lipid.
Immediately subsequent to labeling and infection/mock infection, fresh DLS-D10 was added to each culture well and the cultures returned to the incubator until infected cell lysis. The uninfected, scraped monolayers or infected culture debris was forced sequentially through 20 G and 25 G needles. One set of uninfected hosts was reserved as the host sample until the following centrifugation step. In order to provide a mock-infected (ctl) sample representing host cell lipids contaminating the purified parasite (TL and LT) samples, another set of uninfected hosts was passed through a 3 µm pore polycarbonate filter and reserved until the following centrifugation step. Finally, infected culture debris was passed through a 3 µm pore polycarbonate filter, resulting in a purified parasite preparation. All samples were pelleted by centrifugation at 1200 g for 10 minutes. The pellets were resuspended in PBS, transferred to microfuge tubes, and pelleted again by centrifugation at 11,000 g in a swinging bucket rotor (10 minutes, 4°C). The resulting pellet was resuspended in either 15 µl (for radioactive experiments) or 100 µl (for fluorescence experiments) of 150 mM NaCl. Samples were then assayed for protein concentration using the BCA protein assay (Pierce, Rockford, IL) according to the manufacturer's instructions. Sample spotting onto silica gel 60 TLC plates was normalized for protein concentration (with the exception of the host cell contaminant samples, which were spotted with a volume equal to that of the lysed-out parasite samples). BODIPY-fatty-acid-labeled samples were resolved by developing TLC plates in hexane/ether/acetic acid (70:30:1), whereas NBD-cholesterol-labeled samples were resolved by developing first with hexane/ether/acetic acid (70:30:1), then with toluene/acetone (70:10), and finally with chloroform/acetic acid (96:4), allowing the plates to dry between each development. The dried fluorescent plates were imaged using a gel documentation camera (Alpha Innotech, San Leandro, CA) and a UV transilluminator following the completion of each development step. Plates spotted with radioactive samples were developed in chloroform/methanol/acetic acid/water (25:15:4:2), dried, sprayed with the fluorographic agent En3Hance (Perkin Elmer, Boston, MA), and exposed to X-ray film at -80°C. Densitometric analyses of digitalized autoradiograms were performed using AlphaImager 2000 software (Alpha Innotech, San Leandro, CA).
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Results |
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Host cell neutral lipids are metabolized by intracellular T.
gondii
The concentration of cholesterol and fatty acids by T. gondii
(Fig. 1; and data not shown)
raised the question of whether stored lipids were substrates for modification.
To investigate the metabolism of acquired neutral lipids, potential
metabolites of NBD-cholesterol and the BODIPY-conjugated fatty acids C4C9 and
C12 (differing principally in the position of the fluorophore) were resolved
by thin layer chromatography (TLC) (Fig.
2). Although integrated into host cell membranes, neither
NBD-cholesterol nor BODIPY-C12 served as metabolic substrates
(Fig. 2, cholesterol and C12
host lanes). Remarkably, T. gondii not only acquired these probes
from its host, but also metabolized them
(Fig. 2, TL and LT lanes).
Host-derived cholesterol was modified by the parasites to generate two faster
migrating species that probably represent one or more cholesteryl esters
(Fig. 2, cholesterol TL and LT
lanes, asterisks), consistent with the recent discovery of
acyl-CoA:cholesterol acyltransferase (ACAT) activity in T. gondii
(Sonda et al., 2001). While
parasites contained C4-BODIPY-C9 lipids that migrated similarly to those of
host cells (Fig. 2, C4C9 lanes,
arrowheads), BODIPY-C12 was perceptibly altered only by the parasites to
generate two faster migrating products
(Fig. 2, C12 lanes,
arrowheads). These experiments therefore establish that Toxoplasma
can metabolize some neutral lipids acquired from the host.
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T. gondii sequester host-cell-derived lipids in a
non-secretory compartment
The prospect that lipid constituents of the large puncta were mobilized by
T. gondii was investigated by confocal microscopy. The profile of the
parasite secretory organelles was compared with that of C4-BODIPY-C9 in
parasites freshly egressed from host cells, those very briefly established in
an intracellular niche, and those that had modified their parasitophorous
vacuole and replenished their secretory organelles
(Fig. 3). Whereas the lipid
puncta were dispersed throughout the parasites, the microneme protein MIC4 in
freshly egressed, newly invaded, and established intracellular parasites was
polarized to the parasite apex (Fig.
3, MIC4 panels). When the relationship between the rhoptry protein
ROP2 and the lipid puncta was assessed, under no circumstance did ROP2
colocalize with C4-BODIPY-C9; instead ROP2 contoured the apical region of
extracellular parasites or delineated the PVM of intracellular parasites
(Fig. 3, ROP2 panels).
Likewise, C4-BODIPY-C9 staining was in all cases distinct from that of the
dense granule protein GRA2, which demarcated dense granules in extracellular
parasites and PVs surrounding intracellular parasites
(Fig. 3, GRA2 panels). Notably,
within parasites that had replicated intracellularly, the fluorescent lipids
appeared in organelles reminiscent of the ER and Golgi
(Fig. 3, 6h infection panels).
These observations established that the puncta represent a unique
non-secretory organelle, that the concentrated lipids were not redistributed
en masse to replenish the secretory organelles, and that lipids were gradually
mobilized from the puncta to other organelles.
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Given the discrete staining pattern of C4-BODIPY-C9, it was important to
determine whether these puncta represented the apicoplast, a site of fatty
acid metabolism in apicomplexans (Waller
et al., 1998; Waller et al.,
2000
). When apicoplast DNA was visualized using the nucleic acid
probe TOPRO3, none of the C4-BODIPY-C9 puncta coincided with the singular
apicoplast (Fig. 3, DNA
panels).
Nile Red has been widely used to detect neutral lipid stores, and is
considered the hallmark of lipid bodies in higher eukaryotes
(Greenspan et al., 1985). When
the staining patterns of Nile Red and C4-BODIPY-C9 were examined, partial
overlap under all circumstances was conspicuous
(Fig. 3, Nile Red panels). Nile
Red prominently decorated lipid bodies and to a lesser extent the ER membranes
of both host cells and parasites, the PVM and plasma membrane of briefly
intracellular parasites, and the plasma membrane of established parasites.
Based upon the colocalization of C4-BODIPY-C9 with the sites most intensely
stained by Nile Red, the C4-BODIPY-C9-containing puncta were judged to be
lipid bodies. Lipid bodies are found in a variety of other eukaryotes, where
they are becoming appreciated for their role in cellular fat metabolism
(Ashtaves et al., 2001
;
Blanchette-Mackie et al., 1995
;
Brasaemle et al., 1997
;
Servetnick et al., 1995
).
The T. gondii lipid body was next examined by electron microscopic
(EM) analysis of freshly invaded host cells harboring parasites prelabeled
with NBD-cholesterol liposomes. Upon photoactivation of the NBD moiety in the
presence of diaminobenzidine (Pagano et
al., 1989), an electron-dense precipitate was formed, facilitating
the visualization of the organelle storing NBD-cholesterol. The
low-magnification view revealed a single, intact lipid body in a parasite
immediately surrounded by liposomes and the PVM
(Fig. 4, top-left panel). Upon
closer inspection, this spherical lipid body appeared to be entirely
surrounded by the reaction product (Fig.
4, top-right panel). More often, reaction precipitate surrounded
lipid bodies that were irregular in shape, appeared empty [as previously
reported (Blanchette-Mackie et al.,
1995
)], fractured, and largely extracted, and contained a single
region of precipitate associated with the outer limits of the body
(Fig. 4, bottom panels). These
variations are probably due in part to the preferential incorporation of
NBD-cholesterol in the surface monolayer and the difficulty in preserving
neutral lipid content during EM processing. Regardless of the morphological
variety observed, from this examination it is apparent that
Toxoplasma parasites house lipid bodies that serve as depots for
acquired neutral lipids.
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Intracellular T. gondii preferentially accrue
choline-containing phospholipid species
The diversion of host cell phospholipids, considered together with the
metabolism of host-derived neutral lipids by intracellular T. gondii,
raised the possibility that parasites use host phospholipids/phospholipid
precursors as a source of membrane materials. This prospect was investigated
using three classes of radioactive phospholipid precursors: the head groups
(ethanolamine, serine and choline), phosphatidic acid, and two short-chain
fatty acids (acetic and butyric acids).
A bias of phospholipid/phospholipid precursor diversion by T. gondii was immediately evident upon examination of TLC lanes in which host and parasite lipids were resolved (Fig. 5). Host cells fed [14C]choline accumulated lipids that co-migrated with the PtdCho standard (Fig. 5, choline host lane). Strikingly, parasites isolated from these hosts were comprised of substantial quantities of choline-containing phospholipids (Fig. 5, choline TL and LT lanes). Based on co-migration with the host cell PtdCho as well as the PtdCho standard, these species were tentatively considered to be one or more species of PtdCho. The phosphatidylethanolamine synthesized by hosts fed [14C]ethanolamine was not included in parasite membranes, whereas two spots that co-migrated with PtdCho, presumably formed by the methylation of phosphatidylethanolamine, were noted (Fig. 5, ethanolamine lanes). Parasites grown in [14C]serine-fed hosts also contained similarly migrating species, which were likely generated by the enzymatic modification of serine-labeled host cell phospholipids (Fig. 5, serine lanes). The biased acquisition/metabolism of host cell phospholipids was further indicated upon labeling of cultures with phosphatidic acid, acetic acid and butyric acid. Whereas the efficient uptake of [14C]phosphatidic acid was not coupled with a marked conversion into other phospholipids by hosts, phosphatidic acid was undetectable in parasites, which instead contained a spot co-migrating with PtdCho as the predominant phospholipid species (Fig. 5, phosphatidic acid lanes). Finally, while both [14C]acetic and [14C]butyric acids were anabolized into multiple host phospholipids, they were integrated solely into spots co-migrating with PtdCho in the parasite membranes (Fig. 5, acetic acid and butyric acid lanes). T. gondii thus appears to be particular in its scavenging of lipids and/or precursors from its host.
|
Parasite PtdCho is derived from both parasites and hosts
The relatively low level of diversion of BODIPY-PtdCho from host cells
(Fig. 1), in concert with the
preponderance of radiolabeled PtdCho lipids in parasite membranes
(Fig. 5) intimated that T.
gondii may be competent to metabolize precursors into PtdCho. This was
tested by TLC analysis of host-free parasites incubated at either 2°C or
37°C with radiolabeled phosphatidic acid, choline or acetic acid
(Fig. 6). As expected from its
energy-independent insertion into plasma membranes,
[14C]phosphatidic acid was associated with parasite membranes at
both 2°C and 37°C (Fig.
6, phosphatidic acid panel, EC lanes). However, extracellular
parasites failed to convert [14C]phosphatidic acid into more
complex phospholipids, while a species co-migrating with PtdCho was again
observed within parasites labeled intracellularly
(Fig. 6, phosphatidic acid
panel, compare the 37C lanes). Intracellular parasites fed
[14C]choline converted it into several lipids that co-migrated with
PtdCho standards and which are hence tentatively identified as forms of PtdCho
(Fig. 6, choline panel, IC
lane). Remarkably, host-free parasites not only internalized
[14C]choline at physiological temperature
(Fig. 6, choline panel, EC
lanes), but converted it into one of these lipid species
(Fig. 6, choline panel, compare
the 37C lanes). Moreover, the diffusion of [14C]acetic acid into
extracellular parasites (Fig.
6, acetic acid panel, EC lanes) was succeeded by the incorporation
of two of the three spots co-migrating with PtdCho
(Fig. 6, acetic acid panel,
compare the 37C lanes). These results suggest that T. gondii is able
to synthesize PtdCho from choline and a fatty acid unit.
|
Although extracellular Toxoplasma appears able to manufacture
PtdCho, the extent to which parasites scavenge versus synthesize PtdCho when
they are intracellular remained in question. To address this question, mutant
fibroblasts (89.1) or parental Chinese hamster ovary (CHO) cells infected with
T. gondii were pulsed with [14C]choline and the kinetics
of PtdCho synthesis monitored. 89.1 cells have a moderate deficiency in the de
novo synthesis of PtdCho, owing to a defect in the first enzymatic reaction in
this pathway, the phosphorylation of choline
(Nishijima et al., 1984). This
deficiency was evident in the TLC profiles of uninfected host cells: 89.1
cells were retarded in their synthesis of PtdCho
(Fig. 7, compare H lanes in the
CHO and 89.1 panels). The accumulation of a PtdCho-co-migrating species in
parasites grown in CHO and 89.1 cells also differed
(Fig. 7, compare T lanes in the
CHO and 89.1 panels). Surprisingly, parasites within 89.1 cells actually
accumulated more (two- to threefold, quantitatively) of the radiolabeled
PtdCho than those within CHO cells (Fig.
7, compare H and T lanes in the CHO panel). These results indicate
that when environmental PtdCho resources are limited, resident parasites
adeptly scavenge host cell choline and probably synthesize PtdCho.
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Discussion |
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The Toxoplasma lipid body
The present study substantiates the existence of lipid bodies within T.
gondii and suggests that this organelle has a role in Toxoplasma
lipid homeostasis. Lipid bodies described in plants, animals and yeast are
composed of a monolayer of proteins and polar lipids circumscribing a core of
neutral lipids (reviewed by Murphy and
Vance, 1999). The polar lipids that shuttled from host cell
organelles to the Toxoplasma lipid body (i.e. NBD-cholesterol and the
BODIPY-fatty acids and derivative polar metabolites) are thus predicted to be
stored within the surface monolayer until modified or transported elsewhere.
An active role for the lipid body in the metabolism and trafficking of lipids
has been implicated by the enrichment of several lipid enzymes and
lipid-binding proteins within previously characterized lipid bodies in other
systems (Ashtaves et al., 2001
;
Blanchette-Mackie et al., 1995
;
Brasaemle et al., 1997
). The
parasite lipid body has not yet been confirmed as the site of the lipid
metabolism noted herein. However, trafficking between Toxoplasma
lipid bodies and ER, as noted in other eukaryotes
(Murphy and Vance, 1999
), is
suggested by the gradual disappearance of C4-BODIPY-C9 from the lipid body and
its appearance in the ER and Golgi upon parasite growth and replication. The
Toxoplasma lipid body thus appears to concentrate diverted host cell
lipids, providing a source for the biogenesis of parasite membranes and
possibly serving as a site of lipid metabolism.
The source of Toxoplasma lipids
Toxoplasma parasites acquired lipid resources from the host
regardless of the order of labeling and infection. The order did, however,
impact the compartmentalization of most acquired lipids. Although the reason
behind this observation has yet to be elucidated, in either case the parasites
readily modified host cell lipids. The esterification of acquired
NBD-cholesterol and the modification of acquired BODIPY-fatty acids into
related lipids demonstrates that parasites metabolize host-derived neutral
lipids. Regarding the phospholipids, intracellular parasites metabolize
scavenged choline and appear to convert scavenged phosphatidylethanolamine and
phosphatidylserine into PtdCho, preferentially accruing choline-containing
phospholipids. The manipulation of diverted host cell lipids almost certainly
represents an important mechanism by which Toxoplasma gondii growth
is accommodated.
Toxoplasma does not merely scavenge lipids, but is also capable of
their biosynthesis. T. gondii expresses at least three components of
the type II fatty acid synthesis (FAB II) pathway
(Waller et al., 1998), and a
recent study indicated that interference with a step in this pathway retards
growth of intracellular T. gondii
(McLeod et al., 2001
). The
current demonstration that T. gondii incorporates labeled acetic acid
into fatty acyl chains indicates a capability for fatty acid biosynthesis,
which is presumably mediated by the FAB II pathway. Furthermore, when
considered together with the use of choline, it is likely that the parasite is
fully able to biosynthesize PtdCho, the predominant phospholipid in
Toxoplasma (Foussard et al.,
1991
). The synthesis of less abundant phospholipids is also
probable although not directly demonstrated here. A Toxoplasma
phosphatidylinositol synthase has been described
(Seron et al., 2000
), and the
T. gondii dbEST database of NCBI contains entries with homology to
key enzymes in the synthesis of phosphatidylethanolamine, phosphatidylserine,
and phosphatidylglycerol
(http://www.ncbi.nlm.nih.gov
; GenBank accession numbers BM132998, AW702647, BG657255) Moreover, the
related apicomplexan Plasmodium falciparum contains a full complement
of enzymes that synthesize phospholipids during its residence in the
biosynthetically inactive erythrocyte
(Vial and Ancelin, 1998
).
Studies using information gleaned from the ongoing Toxoplasma genome
project, in conjunction with preparative-scale lipid biochemistry, are
expected to be instrumental in elucidating the precise pathways involved in
Toxoplasma lipid metabolism.
Potential mechanisms of lipid scavenging
The mechanism by which host-cell-derived lipids are transferred across the
PVM to the parasite remains uncertain. A physical interconnection may be
provided by the intravacuolar network, a system of proteins and membranes
constructed by the parasite shortly after entry
(Mercier et al., 1998;
Sibley et al., 1995
). A role
for this extensive membranous interface as a conduit for the transfer of
materials from the host to the parasite is currently under investigation.
There are five potential mechanisms by which the
Toxoplasma-containing PVM acquires host cell lipids. The first of
these is acquisition during invasion, via the inclusion of host plasma
membrane lipids into the forming PVM. Parasites indeed integrate the host
plasma membrane lipids DiIC16 and GM1 into the PVM during invasion
(Mordue et al., 1999a).
However, this possibility is not favored as the sole means of lipid
scavenging, since in the present study parasites readily acquired lipids
provided after the establishment of intracellular residence. Second, it is
conceivable that T. gondii acquires lipids through the interception
of a vesicular transport pathway(s). This possibility can be tentatively
discarded, as T. gondii is segregated from the vesicular transport
routes responsible for the trafficking of lipids as well as proteins, and
vesicular transport carries neither host plasma membrane lipids nor host
lysosomal NBD-cholesterol to PVs (Coppens
et al., 2000
). The third possibility is that lipids passively
diffuse to the parasite. There are several indications that diffusion may play
only a minor role in Toxoplasma lipid acquisition. Diffusion of
cholesterol and PtdCho through aqueous environments (i.e. the host cell
cytosol) is highly unfavorable. Moreover, ablation of the fatty acid
translocase gene in Saccharomyces cerevisiae markedly reduces the
uptake of BODIPY-C12, arguing against diffusion as the central means of
mobilization of this probe (Faergeman et
al., 1997
). Finally, when energy dependent transport processes
were precluded by fixation, diffusion of subsequently added
BODIPY-phosphatidic and -fatty acids to the Toxoplasma-containing
vacuole was undetectable (data not shown). The fourth potential mechanism of
lipid acquisition involves carrier-protein-mediated lipid transfer, which has
been previously invoked to account for the delivery of lysosomal cholesterol
to Toxoplasma (Coppens et al.,
2000
). Although carrier protein-mediated transport is probably
instrumental in parasite lipid acquisition, the differential
compartmentalization of individual lipids (a function of the sequence of lipid
labeling/infection) implied that PVs are not indiscriminant acceptors of
carrier-coupled lipids.
The close opposition of host cell organelles with the PVM suggests an
additional mechanism for PVM lipid acquisition: direct interorganelle transfer
(Sinai and Joiner, 2001). The
biochemical exchange between the mitochondrial-associated membrane (MAM) and
mitochondria provides an analogy with which to consider direct transfer of
host lipids to the Toxoplasma PVM. Lipids manufactured in the ER must
transit from the ER to mitochondria, organelles that, like the PVM and host
cell compartments, are not coupled by vesicular transport
(Sprong et al., 2001
). This
quandary has been remedied by the specialization of the ER in a region known
as the MAM. Lipid transfer from the MAM takes place via translocators that
deliver phospholipids to the mitochondrial membranes for further modifications
(Shiao et al., 1995
). PVMs
also physically associate with the host cell mitochondria and ER, doing so
immediately after invasion, owing to the anchoring of a secreted parasite
protein into both the PVM and mitochondrial membrane
(Sinai and Joiner, 2001
;
Sinai et al., 1997
). Lipid
mobilization to the parasite may thus be facilitated by the physical
association of the PVM and host cell organelles. If so, the specific
recruitment of host organelles by the PVM may be an important adaptation for
lipid acquisition that facilitates intracellular Toxoplasma
growth.
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Acknowledgments |
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References |
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