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INTRODUCTION |
Upon invasion of a host cell, the apicomplexan parasite
Toxoplasma gondii resides in a specialized compartment, the
parasitophorous vacuole
(PV).1 The PV is unique
because it is predominantly derived from the host cell plasma membrane
(1) but is devoid of host cell transmembrane proteins (2, 3) and does
not fuse with endocytic or exocytotic vesicles of the host cell (4-7).
After invasion, T. gondii rapidly modifies the PV membrane
by secreting proteins stored in rhoptries and dense granules
(7-13).
Despite the apparent segregation of the PV from the host cell endocytic
network, metabolites essential for the parasite are known to exchange
with the intravacuolar space. Aqueous soluble molecules of less than
1300 daltons enter the vacuole through non-selective pores in the PV
membrane, thus satisfying the parasite's auxotrophy for purines and
tryptophan (14, 15).
Much less is known about the uptake and metabolism of lipids by
T. gondii. Since proteins involved in lipid biosynthesis
such as acyl carrier proteins and subunits of type II fatty-acyl
synthase have been shown to localize in the apicoplasts, the plastid
organelle may play a role in the lipid metabolism of T. gondii (16). T. gondii membranes contain cholesterol
and are characterized by a low cholesterol/phospholipid ratio and
consequent high level of fluidity (17, 18). It has been shown recently
that the parasite depends on host cell cholesterol derived from
endocytosed LDL and not from endogenous synthesis (19). However, the
exact mechanism of cholesterol uptake by intravacuolar parasites has not been completely elucidated.
An essential enzyme for the regulation of intracellular cholesterol
homeostasis is acyl-CoA:cholesterol acyltransferase 1 (ACAT-1). ACAT-1
covalently joins excess free cholesterol (FC) with fatty acyl-CoA to
form cholesterol esters (CE), which can then be stored in the form of
cytoplasmic lipid droplets. The enzyme is a transmembrane protein
located mainly in the endoplasmic reticulum and perinuclear region (20)
and functions as a homotetramer (21) with the active site apparently
oriented toward the cytoplasm (22). Whereas ACAT-1 is expressed
ubiquitously in mammalian tissues (23, 24), a related enzyme, ACAT-2,
is expressed in the liver and small intestine and is involved in
absorption of dietary cholesterol (25, 26).
Because the parasite depends on cholesterol acquired by host cell
endocytosis but inhabits a vacuole that does not interact with host
cell endocytic vesicles, one or more intermediate steps of cholesterol
uptake by the parasite must exist and remain to be identified. Because
cholesterol esters formed by ACAT are an effective way to store
cholesterol in the cytoplasm, we reasoned that ACAT activity with its
role in cholesterol ester formation and cellular cholesterol
homeostasis plays a role in the optimal parasite proliferation. To test
this hypothesis, we analyzed T. gondii replication in cells
where ACAT enzyme activity was absent through either genetic deletion
or pharmacologic inhibition.
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EXPERIMENTAL PROCEDURES |
Chemicals--
Unless otherwise stated, all reagents were
purchased from Sigma. The acyl amide ACAT inhibitors SaH 58-035 and CI
976 were obtained from Sandoz Inc., East Hanover, NJ, and Parke-Davis, respectively. Stock solutions of inhibitors were prepared at a concentration of 2 mM in Me2SO and were
freshly diluted to the concentrations required for each experiment.
Radiolabeled lipids were purchased from Amersham Pharmacia Biotech.
Cell Types, Parasite Culture, and Purification--
The cell
types used for infection assays with T. gondii were human
foreskin fibroblasts (HFF), murine peritoneal fibroblasts (MPF), and
murine embryonic fibroblasts (MEF) derived from ACAT-1+/+
and ACAT-1
/ mice, which were obtained by targeted gene
disruption (27). MPF were isolated by a combination of mechanical
separation and collagenase digestion. Under sterile conditions, the
anterior abdominal wall was exposed and removed en bloc to a
Petri dish containing a solution of 0.02% collagenase (Crescent
Chemical Co., Hauppauge, NY) in Dulbecco's modified Eagle's medium
(Mediatech, Herndon, VA). The peritoneal surface was scraped gently
with a razor blade to release lining cells, and the remaining tissue was finely minced. The entire mixture was then transferred to a shaking
water bath at 37 °C for 30 min, pelleted at 1,000 × g, and grown to confluence in 75-cm2 tissue
culture flasks in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Life Technologies, Inc.).
HFF and MPF cultures were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum, 2 mM
glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin at
37 °C, 5% CO2. MEF cultures were maintained in RPMI
1640 medium (Life Technologies, Inc.) with the same supplements
described above.
Cultures were passaged by trypsinization at least once a week, and
cells were used up to the 20th passage.
T. gondii tachyzoites of the 
RH strain were used for
infection. This strain expresses Escherichia coli
-galactosidase under control of the T. gondii sag1
promoter (28) and has been used previously to assay parasite
proliferation (29) Parasites were harvested from host cells, passed
through a 27-gauge needle, and purified as described (30). Purified
tachyzoites were counted in a hemocytometer chamber and used for a new
cycle of host cell invasion at the multiplicity of infection (m.o.i.)
stated in the individual figure legends.
Determination of Parasite Proliferation--
T.
gondii was quantitated in infected cells by colorimetric detection
of -galactosidase activity expressed by the parasite strain RH as
described (29). Briefly, either ACAT-1+/+ MEF,
ACAT-1/ MEF or HFF were grown to confluence in 96-well
plates in medium lacking phenol red, and HFF were treated for 12 h
with different concentrations of SaH 58-035, CI 976, or solvent alone.
Cells were then infected with purified parasites at m.o.i. 0.1. After 72 h, 10 µl of the -galactosidase substrate chlorophenol red -D-galactopyranoside (Roche Molecular Biochemicals) was
added to the culture medium to give a final concentration of 100 µM. Following an additional incubation at 37 °C, 5%
CO2 for 1-4 h until color development, plates were
read at a dual wavelength of 570/630 nm using a Spectra MAX 250 microplate reader (Molecular Devices, Sunnyvale, CA). In some
experiments, parasites were directly counted following nuclear staining
using a Leica DMRB fluorescence microscope (Deerfield, IL). Consistent
results of parasite quantitation were obtained by using both the
colorimetric and the direct counting methods.
T. gondii Invasion Assay--
ACAT-1/ MEF and
HFF were grown to confluence on polylysine-coated glass coverslips, and
HFF were treated with 40 µM SaH 58-035, CI 976, or
solvent alone for 12 h. Purified T. gondii tachyzoites were allowed to infect the cells for 1 h at m.o.i. of 0.1. Extracellular parasites were removed by washing the coverslips in PBS,
and cells were immediately fixed in 4% paraformaldehyde (PFA) or
incubated for an additional 24 h prior the fixation. Parasites
were then counted following nuclear staining using a Leica DMRB
fluorescence microscope (Deerfield, IL).
ACAT Activity Assay--
Cholesterol esterification activity was
assessed by pulse labeling of cells as described (31) using
radiolabeled fatty acid as substrate. Briefly, ACAT-1+/+
MEF, ACAT-1/ MEF, and HFF were grown to confluence in
12-well plates, and HFF were treated with SaH 58-035, CI 976, or
solvent alone. ACAT activity was stimulated for 5 h by addition of
5 µg/ml 25-hydroxycholesterol plus 10 µg/ml cholesterol, and cells
were then pulse-labeled for 2 h with 20 µl of sodium
[14C]oleate-albumin complex (10 mM sodium
[14C]oleate, 120 mg/ml albumin, ~10,000
dpm/nmol). After washing with ice-cold 0.2% albumin in PBS followed by
PBS alone, lipids were extracted at room temperature with
hexane/isopropyl alcohol (3:2) for 30 min, and the organic
solvents were evaporated under a N2 stream. 10 µl of
[3H]cholesteryl oleate (~22,000 dpm) was added as an
internal standard. Lipids were then resuspended in 50 µl of
chloroform, spotted on TLC silica gel G plates (Fisher), and separated
in hexane/ethyl ether/glacial acetic acid (80:20:1). Cholesterol ester
bands were identified with iodine vapors, scraped into scintillation
vials, and counted in 5 ml of scintillation fluid (Research Products Division, Costa Mesa, CA). The amount of cholesterol esters was expressed as pmol 14C/min/mg protein.
For ACAT activity assay in T. gondii, tachyzoites were
harvested from ACAT-1/ MEF after 2 lysis cycles, washed
in PBS to remove cellular debris, and aliquots of 107
parasites tested for incorporation of [14C]oleate into
cholesterol esters as described above.
Different concentrations of SaH 58-035 and CI 976 or solvent alone were
added to the parasites during the ACAT stimulation with
25-hydroxycholesterol and cholesterol; the total time of inhibitor
treatment of T. gondii tachyzoites was limited to 7 h
to maintain the viability of extracellular parasites.
Host Cell Viability Assay--
Cell viability was tested using
the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay
(Promega, Madison, WI). Briefly, HFF were grown to confluence in
96-well plates, treated with different concentrations of SaH 58-035, CI
976, or solvent alone for 72 h, and processed according to the
instructions provided by the manufacturer. Absorbance was measured at
wavelength of 490 nm using the microplate reader described previously.
Determination of ATP Content--
ACAT-1+/+ MEF,
ACAT-1/ MEF, and HFF were grown to confluence in
96-well plates, and HFF were treated with different concentrations of
SaH 58-035, CI 976, or solvent alone for 72 h. Cells were washed in PBS and lysed for 30 min at 4 °C in 75 µl of lysis buffer (0.1 M potassium phosphate, pH 7.6, 1% Triton X-100, 1 mM dithiothreitol, 2 mM EDTA). After
centrifugation at 2600 × g for 20 min, 20 µl of
supernatant was collected and added to 100 µl of firefly
luciferase/luciferin solution (Sigma). Bioluminescence was detected
with a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA), and ATP
concentration of the samples was calculated by comparison with an ATP
standard curve.
Determination of Free Cholesterol and Cholesterol Ester
Content--
ACAT-1+/+ MEF, ACAT-1/ MEF,
and HFF were grown to confluence in 12-well plates, and HFF were
treated for 72 h with different concentrations of SaH 58-035, CI
976, or solvent alone. After washing in 0.2% albumin in PBS, cellular
lipids were extracted and separated by TLC using hexane/ethyl
ether/acetic acid (80:20:1) or chloroform/n-heptane (40:60)
for developing FC and CE, respectively. Bands of lipids were visualized
using a solution of 3% cupric acetate in 15% aqueous phosphoric acid
followed by charring of the plates at 120 °C for 15 min. The
intensity of the bands was analyzed with an Alpha Imager 2000 (Alpha
Innotech Corporation, San Leandro, CA), and the FC and CE amounts were
determined by comparison with standard curve.
LDL Receptor Analysis--
For immunoblot analysis, total
extracts of ACAT-1+/+ MEF, ACAT-1/ MEF, and
ACAT inhibitor-treated HFF were obtained by trypsinizing and
resuspending confluent monolayers in SDS sample buffer in the absence
of reducing agents. Proteins (5 µg) from total extracts were
separated by SDS-polyacrylamide gel electrophoresis in a precast
gradient gel (4-15% acrylamide, Bio-Rad) and transferred to
nitrocellulose filters. After blocking of unspecific binding sites with
TBS containing 5% skim milk and 0.3% Tween 20, bands were incubated
with a rabbit antiserum recognizing the LDL receptor (32) and
visualized using horseradish peroxidase-conjugated anti-rabbit
antibodies and the RenaissanceR Chemiluminescence Reagent
Plus (PerkinElmer Life Sciences). Polyclonal antibodies against annexin
I were used to normalize the amount of proteins present in the gel
(33).
For flow cytometry analysis, confluent monolayers of
ACAT-1+/+ MEF, ACAT-1/ MEF, and ACAT
inhibitor-treated HFF were trypsinized, washed in chilled PBS, and
resuspended in PBS with 0.2% bovine serum albumin. Aliquots of
106 cells were incubated on ice with the previously
described anti-LDL receptor antibodies, followed by incubation with
fluorescein isothiocyanate-conjugated anti-rabbit antibodies. Five
thousand cells were analyzed for LDL receptor expression on a FACSort
flow cytometer with CellQuest software (Becton Dickinson, Mountain
View, CA). Background fluorescence of the secondary antibodies alone
was subtracted, using the CellQuest software, from sample fluorescence
before analyzing the mean fluorescence.
Lipid Staining and Fluorescence Analysis--
For staining of
neutral lipids in T. gondii, purified tachyzoites were
applied to poly-lysine-coated glass coverslips, fixed in 4% PFA, and
incubated at room temperature for 30 min in 5 µg/ml Nile Red in PBS
(stock solution 0.5 mg/ml in acetone). Coverslips were then
rinsed in PBS and mounted in glycerol for fluorescence microscopy
employing a filter set for fluorescein (450-500 nm band pass
excitation filter) for yellow-gold fluorescence.
A similar procedure was used for cytochemical staining of cellular free
cholesterol with the antibiotic filipin at concentration of 50 µg/ml
in PBS (stock solution 2.5 mg/ml in dimethyl formamide), using an
excitation filter of 350-410 nm.
Determination of Protein Concentration--
Protein content was
determined using the BCA Protein Assay Kit (Pierce) according to the
instructions provided by the manufacturer. Bovine serum albumin was
used for the standard curve.
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RESULTS |
Absence or Inhibition of ACAT Activity Decreases Parasite
Replication--
To determine whether T. gondii replication
is affected by alteration in cholesterol ester formation, tachyzoites
were used to infect either MEF deficient for ACAT-1 or HFF treated with ACAT inhibitors SaH 58-035 and CI 976 (34) (Fig.
1A). In
ACAT-1/ MEF, parasite replication was reduced by 60%
compared with that in ACAT-1+/+ MEF. In addition, the two
structurally distinct pharmacological ACAT inhibitors, SaH 58-035 and
CI 976, exerted a dose-dependent inhibition of T. gondii replication in HFF. This anti-T. gondii effect
was also present when the ACAT inhibitor treatment followed the
T. gondii infection of the host cells (Fig. 1B),
suggesting that an aberrant initial formation of the PV is not the
cause of the observed reduction in parasite replication. The lack of cholesterol esterification impaired parasite replication, but it did
not kill the parasites. In both ACAT-1/ and
inhibitor-treated cells, T. gondii tachyzoites were able to
induce host cell lysis. However, this process was delayed by an average
of 48 h compared with that in cells with normal ACAT activity.
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Fig. 1.
Optimal replication of T. gondii
requires ACAT activity. A, MEF from
ACAT-1+/+ or ACAT-1/ mice were incubated
with purified T. gondii tachyzoites of the RH strain at
an m.o.i. of 0.1. After 72 h, live parasites were quantitated by
measuring the parasite -galactosidase activity. HFF were treated for
12 h with the indicated concentrations of ACAT inhibitors SaH
58-035 or CI 976 and then infected and assayed in the same manner as
MEF. B, HFF were treated with 40 µM ACAT
inhibitors beginning 12 h before T. gondii infection
(pretreatment) or 2 h after T. gondii infection
(posttreatment) and assessed as described. Data are expressed as a
percent of control (ACAT-1+/+ MEF or Me2SO
(DMSO)-treated HFF) ± S.E. from three experiments done
in triplicate.
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Absence or Inhibition of ACAT Activity Does Not Affect Parasite
Invasion--
The reduction in parasite number seen in cells lacking
ACAT activity could have been a consequence of decreased invasion
efficiency, decreased parasite replication, or both. In order to assess
whether the alteration in cholesterol ester metabolism compromises the ability of T. gondii to enter host cells, an invasion assay
was performed. The number of intracellular parasites in both
ACAT-1/ MEF (Fig.
2A) and inhibitor-treated HFF
(Fig. 2B) was comparable to the number of parasites in
control cells at 1 h postinfection but was significantly decreased
at 24 h postinfection. Therefore, it appears that ACAT activity is
not required for efficient invasion of host cells by T. gondii but for subsequent intracellular replication. Deficiency or
inhibition of ACAT results in an increase in the doubling time of
intracellular tachyzoites from between 6 and 8 h to over 10 h
(data not shown).
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Fig. 2.
ACAT absence or inhibition does not inhibit
T. gondii invasion. A,
ACAT-1+/+ and ACAT-1/ MEF were grown on
glass coverslips and then infected with purified T. gondii
tachyzoites of the RH strain at an m.o.i. of 0.1. After incubation
at 37 °C for 1 h, free parasites were removed by washing in
PBS, and cells were fixed in 4% PFA immediately or after an additional
incubation of 24 h. 4,6-Diamidino-2-phenylindole nuclear staining
was used for counting intracellular parasites in at least 300 host
cells. Results represent mean ± S.E. (n = 5) from
three different experiments. B, HFF were grown on glass
coverslips and treated for 12 h with 40 µM SaH
58-035, CI 976, or solvent alone prior to infection and assayed as
described for MEF.
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To determine whether the proliferation of parasites in cells with
compromised cholesterol esterification impairs the ability of T. gondii to accomplish additional cycles of invasion, parasites were
isolated from ACAT-1/ MEF or inhibitor-treated HFF
after two lysis passages and allowed to invade and proliferate in
untreated HFF. We found that parasites isolated from cells with
compromised cholesterol esterification were as efficient at invading
and replicating in HFF as were parasites isolated from control cells
(not shown). Therefore, alteration of cholesterol esterification does
not permanently impair intracellular cycles of T. gondii.
SaH 58-035 and CI 976 Inhibit ACAT in HFF Without Host Cell
Toxicity--
While the impairment of T. gondii replication
in both ACAT-1/ MEF and in ACAT inhibitor-treated HFF
implied that the lack of ACAT activity was responsible for the
anti-T. gondii effect, it was necessary to obtain direct
evidence in support of this mechanism. Therefore, we examined the ACAT
enzymatic activity in these cells, measured as incorporation of
radiolabeled fatty acid into cholesterol esters. The amount of newly
formed cholesterol esters was decreased by 
95% in
ACAT-1/ MEF compared with control ACAT-1+/+
MEF. Similarly, the formation of new cholesterol esters was inhibited by 95% in HFF treated with 1.0 µM or higher
concentrations of either SaH 58-035 or CI 976 (Fig.
3). This finding is consistent with the
published observation that SaH 58-035 and CI 976 inhibit cholesterol
esterification in other cell types with IC50 values of
average 0.5 µM (34, 35).
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Fig. 3.
ACAT activity assay.
ACAT-1+/+ and ACAT-1/ MEF were pulsed for
2 h with [14C]oleic acid and the lipids extracted.
After TLC separation, bands corresponding to CE were counted for the
presence of radioactive 14C. HFF were treated for 72 h
with ACAT inhibitors at the indicated concentrations and similarly
analyzed. Data are expressed as percent of control
(ACAT-1+/+ MEF or Me2SO-treated HFF) ± S.E. from three separate experiments done in duplicate.
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The observation that the anti-T. gondii effect of ACAT
inhibitors required significantly higher concentrations of the
inhibitors than those required for maximal inhibition of cellular ACAT
activity raised the possibility that the ACAT inhibitors exerted their effects on parasite replication through nonspecific host cell toxicity.
Although neither visual inspection of the morphology nor trypan blue
staining of HFF indicated obvious host cell toxicity at concentrations
of inhibitors that slowed T. gondii replication, we further
examined host cell toxicity using two distinct biochemical assays.
First, we used a colorimetric assay of cell viability based on
functionality of dehydrogenase enzymes found in metabolically active
cells. This revealed that host cell metabolism was not affected by the
presence of the inhibitors except at the highest dose (100 µM) of CI 976, a concentration much higher than that required to reduce T. gondii replication (data not shown).
Some morphologic alteration of the cells and loss of adherence were also observed at this concentration of CI 976. Second, we assayed the
ATP content of host cells to determine whether ACAT inhibitors reduced
the proliferation of T. gondii by blocking the production of
this host cell metabolite on which T. gondii is known to
depend (15, 36). ATP levels were not reduced in ACAT-1/
MEF or inhibitor-treated HFF, indicating that ATP depletion did not
account for the anti-T. gondii effect (data not shown).
Increases in Intracellular Free Cholesterol Content Are Not
Responsible for the Anti-T. gondii Effect--
The anti-T.
gondii effect observed in the absence of cholesterol
esterification could have resulted from accumulation of free cholesterol that is known to be toxic to mammalian cells (37). Therefore, we examined the free cholesterol content in both
ACAT-1/ MEF and ACAT inhibitor-treated HFF.
Quantitative analysis of free cholesterol by TLC showed that the free
cholesterol content did not increase in the absence of ACAT activity
(data not shown). In addition, cells were stained for free cholesterol
using the fluorescent polyene antibiotic filipin and examined by
fluorescence microscopy. Fluorescence did not increase in cells with
compromised ACAT activity compared with control cells (not shown). This
indicates that homeostatic mechanisms used by mammalian cells to
maintain the cholesterol at physiological levels are effective when
ACAT activity is reduced in cultured cells and provide evidence that the anti-T. gondii effect of deficiency of ACAT is not
mediated by toxicity of excess free cholesterol (38, 39).
Down-regulation of LDL Receptor Is Not Responsible for the Anti-T.
gondii Effect--
Possible cellular mechanisms to maintain the free
cholesterol at physiological levels are efflux of free cholesterol (40, 41) and regulation of the enzymes involved in cholesterol and fatty
acid synthesis, as well as LDL uptake (42, 43).
Since it has been shown that receptor-mediated endocytosis of plasma
LDL in the host cell is a key element for intravacuolar proliferation
of T. gondii (19), we asked whether the inhibition of the
enzymatic ACAT activity might cause a decrease in LDL endocytosis through down-regulation of LDL receptors and consequent decrease in
parasite proliferation. To examine this possibility, the presence of
LDL receptor was analyzed by Western blot in ACAT-1/
MEF (Fig. 4A) and
inhibitor-treated HFF (Fig. 4B). The expression of the LDL
receptor did not significantly decrease in cells lacking ACAT activity
compared with the amount observed in control cells. To assess the
amount of LDL receptor present on the surface of the host cells,
inhibitor-treated HFF were incubated at 4 °C with an antiserum
recognizing the LDL receptor and subsequently analyzed by flow
cytometry (Fig. 4C). Surface LDL receptor expression was similar in control and inhibitor-treated cells, confirming that decreased LDL receptor expression did not account for the anti-T. gondii effect.
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Fig. 4.
LDL receptor in cells lacking ACAT
activity. 5 µg of total extracts from confluent monolayers of
MEF (A) and ACAT inhibitor-treated HFF (B) were
separated in a 4-15% gradient gel, blotted, and probed with
polyclonal antibodies against the LDL receptor and annexin-I. Some
degradation product of the LDL receptor is visible (arrow).
A, lane 1, ACAT-1+/+ MEF; lane
2, ACAT-1/ MEF. B, lane 1, untreated HFF; lane 2, HFF treated with 1 µM
SaH 58-035; lane 3, HFF treated with 40 µM SaH
58-035; lane 4, HFF treated with 1 µM CI 976;
lane 5, HFF treated with 40 µM CI 976. C, inhibitor-treated HFF were also incubated with polyclonal
antibodies against the LDL receptor, followed by fluorescein
isothiocyanate-conjugated anti-rabbit antibodies, and analyzed by flow
cytometry. Mean fluorescence is expressed as percent of control
(Me2SO-treated HFF) ± S.E. from two experiments done
in duplicate.
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Alteration in Cholesterol Ester Content in Cells Lacking ACAT
Activity--
Previously we demonstrated that the treatment with ACAT
inhibitors causes an efficient block in the formation of new
cholesterol esters in the host cell (Fig. 3). However, it is possible
that even in the presence of ACAT inhibitors residual stores of
cholesterol esters are still present in the host cytoplasm in form of
lipid droplets. To determine whether 72 h of inhibitor treatment
was sufficient to deplete the host cells of cholesterol esters, we performed a direct quantitation of CE. As shown in Fig.
5, the amount of CE was virtually absent
as expected in ACAT-1/ MEF, whereas it decreased but
was still detectable in inhibitor-treated HFF. This suggested that the
absence of cholesterol esters in the host cell is likely to be the
direct cause of the anti-T. gondii effect in
ACAT-1/ MEF, but additional mechanisms might be
required for the anti-T. gondii effect in inhibitor-treated
HFF cells.
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Fig. 5.
Cholesterol ester content in cells lacking
ACAT activity. Cellular lipids were extracted from
ACAT-1/ MEF and ACAT inhibitor-treated HFF, separated
on TLC plates, and stained with a solution of 3% cupric acetate in
15% aqueous phosphoric acid. After incubation of the plates at
125 °C the bands co-migrating with standard CE were
densitometrically quantified. Data are expressed as percent of control
(ACAT-1+/+ MEF or Me2SO-treated HFF) ± S.E. from two experiments done in duplicate.
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Evidence of Endogenous ACAT Activity in T. gondii--
The
presence of an ACAT homologue in T. gondii with a lower
sensitivity to the ACAT inhibitors might explain the difference in the
dose-response of the anti-T. gondii effect of the inhibitors and their potency for inhibiting ACAT activity in HFF. To determine whether T. gondii tachyzoites are actually capable of
esterifying cholesterol, we monitored the formation of new cholesterol
esters by incubating cell-free tachyzoites with radiolabeled oleic
acid, followed by lipid extraction and TLC analysis. Purified parasites were also treated with ACAT inhibitors and tested for decreases in
cholesterol esterification. For this experiment, T. gondii tachyzoites were isolated from ACAT-1/ MEF in order to
exclude contamination with ACAT activity derived from the host cell. As
shown in Fig. 6, free T. gondii tachyzoites were clearly able to incorporate radiolabeled
oleic acid into cholesterol esters. Moreover, cholesterol
esterification by free tachyzoites was sensitive to inhibition by SaH
58-035 and CI 976, showing a dose-response closely resembling the
anti-T. gondii effect observed in HFF. The sensitivity to
ACAT inhibitors was strikingly lower in T. gondii
tachyzoites than in HFF cells, whereas inhibitor concentrations 10
µM were required to reduce cholesterol esterification by
the parasites (Fig. 6A), inhibitor concentration of 1 µM was sufficient to inhibit ACAT activity of HFF (Fig.
6B). To test the possibility that ACAT inhibitors impair
parasite replication by inhibiting the parasite ACAT, we treated
peritoneal fibroblasts derived from ACAT-1/ mice with
different concentrations of SaH 58-035 and CI 976. The presence of ACAT
inhibitors further decreased the parasite proliferation in host cells
deficient for ACAT activity, indicating the essential role of the
T. gondii enzyme for the optimal replication of the parasite
(Fig. 6C).
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Fig. 6.
Endogenous ACAT activity in T. gondii tachyzoites. A, parasites were
harvested from ACAT-1/ MEF and treated with the
indicated concentrations of inhibitors for 7 h. Extracted lipids
were separated on TLC plates and bands corresponding to CE counted for
the presence of radioactive 14C. B, for
comparison, HFF cells were similarly treated with inhibitors for 7 h and analyzed for newly esterified cholesterol. Data are expressed as
percent of control (Me2SO-treated parasites) ± S.E.
from three separate experiments done in duplicate. C, MPF
from ACAT-1/ mice were treated for 12 h with the
indicated concentrations of ACAT inhibitors SaH 58-035 or CI 976 and
then infected with T. gondii tachyzoites of the RH strain
at an m.o.i. of 0.1. After 72 h, live parasites were quantitated
by measuring the parasite -galactosidase activity. Data are
expressed as a percent of control (Me2SO-treated MPF) ± S.E. from two experiments done in triplicate.
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Moreover, T. gondii tachyzoites were purified from control
untreated HFF, inhibitor-treated HFF, and ACAT-1/ MEF
and analyzed for fluorescent intracellular lipid droplets after
staining with Nile Red. Parasites harvested from control untreated HFF
showed 2-4 neutral lipid droplets per cell, whereas tachyzoites from
cells grown in the presence of ACAT inhibitors were either devoid of
such droplets or presented a noticeable reduction in their size.
Tachyzoites from ACAT-1/ MEF also showed neutral lipid
droplets without significant difference in their appearance compared
with control cells (Fig. 7). Therefore, T. gondii tachyzoites possess an endogenous ACAT activity,
which can serve as a target of inhibitors that are active against
mammalian ACAT. In addition, a BLAST search in the T. gondii
EST data base revealed the presence of two sequences (GenBank accession
numbers N68690 and N82383) with significant similarity to human ACAT-1
at the amino acid level, providing genetic evidence for the presence of
one or more ACAT homologues in T. gondii.
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Fig. 7.
Presence of neutral lipid droplets in
T. gondii tachyzoites. Parasites were harvested
from HFF (A), HFF-treated with 40 µM CI 976 (B), or ACAT-1/ MEF (C), fixed
and stained with Nile Red. Neutral lipid droplets were visualized using
a filter set for fluorescein.
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DISCUSSION |
ACAT is a key element in the intracellular homeostasis of
cholesterol. This enzyme mediates esterification of cholesterol and
fatty acids and allows the cells to store cholesterol esters in the
cytosol. In the present work, we demonstrated that ACAT plays an
essential role in the intracellular proliferation of T. gondii, using as a model system host cells lacking ACAT activity through either gene disruption or pharmacological inhibition. We found
that the lack of ACAT activity in the host cell does not affect the
parasite processes of host recognition, adhesion, and penetration with
formation of the PV. However, once inside the host cell, T. gondii tachyzoites showed an average reduction in proliferation
rate of 60% in the complete absence of host ACAT and a
dose-dependent decrease in replication in host cells
treated with ACAT inhibitors. The anti-T. gondii effect of
the ACAT inhibitors was not caused by host cell toxicity, as cell
morphology, viability, and cellular ATP content were unaffected at the
concentrations of inhibitors used.
In the absence of ACAT activity, both intracellular free cholesterol
content and LDL receptor expression remained unaltered. Thus it appears
that neither a toxic increase in free cholesterol nor a down-regulation
of LDL uptake is responsible for the anti-T. gondii effect
observed. These results also show that in our experimental conditions
where host cells were not loaded with high doses of exogenous
cholesterol, inhibitor treatment does not decrease LDL receptor
expression and that other compensatory mechanisms regulate the
intracellular cholesterol homeostasis in the absence of ACAT activity.
Surprisingly, the importance of ACAT activity is not restricted to the
host cell enzyme. The most important indication derives from the fact
that the concentrations of ACAT inhibitors required for the observed
anti-T. gondii effect are significantly higher than those
required for inhibition of host cell ACAT. Although this could indicate
that the anti-T. gondii effect of ACAT inhibitors is due to
an unrelated activity of the inhibitors, additional results imply that
this is due to the presence of an endogenous ACAT activity in the
parasite. Evidence of esterified lipids in T. gondii
tachyzoites had been restricted so far to the visualization of
intracellular lipid droplets upon staining with the neutral lipid
specific dye Nile Red (19). Our result showed that (i) an ACAT activity
can be effectively detected in T. gondii tachyzoites and
that (ii) this activity can be modulated by treatment with ACAT
inhibitors thus affecting the parasite proliferation. The effect of SaH
58-035 and CI 976 on T. gondii ACAT does not reach the level
of inhibition that was observed on host cell ACAT, suggesting a lower
efficiency of the pharmacological compounds on the parasite enzyme. A
similar decrease in efficiency has been demonstrated for CI 976 on the
ACAT homologue SAT1 in the budding yeast Saccharomyces cerevisiae where a 95% inhibition of ergosterol esterification could be obtained only in presence of 200 µM inhibitor
(44). Interestingly, null mutations in the S. cerevisiae
SAT1 do not affect the growth phenotype of the vegetative form of the
yeast but decrease the efficiency of budding sporulation in the diploid cells (44).
Considering our results as a whole, the observed reduction in T. gondii replication in either ACAT-1/ MEF or ACAT
inhibitor-treated HFF is likely to be mediated by at least two distinct mechanisms.
In the first case, the absence of ACAT enzyme causes a permanent
deficiency of intracellular cholesterol esters. If host cholesterol esters were used by the parasite as source of cholesterol and/or fatty
acids, the absence of such esters would create a situation of lipid
starvation for T. gondii with consequent reduction in parasite replication. This model of cholesterol esters as lipid shuttle
from the host cytoplasm to the PV is supported by the following: (i)
the PV is located in strict proximity to the host cell
endoplasmic reticulum which is the location of ACAT; (ii) cholesterol esters are an efficient way to incorporate a hydrophobic and membrane-bound element such as cholesterol in structures that are
soluble in the cytoplasm; (iii) the intracellular movement of such
lipid droplets does not require vesicular trafficking, which is
consistent with the observation that the PV acquires host cell
cholesterol without involving the host cell vesicular machinery
(19).
In the second case, the presence of ACAT inhibitors can affect T. gondii proliferation by another mechanism. In this situation, despite the complete blockade of new cholesterol esterification by host
ACAT, residual stores of lipid droplets containing cholesterol esters
are still present in the host cytoplasm, thus providing a lipid source
for the parasite. However, the additional reduction in the activity of
the ACAT homologue in T. gondii may still exert a
detrimental effect on parasite proliferation due to the consequent defect in cholesterol ester storage and/or capacity of free cholesterol detoxification. In this case the dose-dependent inhibition
of T. gondii ACAT could account for the similar
dose-dependent reduction in parasite proliferation even in
the presence of a complete blockade of host cell ACAT. Another
possibility is that the inhibition of the ACAT activity in both host
cells and parasites could result in an alteration of the lipid
composition of the growing PVM, thus affecting the parasite proliferation.
The composition of cytosolic lipid droplets suggests possible
mechanisms for the acquisition of cholesterol by the vacuole resident
T. gondii. The surface of lipid droplets is composed of a
phospholipid monolayer with additional proteins (45-47) and unesterified cholesterol components (48, 49). The cytosolic side of the
PV might contain receptors able to recognize the proteins on the lipid
droplets, thus mediating the docking and the formation of an
interfacial membrane continuum between the lipid droplet monolayer and
the surface of the parasite vacuole. This could allow the movement of
lipids in a way similar to that proposed for the intracellular
diffusion of free cholesterol in adipocytes (49). Alternatively, the
lipid monolayer of the lipid droplets could fuse with or bud through
the PV membrane with consequent budding in the intravacuolar space in a
process similar to the release of lipoproteins in the extracellular
medium. As a third possibility, lipids, including cholesterol esters,
could be shuttled from lipid droplets to the PV as monomers or
oligomers by carrier proteins, without direct contact between lipid
droplets and PV.
In summary our studies provide evidence that cholesterol esterification
mediated by the ACAT enzyme is a crucial element for optimal
intracellular replication of T. gondii. In addition, our results imply that cholesterol esters not only constitute a form of
lipid storage but could have a role in dynamic processes of lipid
transport inside the cell. Moreover, the finding that an ACAT homologue
is active in this apicomplexan parasite gives new insights on the
incompletely understood regulation of lipid metabolism in T. gondii. ACAT inhibitors, by reducing cellular cholesterol esters,
may warrant consideration as antitoxoplasmosis agents.
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ABSTRACT
INTRODUCTION
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