From the Department of Microbiology & Immunology and
the § Division of Pediatric Infectious Diseases, Lucile
Packard Children's Hospital, Stanford University School of Medicine,
Stanford, California 94305
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ABSTRACT |
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Toxoplasma gondii is one of the most
widespread parasites of humans and animals. The parasite has a
remarkable ability to invade a broad range of cells within its
mammalian hosts by mechanisms that are poorly understood at the
molecular level. This broad host cell specificity suggests that
adhesion should involve the recognition of ubiquitous surface-exposed
host molecules or, alternatively, the presence of various parasite
attachment molecules able to recognize different host cell receptors.
We have discovered a sugar-binding activity (lectin) in tachyzoites of
T. gondii that plays a role in vitro in
erythrocyte agglutination and infection of human fibroblasts and
epithelial cells. The ability to agglutinate erythrocytes can be
reversed by a variety of soluble glycoconjugates, of which heparin,
fucoidan, and dextran sulfate were the most effective. Interestingly,
infectivity of tachyzoites for human foreskin fibroblasts, cells that
are commonly used to grow T. gondii in vitro, was increased
by low concentrations of the sulfated glycoconjugates that inhibited
hemagglutination activity (i.e. dextran sulfate and
fucoidan) whereas high concentrations inhibited parasite infection.
Furthermore, inhibition of glycosaminoglycan biosynthesis and sulfation
on the host cells reduced Toxoplasma infectivity. Finally,
Toxoplasma tachyzoites showed a reduced ability to infect
epithelial cell mutants deficient in the biosynthesis of surface
proteoglycans. The probable identity of the hemagglutinin(s) was
investigated by 1) direct binding of red blood cells to filter blots of
Toxoplasma proteins separated by polyacrylamide gel
electrophoresis, and 2) binding of metabolically labeled parasite
proteins to fixed mammalian cells. Three parasite bands were thus
identified as candidate adhesins. These results suggest that attachment
of T. gondii to its target cell is mediated by parasite
lectins and that sulfated sugars on the surface of host cells may
function as a key parasite receptor.
The protozoan parasite Toxoplasma gondii infects about
The T. gondii life cycle includes three major, distinct
stages: sporozoites, which are the product of the sexual cycle, and the
asexually dividing tachyzoites and bradyzoites. The sporozoite and the
bradyzoite reside within distinct cyst structures that are relatively
long-lived and infectious if ingested. On the other hand, the rapidly
growing tachyzoite is responsible for spreading the infection from cell
to cell and probably for most of the disease symptoms. Tachyzoites
multiply inside a parasitophorous vacuole that does not fuse with host
cell lysosomes. Rapid multiplication of the intracellular
parasite is followed by rupture of the host cell and infection of
contiguous cells. At some point during infection, there is a shift from
multiplication of tachyzoites to the formation of tissue cysts filled
with bradyzoites (5).
T. gondii tachyzoites have the ability to interact with and
invade a diverse array of cells, such as fibroblasts, epithelial cells,
endothelial cells, macrophages, and cells of the central nervous
system. Ultrastructural studies indicate that invasion is a very rapid
(15-40 s) and complex event in which specialized organelles located in
the anterior pole of the tachyzoite seem to be involved (6-8). The
process of entry is initiated by the parasite contacting the host cell
with its anterior region; this is followed by protrusion of the conoid,
modification of the host cell membrane, and secretion of the microneme
and rhoptry contents. As the parasite enters, host cell pseudopods
extend along the parasite, so that the tachyzoite becomes enclosed in
an intracellular vacuole separated from the host cytosol by a unit
membrane (6-8).
Attachment is a prerequisite for microbial colonization and invasion of
host cells and is usually mediated by microbial surface proteins called
adhesins that bind to protein or carbohydrate epitopes present on the
host cell surfaces. Several lines of evidence indicate that
Toxoplasma tachyzoites interact with host cells by specific
receptor-ligand types of interactions (9-11). For instance, invasion
of host cells by tachyzoites can be inhibited by treating the parasite
with rabbit polyclonal and mouse monoclonal antibodies against the
Toxoplasma major surface protein, SAG1 (9). However, the
fact that a SAG1-deficient mutant is still able to infect host cells
(9) suggests that additional parasite molecules may be available to
allow host cell infection. That components of the extracellular matrix
may be important participants in this process has also been shown in
experiments demonstrating that the glycoprotein laminin was able to
enhance tachyzoite attachment to a macrophage-like cell line in
vitro (10). Subsequently, it was reported that host cell laminin
bound to the parasite surface mediates binding of the tachyzoite to the
laminin receptor The knowledge that T. gondii possesses the ability to attach
to and replicate within almost any type of nucleated cell prompted us
to look for clues that may explain this promiscuous and efficient parasite conduct. One possible interpretation for this seemingly indiscriminate behavior may be the presence of multiple adhesion molecules on the Toxoplasma surface that interact with cells
of dissimilar background and surface composition. However, it is also
possible that a single Toxoplasma adhesin may be able to recognize a host cell surface receptor common to most vertebrate cells.
Because carbohydrates are found modifying most cell surface proteins
and have the greatest potential for structural variety, and because
there is good evidence to believe that they act as carriers of
biological information, our studies were designed to identify a
potential T. gondii sugar-binding protein that might be
involved in the process of host cell recognition and attachment. We
have discovered a Toxoplasma surface lectin with specificity for sulfated polysaccharides that appears to be important in the process of attachment to the host cell.
Cell Lines and Reagents--
Animal cell mutants defective in
glycosaminoglycan biosynthesis were derived from Chinese hamster ovary
(CHO)1 K1 cells and were
kindly provided by Jeffrey D. Esko (University of California, San
Diego) (12-14). These mutants, pgsA-745,
pgsB-761, pgsD-677, pgsE-606, and
pgsB- 650, are defective in xylosyltransferase, galactosyltransferse I, heparan sulfate synthesis, heparan sulfate N-sulfotransferase, and galactosyltransferase I,
respectively. Cells were grown at 37 °C in a humidified atmosphere
containing 5% CO2 in Ham's F-12 medium supplemented with
10% fetal bovine serum, containing 2 mM glutamine, 100 µg/ml penicillin and 100 units/ml streptomycin. Dextran sulfate
(Mr 8,000, 50,000, and 500,000), fucoidan;
chondroitin sulfate A, B, and C; heparin; heparan sulfate; hyaluronan;
dextran (Mr 500,000);
p-nitrophenyl- Parasite Cultures--
Toxoplasma strains RH and PDS
were maintained by serial passage every 2-3 days in confluent
monolayers of primary HFF cells as described previously (15). HFF cells
were grown at 37 °C in Dulbecco's modified essential medium
supplemented with 5% Nu-serum, containing 2 mM glutamine,
100 µg/ml penicillin and 100 units/ml streptomycin. Extracellular
tachyzoites were isolated by filtration of freshly lysed-out
tachyzoites through 3 µm polycarbonate filters (Nucleopore), followed
by centrifugation at 500 × g for 10 min at 4 °C.
Alternatively, parasites were obtained by scraping the infected
monolayer prior to natural lysis, syringed through a 27 gauge needle to
release intracellular parasites and filtering as above. Organisms were
washed by centrifugation in DMEM, resuspended in DMEM containing 0.5%
BSA, and used for infection experiments.
Assay for Hemagglutinating Activity--
Blood from BALB/C mice
and New Zealand White rabbits was drawn into Alsever's solution.
Erythrocytes were separated from platelet-rich plasma and the buffy
coat by differential centrifugation at 150 × g for 15 min. The erythrocytes were then pelleted by centrifugation at 350 × g, washed three times in PBS, pH 7.2, and resuspended in
the same buffer at a final concentration of 2% (v/v). A fraction of
erythrocytes was fixed by addition of glutaraldehyde to 1% for 1 h at 4 °C, washed twice in PBS, pH 7.2, and further incubated for
1 h at 4 °C in 0.2 M ethanolamine, pH 7.5. Fixed
cells were washed twice and resuspended to 2% in PBS. Hemagglutination
assays were performed as described (16) using 96-well round-bottom microtiter plates (Falcon, 3911). Briefly, extracellular tachyzoites were harvested, filtered, washed three times in PBS, pH 7.2, resuspended at 1 × 108 tachyzoites/ml, and used for
the agglutination assay. Alternatively, Toxoplasma
subcellular fractions prepared by sonication, ultracentrifugation, and
detergent solubilization were used in hemagglutination assays with
glutaraldehyde-fixed erythrocytes. Thus, a sample (25 µl) of live
Toxoplasma organisms or lysate was serially diluted (2-fold) using PBS, 0.5% BSA as diluent. The assay was initiated by the addition of 25 µl of a 2% suspension of fresh or
glutaraldehyde-fixed rabbit erythrocytes. After 1 h at room
temperature, each well was examined for agglutination. Lectin titer was
defined as the reciprocal of the highest dilution of parasites capable
of causing detectable agglutination of erythrocytes, as determined by
visual inspection. For inhibition experiments, serial dilutions of
potential inhibitors (25 µl) were mixed with an equal volume of a
concentration of parasites 4-fold higher than the minimum concentration
required to give complete agglutination, for 30 min at room
temperature. At the end of this time, 25 µl of a 2% suspension of
erythrocytes was added, and the minimum concentration causing
inhibition recorded after 60 min at room temperature.
T. gondii Infection Assay--
HFF cells were harvested by
trypsinization, resuspended in DMEM supplemented with 5% Nu-Serum,
plated at ~1 × 105 cells/well in 24-well plates
(Falcon), and incubated overnight at 37 °C. The next day, cell
monolayers were washed twice with serum-free DMEM and incubated at
37 °C in 0.25 ml of 0.5% BSA, DMEM until needed. Tachyzoites were
harvested, resuspended at ~4 × 105 organisms/ml and
0.5 ml added to duplicated monolayers, and incubated at 37 °C. One h
after inoculation, cells were washed three times with serum-free DMEM
and incubated in DMEM supplemented with 5% Nu-serum.
Toxoplasma invasion was determined from the selective incorporation of [3H]uracil by replicating parasites as
described previously (17). For inhibition experiments, tachyzoites were
preincubated with various concentrations of inhibitor for 30 min at
37 °C in 0.5% BSA, DMEM. At the end of the incubation time, the
mixture was added to cell monolayers and incubated at 37 °C for
1 h. Then, cell monolayers were washed three times with serum-free
DMEM to remove unattached organisms and further incubated in DMEM
supplemented with 5% Nu-Serum overnight at 37 °C, and infection
rate was determined as described above. To assess the role of host cell
surface proteoglycans, T. gondii infectivity was determined
using confluent monolayers of animal cell mutants pgsA-745,
pgsB-761, pgsD-677, pgsE-606, and
pgsB-650, which are defective in glycosaminoglycan
biosynthesis, and wild type K1 cells as described (18).
T. gondii Attachment Assay--
T. gondii attachment
was determined using confluent monolayers of animal cell mutants
pgsA-745 and pgsB-761, which are defective in
glycosaminoglycan biosynthesis, and wild type K1 cells grown in 24-well
tissue culture plates (Falcon) fixed by addition of 1% glutaraldehyde
(19). Toxoplasma tachyzoites were harvested, preincubated in
methionine-free DMEM for 30 min at 37 °C, and metabolically labeled
by addition of 250 µCi/ml of [35S]methionine for 1 h at 37 °C. At the end of the incubation time, organisms were washed
three-times in DMEM and resuspended at ~2 × 105
cells/ml in 0.5% BSA, DMEM. To assay binding, 0.5 ml was added to
duplicated monolayers, centrifuged at 500 × g for 5 min at 4 °C to increase binding efficiency, and incubated at
37 °C. After 5 and 15 min of incubation, cell monolayers were washed
three times with DMEM to remove unattached organisms, and the
monolayer-associated radioactivity was determined by addition of 0.1 N NaOH and liquid scintillation counting.
Binding of FITC-conjugated Heparin--
Freshly harvested
organisms were washed in PBS, incubated with increasing concentrations
of FITC-heparin for 1 h at 4 °C, and washed three times with
ice-cold PBS, and organism-associated fluorescence was determined using
an Olympus BX60 fluorescence microscope. Alternatively, live organisms
were fixed in suspension immediately after harvesting by addition of
3% buffered-formalin for 10 min at 4 °C, incubated in 0.1 M glycine, PBS, pH 7.2, washed in PBS, pH 7.2, and used
intact or after permeabilization with 0.25% Triton X-100. In another
set of experiments, parasite aliquots were dried on glass slides, fixed
with buffered-formalin as above, and used for binding assays. Binding
specificity was evaluated by using 0.5 µg/ml of FITC-labeled heparin
and competition with varying concentrations of unlabeled potential inhibitors.
Indirect Fluorescent Antibody Staining--
Cellular
localization of glycosaminoglycans was examined using monoclonal
antibodies specific for heparin (MAB570, IgG2b; MAB2040, IgM), heparan
sulfate proteoglycan core protein (MAB458, IgG1), chondroitin-4-sulfate
(MAB2030, IgG1), and chondroitin-6-sulfate (MCA277, IgM). For
evaluation of extracellular organisms, tachyzoites were dried on glass
slides, fixed by incubation for 10 min at room temperature in 3%
buffered-formalin, washed in 0.1 M glycine, PBS, pH 7.2, and incubated in the appropriate concentration of antibodies in 1%
BSA, PBS, pH 7.2. For intracellular parasites, confluent monolayers of
HFF were grown in 8-well chamber slides overnight, infected by addition
of tachyzoites, and incubated for 48 h. Infected cell monolayers
were rinsed with PBS, pH 7.2, fixed in 3% buffered-formalin as above,
permeabilized by preincubation in 0.25% Triton X-100, PBS, pH 7.2, and
probed using ~5 µg/ml of specific monoclonal antibodies, followed
by a goat anti-mouse IgG or IgM conjugated to fluorescein
isothiocyanate (Sigma) diluted 1:50 in PBS, 1% BSA. Negative control
included normal mouse serum (1:100 dilution) or incubation with a goat
anti-mouse IgG or IgM conjugated to fluorescein isothiocyanate.
Inhibition of Proteoglycan Biosynthesis and Sulfation by HFF
Cells--
To inhibit the synthesis of host cell proteoglycans, live
HFF cell monolayers grown in 24-well plates were preincubated with 2 mM
p-nitrophenyl- Identification of Hemagglutinating
Factors--
Toxoplasma tachyzoites were harvested as
explained above, washed once in serum-free DMEM, resuspended in
methionine-free DMEM supplemented with 2% dialyzed fetal bovine serum,
and incubated for 30 min at 37 °C. Then, parasite proteins were
metabolically labeled by addition of 250 µCi/ml of
[35S]methionine for 1 h at 37 °C. At the end of
the incubation time, organisms were washed three times in PBS, pH 7.2, resuspended at ~2 × 108 cells/ml, and sonicated at
4 °C (three cycles of 30 s at 25 W). Cell debris was pelleted
by centrifugation at 500 × g for 10 min at 4 °C.
Subcellular fractions were obtained by centrifuging the sonicated
supernatant at 100,000 × g for 1 h at 4 °C to
give a vesicular membrane pellet and soluble cytoplasmic components. The membrane pellet was then solubilized in 1% Triton X-100 for 1 h at 4 °C in the presence of antiprotease mixture (1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml of aprotinin, leupeptin,
soybean trypsin inhibitor, and pepstatin) and centrifuged as above to
give a cytoskeleton pellet and soluble membrane fraction. Finally, the
cytoskeleton pellet was solubilized in 1% SDS for 1 h at 20 °C
and centrifuged as above to obtain a soluble cytoskeleton fraction.
For identification of hemagglutinating factors, a 100-µl aliquot of
the different labeled-parasite fractions was mixed with 100 µl of a
25% (v/v) suspension of 1% glutaraldehyde-fixed rabbit red blood
cells or HFF cells and 100 µl 1% BSA in PBS and incubated for 1 h at room temperature. The cytoskeleton fraction in addition received
700 µl of 2% Triton X-100 in PBS as to make the concentration of SDS
0.1%. At the end of the incubation time, the cells were washed three
times in washing buffer (0.25 M NaCl, 0.1% SDS, 0.25% Triton X-100), and bound proteins were released by solubilization in 50 µl of SDS-PAGE sample buffer, analyzed by electrophoresis in a 10%
polyacrylamide gel, and detected by autoradiography. Alternatively,
bound proteins were eluted by incubation of washed cells with soluble
glycoconjugates and released proteins mixed with an equal amount of 2×
SDS-PAGE sample buffer and detected as described above. For inhibition
experiments, radiolabeled parasite fractions were preincubated with
different concentrations of the potential inhibitors for 30 min at room
temperature before incubation with fixed cells.
SDS-Polyacrylamide Gel Electrophoresis and Western
Blotting--
SDS-PAGE was performed according to Laemmli (21) using a
10% separating gel. Tachyzoites of the RH strain were harvested, washed in PBS, pH 7.2, and solubilized in 1% Triton X-100 for 1 h
at 4 °C in the presence of antiprotease mixture. Next, the lysate
was mixed with an equal volume of solubilization buffer containing 10%
Identification of a T. gondii Sugar-binding Protein--
The
ability to agglutinate erythrocytes has been widely used to detect and
characterize microbial factors that mediate host cell attachment (22,
23). The specificity of this interaction can be assessed by exploiting
the large variability of glycoproteins and glycolipids on the surface
of erythrocytes of various animal species (23, 24). The method is
semiquantitative because it measures hemagglutination titers by
doubling dilutions, but is very reproducible, easy to perform, and
highly flexible (22, 23). In preliminary experiments, hemagglutination
assays were performed at room temperature using purified, live T. gondii as the source of presumptive lectin; the results showed
that the parasites were very effective in agglutinating fixed rabbit
red blood cells, with a minimum of ~103 parasites
required to produce visible hemagglutination (data not shown).
The fact that significant activity was observed during incubation at
4 °C (which should prevent parasite secretory/excretory activity)
suggested that the hemagglutinating activity was associated with the
parasite surface. However, a hemagglutinating activity was also
detected in a soluble, secreted material, because parasite-free supernatants, obtained by incubating tachyzoites in serum-free DMEM for
1 h at 37 °C, followed by centrifugation at 500 × g for 10 min and filtration through a 0.45 µm filter
showed strong hemagglutinating activity in the conditioned media.
Subsequent experiments were performed using glutaraldehyde-fixed
erythrocytes and lysates of T. gondii in an attempt to
extend the range of conditions to be tested. Subcellular fractionation
(Fig. 1) showed that the strongest
hemagglutinating activity was located in the cytoskeleton fraction with
a lesser amount in the soluble membrane material, whereas the cytosol
had no detectable activity. This result implies that part of the
hemagglutinin activity is localized in the membrane fraction.
Interestingly, detergent lysates of the cytoskeleton fraction were more
active than total lysates, despite the presence of a relatively high
concentration (~ 0.4%) of the cationic detergent SDS. This suggests
that the active site of the hemagglutinin is highly stable, that by
denaturing the molecule a second binding domain is exposed, and/or that
some of the hemagglutinin is contained within detergent-soluble
constituents. No differences in hemagglutinating activity were observed
when the assay was performed at 20, 37, or 40 °C, but a decline of
50% in activity was observed after incubation at 56 °C for 5 min,
and heating at 100 °C almost completely abolished
hemagglutination (~90% decrease).
If the hemagglutinin described above is a sugar-binding protein, it
would be expected that red cell agglutination should be inhibited by
soluble glycoconjugates. A total of 33 simple (mono- and disaccharides)
and complex (oligosaccharides, glycoproteins, and proteoglycans)
saccharides were tested for their ability to inhibit hemagglutination.
The relative potency of the inhibitors is shown in Table
I. Of the 33 examined, 28 showed no
inhibition, even at the highest concentrations tested (5-50 mg/ml).
The remaining 5 showed various degrees of inhibition, with minimal
inhibitory concentrations ranging from 0.0006 to 0.156 mg/ml. The most
striking feature of this analysis was that sulfated sugars, including
heparin, fucoidan, and dextran sulfate, were the most effective
inhibitors of hemagglutination. Uncharged and carboxylated polymers, as
well as most glycoproteins, were ineffective or comparatively
poor inhibitors. The fact that asialofetuin was as potent an inhibitor as fetuin suggests that sialic acid is not involved in the attachment site. In addition, none of the mono- and disaccharides tested, including L-fucose, D-mannose,
D-galactose, Glc, GalNAc, GlcNAc, N-acetylneuraminic acid, and lactose, showed any inhibitory
activity, indicating that the active site is probably extended and able to accommodate a structure larger than a disaccharide.
The inability of dextran to inhibit hemagglutination suggests that
sulfation of the polysaccharide is critical for agglutination to occur.
Evidence for a specific steric requirement is based on the fact that
inhibition of hemagglutination was not related to the charge density on
the polysaccharides. Fucoidan, for example, inhibited agglutination at
600 ng/ml and has a negative charge density of 0.3/monomer, whereas
chondroitin sulfate and dermatan sulfate with a negative charge density
of 1.0/monomer, did not inhibit agglutination at >5 mg/ml (Table
II). In fact, the inability of heparan
sulfate, closely related to heparin, to inhibit hemagglutination even
at 1 mg/ml, suggests that the spatial orientation of sulfated groups
and the composition of the polymer chain strongly affect the affinity
of binding. Several other glycoconjugates, including bovine
submaxillary mucin, hog gastric mucin, thyroglobulin, and ovalbumin,
did not inhibit hemagglutination. In addition, neither EDTA nor EGTA at
200 mM inhibited hemagglutination, suggesting that
exogenous divalent cations are not important for lectin activity to
occur or that Ca2+ is tightly bound and is not released in
the presence of the chelating agents. All the strains tested so far,
including the RH and ME49 strains and a SAG1-deficient mutant of ME49,
have shown hemagglutinating activity and similar sugar specificity,
indicating that the activity is not a peculiarity of one strain (data
not shown).
Infection of HFF Cells by T. gondii Is Stimulated by
Glycoconjugates--
Incubation of T. gondii tachyzoites
with HFF monolayers leads to rapid invasion by the parasite, a process
that can be readily evaluated by light microscopy and quantitated by
staining the host cells and counting the number of infected cells and
intracellular organisms. Investigators in the field have also exploited
the ability of the parasite, but not the host cell, to incorporate [3H]uracil, to more rapidly quantitate the number of
intracellular T. gondii (17). However, because this assay
does not discriminate between failure to invade and failure to grow
once inside the cell, specific methods that do make this distinction
must also be used (see below).
To test the hypothesis that T. gondii binding to host cells
is mediated by a sugar-protein type of interaction and given our preliminary findings using erythrocytes as target cells, we attempted to block the infectivity of Toxoplasma (defined as invasion
plus growth) to HFF cells with soluble glycoconjugates of known
structure. Using a limited number of compounds, we found that the
infectivity was not inhibited by any of the glycoconjugates tested
except at exceptionally high concentrations (see below). On the
contrary, we observed that addition of fucoidan and dextran sulfate
increased the infectivity of the parasite by 2.8- and 1.8-fold,
respectively, when tested at 100 µg/ml (Fig.
2), and that the effect was
dose-dependent with maximal stimulation in the 10-100
µg/ml concentration range, but, at least for fucoidan, the
enhancement was lost at 500 µg/ml. Increase in infection was
specific, as other glycoconjugates, such as heparin, chondroitin
sulfate, and keratan sulfate, were not effective in stimulating
Toxoplasma infectivity (Fig. 2 and data not shown).
Interestingly, the glycoconjugates that showed the
infectivity-enhancing activity were also two of the most effective inhibitors of the hemagglutinating activity. The results in Fig. 2 can
be interpreted on the basis of a lectin activity present on the surface
of T. gondii and HFF cells that shares the same carbohydrate
specificity. Therefore, at low concentrations, simultaneous binding of
the same ligand will promote cell-cell interaction and increase
infectivity. Whereas additional increase in the dose of heparin and
dextran sulfate (1.25-5 mg/ml) produced a marked decrease (80%) in
Toxoplasma infectivity (data not shown), interpretation of
the results at these high concentrations is very tentative because of
the possibility of nonspecific effects on the cultures. Interestingly,
even the highest concentrations of fucoidan (5 mg/ml) failed to inhibit
Toxoplasma infectivity suggesting a large number of surface
receptors or coaggregation of the polysaccharide on the tachyzoite cell
surface, as has been suggested for other organisms (25).
Proteoglycan Biosynthesis and Sulfation Are Required for Maximal
Infectivity of Mammalian Cells by T. gondii--
The observation that
only sulfated polysaccharides inhibited cell agglutination and modified
infectivity suggested that sulfation of the polysaccharide was
important for parasite recognition. We first tested the ability of
tachyzoites to infect HFF cells grown in the presence of
p-nitrophenyl-
To further evaluate the involvement of glycosaminoglycans in
Toxoplasma infection, we next used sodium chlorate to
interfere with sulfation reactions (27, 28). Incubation of cell
monolayers in the presence of chlorate reduced the infectivity by 59 or
68% as compared with cells grown in media containing NaCl at similar ionic strength or normal DMEM, respectively (Fig. 3B).
Removal of chlorate from the culture medium of cells just prior to
infection resulted in essentially full restoration of host cell
susceptibility (data not shown).
Reduced T. gondii Infectivity for Epithelial Cell Mutants Deficient
in Proteoglycan Biosynthesis--
Because selectivity is not easily
achieved with any of the available pharmacological inhibitors, we next
examined the interaction of Toxoplasma with cell lines
carrying genetic defects in glycosaminoglycan assembly. Such animal
cell mutants have been derived from CHO cells by Esko and co-workers
(12-14) and offer the sought selectivity. Thus, on the basis that
Toxoplasma expresses a lectin activity that is best
inhibited by the proteoglycan heparin and sulfated polysaccharides
(i.e. fucoidan and dextran sulfate), we used five CHO cell
mutants (pgsA-745, pgsB-761, pgsD-677,
pgsE-606, and pgsB-650) defective in proteoglycan
biosynthesis to test for the role of those molecules in mediating host
cell infection by this organism. All the mutants lack or have reduced
amounts of heparan sulfate and other heparin-like sequences present in
wild type cells. The results showed that all five
proteoglycan-deficient mutants supported T. gondii infection
to a much lesser extent than wild type cells (Fig.
4). Four independent experiments
performed in triplicate have consistently showed a reduction of about
63-80% compared with parental cells. Mutants pgsA-745,
pgsB-761, and pgsB-650 make 5, 8, and 32%,
respectively, of the amount of proteoglycan synthesized by wild type
cells (12). The fact that mutant pgsD-677 does not
synthesize any heparan sulfate but contains about three times more
chondroitin sulfate that the wild type cells suggests that heparan
sulfate, but not chondroitin sulfate, mediates infection of
Toxoplasma. Because mutant pgsE-606 produces
heparan sulfate that is 2-3-fold undersulfated but normal amounts of
fully sulfated chondroitin sulfate (13), we conclude that sulfation of
the heparan chain is important for optimal infection. These findings support the hypothesis that proteoglycan-mediated infection plays an
important role during Toxoplasma infection.
Attachment of Toxoplasma to Cells Deficient in Proteoglycan
Synthesis Is Reduced--
Two different approaches were taken in order
to evaluate whether the differences observed in Toxoplasma
infectivity of epithelial cell mutants deficient in proteoglycan
biosynthesis were related to early events in the infection process
(i.e. attachment/invasion) or to altered intracellular
growth. First, metabolically labeled tachyzoites were used to infect
live, confluent cells and, after extensive washing and only a short (1 h) incubation at 37 °C, the monolayer-associated radioactivity was
then measured. A significant reduction (61-64%) was observed in the
ability of Toxoplasma to attach/invade mutant
pgsA-745 cells as compared with wild type K1 cells at all
the doses evaluated (Fig. 5A),
supporting the idea that the differences observed occur early in the
infection process. Second, glutaraldehyde-fixed monolayers
(i.e. cross-linked cell surface membrane) were evaluated for
their ability to support attachment of metabolically labeled parasites.
Organisms were gently centrifuged on top of the monolayers to increase
the efficiency of binding and the radioactivity associated to the cells
was quantitated after 5 and 15 min of incubation at 37 °C. Binding
of tachyzoites to mutant pgsA-745 cells was reduced by about
60% in both cases as compared with wild type cells (Fig.
5B), further supporting the idea that diminished attachment
accounts for the differences in infectivity.
Fluoresceinated Heparin Binds to Toxoplasma
Tachyzoites--
Direct binding of heparin to Toxoplasma
was tested using FITC-labeled heparin. In preliminary experiments, we
found that live, fresh organisms were unable to bind detectable amounts
of FITC-heparin at 4 °C (Fig.
6A). Note the presence of a
few dead parasites in this preparation that do stain, consistent with
the results using permeabilized organisms to be discussed below. After
formalin fixation of live organisms and permeabilization with 0.2%
Triton X-100, discrete intracellular binding of fluoresceinated heparin was observed within elongated organelles in the anterior region (Fig.
6B). Interestingly, parasites dried on glass slides and fixed with buffered-formalin or methanol showed an intense, peripheral fluorescence (Fig. 6C). This suggests that stimulation from
attachment results in lectin activity appearing on the surface of the
parasites. In this context, when freshly harvested, live tachyzoites
were added to tissue culture-treated chamber slides for 15 min at
37 °C, washed, and fixed with formalin, strong binding of
FITC-heparin to the entire parasite periphery was observed, supporting
the possibility of induced release once the organism interacts with a
substratum (data not shown) (29).
To determine whether there was a single promiscuous glycosaminoglycan
receptor or different receptors for heparin, dextran sulfate, and
fucoidan, competition experiments were performed in the presence of a
fixed amount of FITC-heparin (0.5 µg/ml) and varying amounts of each
putative competitor (Fig. 7). Heparin, dextran sulfate, and fucoidan completely inhibited FITC-heparin binding
to fixed tachyzoites at 100 µg/ml. The inhibition was specific,
inasmuch as neither dextran nor chondroitin sulfate (Fig. 7) inhibited
FITC-heparin binding at 100 µg/ml or even at 1 mg/ml (data not
shown). These findings are consistent with a common receptor for
heparin, dextran sulfate, and fucoidan and with the possibility that
Toxoplasma contains a large pool of intracellular
heparin-binding molecules that, once released, interact with the
parasite surface.
Anti-proteoglycan Antibodies Bind to Toxoplasma
Tachyzoites--
Because FITC-heparin failed to bind to the
surface of live Toxoplasma in detectable amounts, we next
asked whether proteoglycans were found spontaneously interacting with
tachyzoites in culture. Using monoclonal antibodies against heparin
(MAB570 and MAB2040) to probe live extracellular tachyzoites, no
staining was observed. However, using the same antibodies, a bright,
punctate fluorescence was observed on tachyzoites dried on glass slides
and fixed with buffered formalin (Fig.
8A).
Anti-chondroitin-6-sulfate antibody (MCA277) also interacted with the
fixed tachyzoites, but in this case, an intense, diffuse fluorescence
was observed over the whole organisms (Fig. 8C). We failed
on repeated occasions to observe binding of monoclonal antibodies
against heparan sulfate proteoglycan core protein (MAB458) (Fig.
8B) and against chondroitin-4-sulfate (MAB2030) on both live
and fixed tachyzoites (data not shown). When the anti-heparin
monoclonal was used to probe fixed, infected HFF cells, a bright,
intense, and homogeneous fluorescence was observed associated with the
intracellular organisms filling the parasitophorous vacuole (Fig.
9A). In contrast, the
anti-chondroitin-6-sulfate antibody produced a fine granular pattern on
individual organisms (Fig. 9B). As with extracellular
organisms, no fluorescence was observed when monoclonal antibodies
against heparan sulfate proteoglycan core protein (MAB458) (Fig.
9C) or anti-chondroitin-4-sulfate (MAB2030) (Fig.
9D) were used with intracellular organisms. The origin of
the parasite-associated proteoglycan-like molecule is currently
unknown, but its presence is in agreement with the hypothesis that
proteoglycans may play an active role in Toxoplasma-host cell interaction.
T. gondii Proteins Bind to Erythrocyte and HFF Cell
Surfaces--
In an attempt to identify the putative
Toxoplasma lectin(s), we took advantage of the known ability
of parasite lysates to agglutinate rabbit erythrocytes. Thus, total
tachyzoites proteins were separated by SDS-PAGE and transferred to
nitrocellulose filters. Filters were equilibrated in Triton X-100,
followed by BSA, PBS, pH 7.2, and incubated with a suspension of
erythrocytes at room temperature. After gentle rinsing of the filter
membrane with PBS, pH 7.2, we observed binding of erythrocytes to three
distinct bands of 45, 65, and 71 kDa (Fig.
10A). Preincubation of the
filter with soluble fucoidan (1 mg/ml) prevented binding of the 65- and 71-kDa bands (data not shown) but not the 45-kDa band. When
nitrocellulose replicas of Toxoplasma subcellular fractions
were tested by the erythrocyte binding assay, the pattern of binding
closely mimicked the relative hemagglutinating activity observed in
lysates, with prominent bands in the membrane and cytoskeleton
fractions and a cytosolic fraction almost devoid of binding activity
(Fig. 10B).
To gain further insights on the possible identity of the lectin(s), we
tested whether metabolically labeled, solubilized T. gondii
proteins were able to bind to glutaraldehyde-fixed erythrocytes. For
these experiments, parasites were labeled with
[35S]methionine, and subcellular fractions were prepared
by sonication as described above and used for ligand binding
experiments. In preliminary experiments, incubation of fixed
erythrocytes with Toxoplasma lysates showed that a limited
number of radioactive proteins bind to the surface of fixed cells (Fig.
11). Particularly, proteins with
molecular masses of 26, 45, and 65 kDa were eluted from the cell
surface and easily detected by SDS-PAGE and autoradiography. Interestingly, binding of the 26- and 65-kDa proteins can be inhibited by soluble heparin and fucoidan (Fig. 11) and dextran sulfate (data not
shown), in accordance with the ability of these compounds to inhibit
hemagglutination. In contrast, no inhibition of binding for these two
bands was observed when many other polysaccharides were tested,
including chondroitin sulfate, dextran (Fig. 11), and dermatan sulfate
(data not shown). The 45-kDa protein bound with such a high affinity to
the surface of the cell that its binding could not be inhibited by any
of the glycoconjugates used at the doses tested. When the
glutaraldehyde-fixed erythrocytes were substituted with fixed HFF or
epithelial (CHO K1) cells in the adsorption experiment, a similar
pattern of bands was observed, with the 26-, 45-, and 65-kDa bands
binding with equal relative amounts to all cells (data not shown).
These combined data indicate that 26- and 65-kDa proteins are strong
candidates for the Toxoplasma glycosaminoglycan-binding
molecules. The role of the 45-kDa protein awaits further biochemical
and immunological studies.
Cell surface lectins are increasingly being considered as
mediators of cell-cell interaction in a range of biological systems (30-32). In the microbial world, lectins help microorganisms in the
process of recognition and attachment to host surfaces, a critical step
in successful colonization and, ultimately, production of disease (33).
In the case of Apicomplexa parasites, there is evidence that the homing
of malaria sporozoites to the liver is based on the recognition of
hepatocyte cell surface heparan sulfate (reviewed in ref. 34), whereas
infection of the host erythrocytes by Plasmodium merozoites
appears to be mediated at least in part by sialic acid (35), whereas
placental chondroitin sulfate A is a cell surface receptor for infected
erythrocytes (36). We thus sought to determine whether a
Toxoplasma lectin or family of lectins that recognize
different sugar residues might modulate Toxoplasma tropism
and infection.
For our initial experiments, we chose the method of mixed agglutination
using whole tachyzoites and detergent lysates of the parasite plus
rabbit erythrocytes and found an agglutinating activity inhibitable by
the sulfated polysaccharides heparin, dextran sulfate and fucoidan.
None of the tested mono- and disaccharides inhibited agglutination,
suggesting that a polysaccharide is required to span two or more active
sites on the sugar binding domain or may be a reflection of the fact
that the binding mechanism involves a large number of weak
interactions. An important question is whether inhibition of
hemagglutination by heparin, dextran sulfate and fucoidan is related to
electrostatic interactions or a specific steric configuration, because
this would give an insight into the molecular mechanisms involved in
these events. Because the glycosaminoglycans, heparan sulfate,
hyaluronic acid, chondroitin sulfate, dermatan sulfate, and keratan
sulfate have charge densities that are comparable to or higher than
fucoidan, and yet they display no inhibitory activity, the inhibitory
mechanism must depend on more than the electrostatic interaction
between charged molecules of different polarities.
The structural specificity required for inhibition is also exemplified
by the difference in potency of heparin and heparan sulfate.
Structurally, heparin and heparan sulfate molecules consist of repeated
disaccharide units that are sulfated differently. In general, heparin
contains a higher proportion of sulfation and higher
L-iduronic acid:D-glucuronic acid ratio than
heparan sulfate, suggesting that although a relatively high sulfation density appears essential, it is possible that the composition of the
carbohydrate backbone and the position of sulfated groups also play a
crucial role in the interaction of the Toxoplasma lectin and
its inhibitors. Furthermore, a range of effects on Toxoplasma-host cell interaction, not directly related to
their charge density (Table II), was observed with the different
sulfated polysaccharides. Therefore, a unique specificity of binding,
probably related to the conformation of the native ligand, dictates the biological effect and suggests that the location of the sulfated residues is as important as the charge density per se.
It would be tempting to propose a model on the basis of some common
structural motif. For fucoidan, a sulfated polymer of Competition experiments using FITC-heparin and unlabeled inhibitors
(e.g. heparin, dextran sulfate, and fucoidan) argue for the
existence of a nonspecific parasite receptor capable of interacting with a range of sulfated polysaccharides able to inhibit
hemagglutination and alter parasite infection.
The identity of the host cell molecule(s) recognized by this parasite
receptor is not known. Among the prominent sulfated macromolecules
found in higher cells are the proteoglycans, a class of glycoproteins
that contain a core protein with one or more covalently attached
glycosaminoglycan chains. Glycosaminoglycans consists of linear
polymers composed of disaccharide repeating units of uronic acid and
hexosamine. There are four general classes of glycosaminoglycans: 1)
hyaluronic acid, 2) chondroitin/dermatan sulfate, 3) keratan sulfate,
and 4) heparan sulfate/heparin (39, 40). As the chains polymerize, they
undergo various sulfation and epimerization reactions that, together
with variations in the length of the polymer, confer proteoglycans with
a tremendous structural heterogeneity. Recent studies support the role
of cell surface proteoglycans as adhesion receptors for many pathogenic microorganisms (reviewed in Ref. 41), including viruses, protozoan parasites, and bacterial pathogens (19, 25, 42-44). These interactions fall into two classes: (a) Trypanosoma cruzi
(19), Leishmania donovani (43), Borrelia
burgdorferi (20, 44), and some herpesvirus (42) bind directly to
mammalian cell surface proteoglycans; and (b)
Chlamydia trachomatis (25) employs a different mechanism in
which attachment to, and subsequent infectivity of, eukaryotic cells is
dependent on a heparan sulfate-like ligand on the surface of the
organism that bridges acceptors on the microorganism and host cell
surface membranes, facilitating cell-cell contact. It has been proposed
that the heparan sulfate-like adhesin is synthesized by
Chlamydia (25). Our finding that low doses of soluble,
sulfated polysaccharides induce a significant increase in tachyzoite
infectivity suggests that the Toxoplasma receptor belongs to
the second class of molecules. This was further supported by the fact
that increasing doses of soluble proteoglycans and sulfated
polysaccharides not only abolished such an increase, but at the highest
concentration produced a significant inhibition, as expected when
receptors on both cell surfaces are saturated, inhibiting the ternary complexes.
The availability of mutant cell lines with genetic defects in
glycosaminoglycan assembly provides us with additional data to support
the idea that sulfated polysaccharides participate in host cell
infection. Our results with these cell lines indicate that heparan
sulfate may be the host-derived ligand for the parasite lectin.
However, heparin but not heparan sulfate was found to inhibit the
lectin activity as assessed by inhibition of hemagglutination and
modified infection of HFF cells. Because the structural analysis of the
glycosaminoglycan chain of heparan sulfate has demonstrated the
presence of heparin segments in its structure (45), we hypothesize that
these heparin-like regions may mimic the natural carbohydrate ligand
for the parasite lectin. This suggestion is further supported by the
fact that preliminary results from experiments using glycosaminoglycan lyases indicated that treatment of HFF cells with heparinase (specific for heparin) but not heparitinase (specific for heparan sulfate) (45)
or chondroitinase ABC resulted in about a 25% decrease in the ability
of Toxoplasma to infect the host cell (data not shown). Taken together,
the evidence presented supports the hypothesis that T. gondii utilizes cell surface sulfated sugars, including a
heparin-like molecule, to interact with the host cell.
To gain insight into the identity of the Toxoplasma lectin
activity, we exploited its ability to bind a variety of eukaryotic cells. First, by overlaying nitrocellulose filters, containing separated Toxoplasma proteins, with a suspension of red
blood cells, we identified putative adhesins of 45, 65, and 71 kDa. The
fact that preincubation of the filter with fucoidan abolished the
binding of the 65- and 71-kDa proteins provided evidence of their
sugar-binding ability (data not shown). We next asked whether intrinsically radiolabeled Toxoplasma proteins were able to
bind to fixed cells and found that molecules of 26 and 65 kDa bound in
a sugar inhibitable manner to the surface of the erythrocytes, whereas
a protein of 45 kDa resisted elution by all the inhibitors tested. The
relative participation of these proteins in the process of infection is
currently unknown, but their diversity and specificity indicates that
Toxoplasma, like other microorganisms, may employ various
molecules to interact with the host.
Several groups have pursued the study of sugar-binding proteins on
Toxoplasma (9, 46-48). Specific binding of neoglycoproteins (i.e. BSA-GlcN) to live tachyzoites was observed by Robert
et al. (46). Lectins and neoglycoproteins (BSA-GlcNAc and
BSA-D-galactose) labeled with colloidal gold particles were
used by de Carvalho et al. (47) to probe thin sections of
tachyzoites with the result that significant labeling of the rhoptries
was seen. In neither case, however, were the authors able to identify
or determine the function of the parasite molecule responsible for the
sugar-binding activity. In another study, inhibition of infection of
fibroblast by preincubation of tachyzoites with BSA-GlcN was observed
(9). Because this effect was significantly higher in a strain of
T. gondii expressing SAG1 than in a SAG1-deficient mutant,
Mineo et al. (9) concluded that SAG1 likely binds a
glycosylated host cell receptor. More recently, a 15-kDa fetuin-binding
protein was identified using affinity chromatography on fetuin-agarose (48). Differences in carbohydrate specificity, biological effect observed, and molecular mass distinguish these sugar-binding proteins from the sulfated polysaccharide-specific lectin(s) described in this
paper. Determining the relative contribution of all these molecules to
parasite infectivity is essential to a better understanding of the
pathogenic process in toxoplasmosis. They also provide specificities
and tentative identities (by size) for the lectins involved, making
their further characterization now possible.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
of the adult population of the world (1). Although toxoplasmosis is generally asymptomatic in healthy adults, the infection may cause severe sequelae in neonates and life-threatening lesions in AIDS patients (2-4). Furthermore, toxoplasmosis is also of
considerable importance in domestic animals, given its association with
abortion in sheep and swine and as a major source of infection for
humans (5).
6
1 on eukaryotic cells (11).
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
-D-xylopyranoside; p-nitrophenyl-
-D-xylopyranoside; and
glutaraldehyde; as well as all monosaccharides and glycoproteins, were
purchased from Sigma. FITC-conjugated heparin was obtained from
Molecular Probes. Monoclonal antibodies against heparin (MAB570 and
MAB2040), heparan sulfate proteoglycan core protein (MAB458), and
chondroitin-4-sulfate (MAB2030) were obtained from Chemicon
International Inc. Monoclonal antibody against chondroitin-6-sulfate
(MCA277) was obtained from Serotec. All other reagents were analytical
grade or the best available commercial grade.
-D-xylopyranoside DMEM in 2%
dialyzed fetal calf serum overnight at 37 °C and then used for
infection assays. As a control, monolayers were incubated with 2 mM
p-nitrophenyl-
-D-xylopyranoside, which does
not inhibit proteoglycan biosynthesis. To inhibit sulfation of
glycosaminoglycans, HFF cells were cultured overnight in 24-well plates
in DMEM supplemented with 2% dialyzed fetal bovine serum and
NaClO3, a reversible inhibitor of adenosine
3'-phosphoadenylylphosphosulfate and proteoglycan sulfation or NaCl as
a control to a final concentration of 60 mM, as described
(20). Monolayers were washed once with cold DMEM and analyzed as
described above for T. gondii infection assay.
-mercaptoethanol, and unheated proteins were separated by SDS-PAGE
and transferred to nitrocellulose by semidry electroblotting. The
nitrocellulose sheet was incubated for 1 h at room temperature in
1% Triton X-100, PBS, pH 7.2, followed by 1 h in 1% BSA, PBS, pH
7.2. It was then incubated with a 1% suspension of fresh rabbit erythrocytes in 1% BSA, PBS, pH 7.2 and allowed to settle for 1 h
at room temperature. After gentle washing, the sheet was fixed for 10 min in 3% buffered formalin, pH 7.2, and rinsed with PBS, pH 7.2, and
binding of the erythrocytes was recorded by black and white photography.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Lectin activity associated to
Toxoplasma fractions. Subcellular fractions prepared
by sonication, ultracentrifugation, and detergent lysis were double
diluted in microtiter plates and mixed with 2% washed erythrocytes,
and hemagglutination titers were scored after 60 min at room
temperature. The lectin titer is the greatest fold dilution of
parasites causing visible hemagglutination.
Sugar specificity of T. gondii lectin
Structural features of polysaccharides
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Fig. 2.
Stimulation of T. gondii
infectivity by soluble proteoglycans. Tachyzoites were
harvested, mixed with proteoglycans at the indicated concentrations,
and allowed to infect a monolayer of HFF for 1 h at 37 °C.
Then, monolayers were washed, and the number of intracellular organisms
was determined by the selective incorporation of
[3H]uracil over 16 h. Values shown are the mean ± S.E.
-D-xylopyranoside, a soluble acceptor for glycosaminoglycan polymerization that competes with the
endogenous proteoglycan core protein acceptor, resulting in diminished
cell surface proteoglycans (26). Under these conditions we observed a
reduction of 60% in the ability of Toxoplasma to invade
and/or grow in HFF monolayers as compared with control cells grown in
normal media (Fig. 3A).
Controls treated with the inactive a-form of the drug showed little, if
any, reduction in infectivity (Fig. 3A).
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Fig. 3.
Infection of HFF cells treated with
inhibitors of proteoglycan synthesis and sulfation. A,
HFF cells were grown in the presence of 2 mM
p-nitrophenyl- -D-xylopyranoside or
p-nitrophenyl-
-D-xylopyranoside in 2%
dialyzed fetal calf serum and DMEM overnight at 37 °C and used in
Toxoplasma infection assays. B, HFF cells were
treated overnight with 60 mM NaClO3 or NaCl in
2% dialyzed fetal calf serum and DMEM and used in
Toxoplasma infection assays as described. Each value
represents the mean ± S.E.
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Fig. 4.
Proteoglycans are required for efficient cell
infection by T. gondii. Cell monolayers (~75% confluence)
were incubated with tachyzoites (cell:parasite ratio, 1:2) for 1 h
at 37 °C. At the end of the incubation time, monolayers were washed
three times, and the number of intracellular organisms was determined
by the selective incorporation of [3H]uracil. Each value
represents the mean ± S.E.
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Fig. 5.
Attachment of Toxoplasma to
proteoglycan deficient cells. A, intrinsically
radiolabeled tachyzoites were incubated with live confluent monolayers
of CHO K1 or pgsA-745 cells. After 1 h at 37 °C,
unbound organisms were washed, and the amount of radioactivity
associated with the monolayer was determined by liquid scintillation.
B, intrinsically radiolabeled tachyzoites were centrifuged
on glutaraldehyde-fixed monolayers of CHO K1 or pgsA-745
cells. After 5 and 15 min at 37 °C, unbound organisms were washed,
and the amount of radioactivity associated with the monolayer was
determined as described. Values shown are the mean ± S.E.
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Fig. 6.
Binding of FITC-heparin to tachyzoites.
Live tachyzoites were incubated at 4 °C in the presence of 0.5 µg/ml FITC-heparin and washed in cold PBS, and the associated
fluorescence was evaluated (a). Live tachyzoites were fixed
by addition of 3% buffered formalin, washed, permeabilized with 0.25%
Triton X-100, and evaluated for binding of FITC-heparin (b).
Tachyzoites were dried on glass slides, fixed with 3% buffered
formalin, and evaluated for binding of FITC-heparin (c) as
described under "Experimental Procedures."
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Fig. 7.
Binding of FITC-heparin to tachyzoites is
inhibited by sulfated polysaccharides. For competition
experiments, fixed tachyzoites were preincubated in an excess of the
putative inhibitors (100 µg/ml) (a, noninhibitor;
b, heparin; c, chondroitin sulfate; d,
fucoidan; e, dextran sulfate; f, dextran)
followed by addition of 0.5 µg/ml FITC-heparin, washed, and evaluated
for binding of fluoresceinated heparin.
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Fig. 8.
Anti-proteoglycan antibodies bind to
Toxoplasma. Tachyzoites were dried on glass slides,
fixed with 3% buffered-formalin, and incubated with anti-heparin
(a), anti-heparan sulfate proteoglycan core protein
(b), or anti-chondroitin-6-sulfate (c) monoclonal
antibodies (~5 µg/ml). After incubating with fluoresceinated
anti-mouse immunoglobulins, organisms were evaluated under fluorescence
microscopy.
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Fig. 9.
Anti-proteoglycan antibodies bind to
intracellular Toxoplasma. Monolayers of infected HFF
cells were dried on glass slides, fixed with 3% buffered-formalin,
permeabilized with 0.2% Triton X-100, and incubated with anti-heparin
(a), anti-chondroitin-6-sulfate (b), anti-heparan
sulfate proteoglycan core protein (c), or
anti-chondroitin-4-sulfate (d) monoclonal antibodies (~5
µg/ml). After incubating with fluoresceinated anti-mouse
immunoglobulins, organisms were evaluated under fluorescence
microscopy. t, tachyzoites.
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Fig. 10.
Rabbit erythrocytes bind to nitrocellulose
immobilized Toxoplasma proteins. A,
tachyzoite total proteins were separated by SDS-PAGE, transferred to
nitrocellulose filters, equilibrated in Triton X-100 followed by 1%
BSA, PBS, pH 7.2, and incubated with a suspension of
glutaraldehyde-fixed erythrocytes at room temperature. After gentle
rinsing of the filter membrane with PBS, pH 7.2, erythrocyte binding
was evaluated by visual inspection. Lane 1, Coomassie stain
of total proteins; lane 2, binding of erythrocytes to
nitrocellulose transferred proteins. B, tachyzoites were
sonicated and fractionated by ultracentrifugation and detergent lysis.
Proteins were separated by SDS-PAGE, transferred to nitrocellulose
filters, and probed as described under "Experimental Procedures."
Lane 1, total proteins; lane 2, membrane
fraction; lane 3, cytosol; lane 4, cytoskeleton.
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Fig. 11.
Intrinsically radiolabeled Toxoplasma
proteins bind to eukaryotic cells. Tachyzoites were
metabolically labeled, solubilized in Triton X-100, and incubated with
glutaraldehyde-fixed rabbit erythrocytes. After being washed three
times, erythrocytes were boiled in 2× sample buffer, and proteins were
separated by 10% SDS-PAGE. Eluted radioactivity was evaluated by
autoradiography as described under "Experimental Procedures."
t, total proteins; m, membrane fraction. For
inhibition experiments, intrinsically radiolabeled
Toxoplasma proteins were preincubated with BSA (lane
1), heparin (lane 2), dextran (lane 3),
chondroitin sulfate (lane 4), or fucoidan (lane
5) before incubation with fixed erythrocytes.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-(1
3)-linked L-fucose, it has been estimated that
most of the sulfated esters are located on the C-4 position (37).
However, in the case of heparin the carbohydrate backbone consists of
hexuronic acid (D-glucuronic or L-iduronic)
-(1
4)-linked to D-glucosamine units, with variable
location of the sulfated substituents, encompassing: N-sulfation of free amino groups at C-2 of glucosamine,
O-sulfation at C-2 of iduronic acid residues, and
O-sulfation at C-6 of glucosamine units (38). Dextran
sulfate is a
-(1
6)-linked D-glucose polymer that is
highly O-sulfated. Therefore, the available information does
not justify modeling on the basis of their constituent sugars and
glycosidic linkage. It is possible that the role of the polysaccharide backbone may be to determine the specificity of binding by providing a
correct spatial orientation of the N-acetylated,
N-sulfated, and O-sulfated groups, mimicking the region on
the host cell recognized by the Toxoplasma lectin.
Alternatively, it is possible that those agents with the highest
sulfate content may make it more likely that a sulfate group is in a
favorable context for binding in a substantial fraction of the molecules.
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ACKNOWLEDGEMENTS |
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We thank Drs. Adrian Hehl, Ian Manger, and other members of this laboratory for helpful discussions and advice. We also thank Dr. David Sibley for exchange of information before publication.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AI 21423 and K08 AI 01286.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Dept. of Parasitology, Gorgas Memorial Health Research & Information Center, Apartado 6991, Panama 5, Panama.
To whom correspondence should be addressed: Dept. of
Microbiology & Immunology, Sherman Fairchild Science Building, D-305, Stanford University School of Medicine, Stanford, CA 94305. Tel.: 650-723-7984; Fax: 650-723-6853; E-mail:
jboothr{at}leland.stanford.edu.
The abbreviations used are: CHO, Chinese hamster ovary; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; FITC, fluorescein isothiocyanate; GalNAc, N-acetyl-D-galactosamine; Glc, D-glucose; GlcN, D-glucosamine, GlcNAc, N-acetyl-D-glucosamine; HFF, human foreskin fibroblast; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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