Previous Article | Next Article 
Infection and Immunity, December 2000, p. 7198-7201, Vol. 68, No. 12
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Attachment of Toxoplasma gondii to a
Specific Membrane Fraction of CHO Cells
Chaitali
Dutta,
Jane
Grimwood,
and
Lloyd H.
Kasper*
Department of Medicine and Microbiology,
Dartmouth Medical School, Lebanon, New Hampshire 03756
Received 5 June 2000/Returned for modification 6 July 2000/Accepted 16 August 2000
 |
ABSTRACT |
We have observed previously that attachment of Toxoplasma
gondii to synchronized host cells is considerably increased at
the mid-S phase (4 h postrelease). Synchronized CHO host cells at the
mid-S phase were fractionated by molecular weight, and the antigens
were used to produce a panel of polyclonal mouse antisera. The
polyclonal antisera raised against fraction 4 with molecular mass
ranging approximately from 18 to 40 kDa significantly reduced attachment to mid-S-phase host cells. Immunofluorescence assays demonstrated strong reactivity to mid-S-phase host cells and identified a number of potential receptors on Western blots. These data indicate that there is a specific host membrane receptor for parasite attachment that is upregulated during the mid-S phase of the host cell cycle.
| |
TEXT |
Toxoplasma gondii is an
obligate intracellular parasite that causes one of the most common
parasitic infections of humans and other mammals. Parasite invasion of
the host cells is essential in order for the parasite to undergo a
replicative phase. First, recognition and attachment of the parasites
to the host cells must occur. Following the attachment process, the
parasite initiates the invasion through the plasma membrane. During
penetration through the cell plasma membrane, a constricted site, which
moves toward the posterior end of the parasite, forms. Once the
parasite is inside, a small cytoplasmic projection closes the host cell
plasma membrane, leaving the parasite within a parasitophorous vacuole.
Several candidate ligands have been evaluated in the attachment
process. Both surface antigens, in particular SAG1 (p30) (2, 3, 4,
5, 9, 14), and microneme proteins (such as MIC2) have been
implicated as important in the process of attachment (5).
Alteration of these ligands results in decreased infectivity of the
parasite. Recent data would suggest that at least some of the microneme
proteins are principally involved in the process of host cell
penetration (1, 2).
That T. gondii can infect a wide range of host cells would
imply the presence of a common receptor(s) (6, 7).
Competition assays have demonstrated that parasite attachment can be
saturated (14). Moreover, attachment by the parasite to the
host cells may be specific in that a receptor-mediated interaction
occurs with higher frequency during the mid-S phase of the host cell cycle (8). Murine polyclonal antibody raised against host
cells harvested at mid-S phase showed more significant inhibition of attachment of host cells by toxoplasmas than did polyclonal antibody raised against host cells harvested at the beginning of the S phase.
This observation indicates that the potential receptor for attachment
to the host cell by toxoplasmas is abundant during the mid-S phase. In
this report, we characterize and partially identify the host cell
receptor for parasite attachment that is upregulated during the mid-S
phase of the cell cycle.
Murine polyclonal antibody against a specific host cell membrane
fraction inhibits parasitic attachment.
The parasites (RH strain)
were maintained by serial passage in a human foreskin fibroblast
confluent monolayer (11, 15) cultured in modified Eagle's
medium with 10% heat-inactivated fetal calf serum and antibiotic as
described previously (10). Chinese hamster ovary cells
(CHO-pro 5; ATCC CRL-1781) were used as host cells in all experiments.
These cells were maintained in alpha minimum Eagle medium supplemented
with 10% heat-inactivated fetal calf serum and antibiotics.
CHO cells were synchronized by a combination of serum starvation
and hydroxyurea block (12, 16, 17). Cells were seeded at low
density with medium containing 10% fetal calf serum for 24 h at
37°C. The monolayers were serum starved for 48 h and then treated with 1 mM hydroxyurea, 10% fetal calf serum, and antibiotics for 12 h. Cells were washed with warm medium and allowed to
progress in synchrony to the S phase by incubation in medium containing 10% serum. Cell synchronization and passages through the S phase were
monitored by fluorescence-activated cell sorter analysis. At the
appropriate time points, cells were fixed in 95% ethanol overnight.
The ethanol was washed off with phosphate-buffered saline (PBS). Cells
were resuspended in 400 µl of PBS, and 50 µl of RNase A (5 mg/ml)
and 500 µl of propidium iodide (50 µl/ml in 50 mM sodium citrate)
were added. Data were analyzed by the Modfit program.
The synchronized cells were harvested 4 h postrelease (H-4
antigen) by scraping the cells off the flask and washing twice
with
ice-cold PBS. They were incubated with Dounce buffer with
protease
inhibitor (10 mM Tris-Cl [pH 7.6], 0.5 mM MgCl
2, 10 µg
of leupeptin/ml, 10 µg of aprotinin/ml, 1 mM phenylmethylsulfonyl
fluoride in 100% ethanol, 1.8 mg of iodoacetamide/ml) for 10 min
at
4°C. After the cells were pressurized in a nitrogen cavitation
device
to 100 lb/in
2 for 10 min, they were homogenized with
tonicity restoration buffer
(10 mM Tris-Cl [pH 7.6], 0.5 mM
MgCl
2, 0.6 M NaCl, and protease
inhibitors) and nuclear
fractions were removed by low-speed centrifugation.
The supernatant was
centrifuged with 0.5 M EDTA at a final concentration
of 5 mM for 45 min
in a Beckman Ti 70-I rotor at 100,000 to 150,000
×
g
at 4°C. The membrane pellet was dissolved in Triton X-100
lysis
buffer (300 mM NaCl, 50 mM Tris-Cl, 0.5% Triton X-100, and
protease
inhibitors) with repeated vortexing for 30 to 45 min.
Insoluble
materials were pelleted for 15 min at 10,000 ×
g, at
4°C, and the supernatant was
saved.
Approximately 100 to 150 µg of whole membrane fraction was
solubilized in sample buffer (nonreducing) for 5 min at 100°C and
electrophoresed on 10% gels without lanes. The bands of gels were
cut
into horizontal strips by comparing the relative mobility
of the
protein sample to the distance traveled by a known
molecular-weight-marker
protein into different segments. The different
fractions were
named according to molecular weight as follows:
fraction 1, 250,000
to 140,000; fraction 2, 140,000 to 70,000;
fraction 3, 70,000
to 40,000; fraction 4, 40,000 to 18,000; and
fraction 5, 18,000
to
0.
Electro-Eluter Model 422 (Bio-Rad) was used to elute the protein from
the gel. BALB/c mice were immunized with different fractions
(1 to 5)
of whole-cell membrane antigen by using Freund's complete
adjuvant for
the first injection and Freund's incomplete adjuvant
for the
subsequent injections. The animals received 10 total injections
over a
12-week period (6 to 10 µg of protein/injection). Serum
was prepared
and stored at
20°C. The immunoglobulin G (IgG) titer
was checked by
enzyme-linked immunosorbent assay before use of
an attachment
assay.
Mice that received either fraction 2 (140,000 to 70,000) or fraction 3 (70,000 to 40,000) died within 7 to 21 days postvaccination.
The sera
obtained from mice immunized with fraction 1 (250,000
to 140,000),
fraction 4 (40,000 to 18,000), or fraction 5 (18,000
to 0) were
evaluated for their ability to inhibit parasite attachment
to a
population of mid-S-phase cells. The asynchronized CHO cells
were
preincubated in antisera diluted 1:100 for 30 min prior to
the addition
of the
tachyzoites.
Parasites (3 × 10
6) were added to the culture and
incubated for 30 min at 37°C. The coverslips were then washed to
remove extracellular
parasites by immersing them ten times in warm
medium. Host cells'
adherent and intracellular parasites were then
fixed for 20 min
with Bouin's fixative and were dehydrated and Giemsa
stained.
Infection was quantitated by counting the number of
intracellular
and attached parasites per 200 host cells on at least
three replicate
coverslips under a
microscope.
In this assay, antisera raised against fractions 1, 4, and 5 inhibited
tachyzoite attachment to both live and fixed asynchronous
host cells.
When compared with controls, the antisera raised against
fractions 1 and 5 blocked parasite invasion by 12.5 and 10.5%
in the fixed assay
and 3.5 and 13.7% in the live assay, respectively.
However, the
inhibition observed for the cells preincubated with
antisera against
fraction 4 was significant in both cases, being
53.2 and 52.8% in the
fixed and live assays, respectively (Fig.
1A and B).
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 1.
Effects of polyclonal mouse antisera raised against
whole-cell-fraction and fraction-1, -4, and -5 MDBK cells on live and
fixed attachment assays. Host cells were incubated with medium only or
preimmune (normal mouse serum [NMS]) or immune antisera for 30 min
prior to the addition of the parasites. Results are expressed as the
number of parasites attached to 200 host cells for three replicate
cultures.
|
|
Rabbit polyclonal antibody against fraction 4 inhibits parasite
attachment.
New Zealand White rabbits were immunized only with
eluted fraction 4 by using Freund's complete adjuvant for the first
injection and Freund's incomplete adjuvant for the subsequent
injections. The animals received 10 total injections over a 12-week
period (40 to 50 µg of protein/injection). They were terminally bled on the fifth day after the 10th immunization. High-titered
anti-fraction 4 IgG (1:6,400) was tested for efficacy by enzyme-linked
immunosorbent and immunofluorescence assays (IFA). To be certain that
the inhibitory activity was specific to the antibody, the IgG for
fraction 4 was purified by affinity chromatography on protein G coupled
to Sepharose (Pharmacia, Piscataway, N.J.) and used at various
concentrations in a blocking assay with asynchronous, fixed CHO cells
(Fig. 2). The antisera against fraction 4 were then evaluated by using the attachment assay with
glutaraldehyde-fixed asynchronous or synchronous CHO cells. Floating
coverslips of nonconfluent, synchronous host cells were washed twice in
medium and fixed in 2% glutaraldehyde (grade 1; Sigma) for 5 min at
4°C. The fixed cells were then washed three times in PBS and
incubated overnight in 0.16 M ethanolamine (pH 8.3). The fixed cells
were then incubated with minimum Eagle medium-0.2% bovine serum
albumin containing either anti-fraction 4 antibodies diluted 1:100 or
25 µg of purified anti-fraction 4 IgG (determined from Fig. 2) per ml
for 30 min. Then parasites (3 × 106) were added to
the culture and incubated for 30 min at 37°C. The coverslips were
then washed to remove extracellular parasites by immersing them ten
times in warm medium. Host cells' adherent and intracellular parasites
were then fixed for 20 min with Bouin's fixative and were dehydrated
and Giemsa stained. Infection was quantitated by counting the number of
intracellular and attached parasites per 200 host cells on at least
three replicate coverslips. When compared to the asynchronous control
culture, antisera against fraction 4 blocked parasite attachment
significantly (72% with anti-fraction 4 and 80% with column-purified
anti-fraction 4 IgG) (Fig. 3).
Anti-fraction 4 IgG also showed significant sequential inhibition of
parasitic attachment in synchronized CHO cells when compared with that
in the control synchronized culture (Fig.
4A and B).
View larger version (40K):
[in this window]
[in a new window]
|
FIG. 2.
Titration of rabbit anti-fraction 4. Shown is the effect
of different concentrations of anti-fraction 4 IgG on the attachment of
the RH strain of Toxoplasma tachyzoites to fixed,
asynchronous CHO cells over a 30-min period. Host cells were
preincubated in various concentrations of purified IgG for 30 min prior
to the addition of parasites. Results are expressed as the number of
parasites attached to 200 host cells.
|
|
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 3.
Effect of anti-fraction 4 polyclonal antisera or
anti-fraction 4 purified IgG on the attachment of the RH strain of
Toxoplasma tachyzoites to asynchronous (live) CHO-pro cells
over a 30-min period. Host cells were used untreated (control) or
treated with preimmune (normal rabbit serum [NRS or NRIgG]) or immune
anti-fraction 4 (F4) serum or purified IgG (F4IgG). Results are
expressed as the number of parasites attached to 200 host cells for
three replicate cultures.
|
|
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 4.
CHO cells were synchronized with serum starvation and
hydroxyurea block, resulting in a halt in cell cycle progression
through the S phase at the G1/S boundary. Synchrony was
then monitored by fluorescence-activated cell sorter analysis; data are
represented as the percentage of cells undergoing the S phase (A). (B)
Shown are the kinetics of the attachment of T. gondii to
synchronous CHO cells fixed at various time points in the S phase in
the presence of polyclonal anti-fraction 4 IgG antibody or normal
rabbit IgG antibody or medium alone. Host cells were preincubated with
the antibodies (25 µg/ml) for 30 min prior to the addition of
parasites. Results are expressed as the number of parasites (mean ± standard deviation) attached to 200 host cells (n = 3).
|
|
Indirect fluorescence antibody labeling.
Purified IgGs, both
normal and anti-fraction 4, were labeled with fluorescein
isothiocyanate (Fluorolink antibody labeling kit; Amersham Pharmacia
Biotech). Synchronous and asynchronous CHO cells were fixed in 50%
methanol and 50% acetone for 2 min. Cells were washed with PBS and
blocked with 5% bovine serum albumin in PBS for 45 m. They were
then incubated with either normal rabbit IgG or anti-fraction 4 IgG at
a concentration of 10 µg/ml in PBS for 90 min at room temperature.
Cells were washed in PBS 3 times and mounted in slow-fade light
antifade buffer (Molecular Probes, Eugene, Oreg.). The cells were
examined with a Zeiss Axiophot epifluorescence microscope (Fig.
5). It is clearly evident that these sera
showed greater reactivity to the cells which were at 4 h
postrelease than to the cells which were at either 0 or 8 h
following release of CHO cells.
View larger version (89K):
[in this window]
[in a new window]
|
FIG. 5.
Localization of rabbit anti-fraction 4 antibodies on
glutaraldehyde-fixed asynchronous and synchronous CHO cells by confocal
microscopy. Cells are asynchronous CHO and normal rabbit serum (A),
asynchronous CHO and anti-fraction 4 (B), synchronous 0 h and
anti-fraction 4 (C), synchronous 2 h and anti-fraction 4 (D),
synchronous 4 h and anti-fraction 4 (E), synchronous 8 h and
anti-fraction 4 (F). Both antibodies were used at 10 µg/ml.
|
|
Western blotting with rabbit polyclonal anti-fraction 4 antibody.
Synchronized CHO cells were harvested at 4 h
following release from hydroxyurea block, and prepared membrane
fractions were separated by sodium dodecyl sulfate-10% polyacrylamide
gel electrophoresis (SDS-10% PAGE). The proteins were transferred
onto a nitrocellulose membrane. The membrane was incubated with primary
antibodies (25 µg of rabbit polyclonal purified anti-fraction 4 IgG/ml) overnight at 4°C and for 1 h with secondary antibodies
(alkaline phosphatase-conjugated anti-rabbit IgG). The membrane was
developed with alkaline phosphatase-conjugated substrate (Bio-Rad)
until color developed at room temperature.
A series of studies were performed in an attempt to identify the
specific antigen(s) recognized by the anti-fraction 4 rabbit
polyclonal
IgG. The most valuable information arose from Western
blot analysis, in
which this antiserum was found to identify four
to six bands with
apparent molecular masses of 115, 100, 68, 30,
and 25 kDa (Fig.
6). There were no obvious bands that
provided
a differential between the control and test antibody (data not
shown). Further studies included biotin labeling of the 4-h-postrelease
host cell surface antigens followed by immunoprecipitation (data
not
shown). None of these assays provided additional information
regarding
the specific character of the receptor for the parasite
ligand.
View larger version (31K):
[in this window]
[in a new window]
|
FIG. 6.
Western blot analysis. Proteins from
synchronized-CHO-cell (at 4 h) membrane were separated by
mini-SDS-10% PAGE, transferred to nitrocellulose, and probed with
rabbit anti-fraction 4 antibodies diluted 1:50. Molecular weights are
indicated in thousands.
|
|
In this report, we have observed that synchronized host cells are most
favorable to attachment when cells are at the peak
of DNA synthesis (4 h postrelease). Attachment can be blocked
by inhibitors of protein
synthesis and partially affected by inhibition
of glycolysis
(unpublished data). This would suggest that the
ubiquitous receptor for
parasite attachment is a complex molecule
or perhaps a number of
molecules. The polyclonal antisera generated
against the
4-h-postrelease cells inhibit attachment to both live
and fixed cells.
Antisera raised against MDBK cells can block
parasite attachment to CHO
cells, indicating that the receptor(s)
is well conserved
(
8).
Several different approaches were utilized to identify the host cell
receptor for parasite attachment. Silver staining of
synchronized
MDBK/CHO cell membrane prepared at different time
points after
hydroxyurea release and separated by SDS-PAGE failed
to demonstrate a
difference in protein expression (data not shown).
A purified IgG
fraction directed at proteins from fraction 4 (18,000
to 40,000) was
reactive by IFA to host cells and demonstrated
considerable
interference with parasite attachment at 4 h postrelease.
The IFA
studies suggested that cells at 4 h postrelease were more
reactive
to the antibody than cells assayed at the time of release
(0 h). We
next attempted to identify expression of novel membrane
antigens by
Western blotting using the anti-fraction 4 sera. For
4-h membrane
preparations, multiple bands were noted on Western
blots. Although the
mice were immunized with antigens from fraction
4, the antisera
recognized a number of bands representing higher
masses, including 115, 100, and 68 kDa. Unfortunately, there was
no clearly demonstrable
expression of unique antigens at the time
point of
interest.
Our findings further indicate that the receptor for parasite attachment
is a complex molecule that has several structural
properties. These
data suggest that this receptor(s) has biochemical
qualities consistent
with being a protein as well as a glycosylated
product. Since none of
the approaches utilized, including protein
or glycosylation inhibition,
antibody blocking, or use of host
cells devoid of proteoglycans,
provided a distinct loss of the
ability for the parasite to attach, it
is possible that there
is more than a single molecule that serves as
the receptor for
the parasite. Just as there is a family of parasite
surface antigen-related
products (the SRS family) (
13), it
is not inconceivable that
there are different receptors expressed with
different frequencies
on a number of host cells. This could perhaps
account for the
ubiquity of the parasite in our environment and its
ability to
infect all mammalian cells. It further allows for successful
attachment
once the host has mounted an immune response against one
surface
ligand. This would be a survival method that would provide the
parasite the opportunity to infect many different cell lines in
the
infected
host.
| |
ACKNOWLEDGMENTS |
This work was supported by grant AI 30000 from the National
Institutes of Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medicine and Microbiology, Dartmouth Medical School, 640E Borwell,
Lebanon, NH 03756. Phone: (603) 650-8787. Fax: (603) 650-6841. E-mail: Lloyd.H.Kasper{at}Dartmouth.EDU.
Present address: Department of OB/GYN, University of California,
San Francisco, CA 94143.
Editor:
S. H. E. Kaufmann
| |
REFERENCES |
| 1.
|
Achbarou, A.,
O. Mercereau-Puijalon,
J. Autheman,
B. Fortier,
D. Camus, and J. Dubremetz.
1991.
Characterization of microneme proteins of Toxoplasma gondii.
Mol. Biochem. Parasitol.
47:223-234[CrossRef][Medline].
|
| 2.
|
Carruthers, V., and L. Sibley.
1997.
Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts.
Eur. J. Cell Biol.
73:114-123[Medline].
|
| 3.
|
Carruthers, V. B., and L. Sibley.
1999.
Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii.
Mol. Microbiol.
31:421-428[CrossRef][Medline].
|
| 4.
|
Carruthers, V. B.,
S. N. Moreno, and L. Sibley.
1999.
Ethanol and acetaldehyde elevate intracellular Ca2+ and stimulate microneme discharge in Toxoplasma gondii.
Biochem. J.
342:379-386.
|
| 5.
|
Channon, Y.,
E. I. Suh,
R. M. Seguin, and L. H. Kasper.
1999.
Attachment ligands of viable Toxoplasma gondii induce soluble immunosuppressive factors in human monocytes.
Infect. Immun.
67:2547-2551[Abstract/Free Full Text].
|
| 6.
|
Furtado, G. C.,
M. Slowik,
H. K. Kleinman, and K. A. Joiner.
1992.
Laminin enhances binding of Toxoplasma gondii tachyzoites to J774 murine macrophage cells.
Infect. Immun.
60:2337-2342[Abstract/Free Full Text].
|
| 7.
|
Furtado, G. de C.,
Y. Cao, and K. A. Joiner.
1992.
Laminin on Toxoplasma gondii mediates parasites binding to the  1 integrin receptor  61 on human foreskin fibroblasts and Chinese hamster ovary cells.
Infect. Immun.
60:4925-4931[Abstract/Free Full Text].
|
| 8.
|
Grimwood, J.,
J. R. Mineo, and L. H. Kasper.
1996.
Attachment of Toxoplasma gondii to host cells is host cell cycle dependent.
Infect. Immun.
64:4099-4104[Abstract].
|
| 9.
|
Grimwood, J., and J. E. Smith.
1992.
Toxoplasma gondii: the role of parasite surface and secreted proteins in host cell invasion.
Int. J. Parasitol.
26:169-173.
|
| 10.
|
Kasper, L. H.,
J. H. Crabb, and E. R. Pfefferkorn.
1983.
Purification of a major membrane protein of Toxoplasma gondii by immunoabsorption with a monoclonal antibody.
J. Immunol.
130:2407-2412[Abstract].
|
| 11.
|
Kasper, L. H., and J. R. Mineo.
1994.
Attachment and invasion of host cells by Toxoplasma gondii.
Parasitol. Today
10:184-188.
|
| 12.
|
Krakoff, I. H.,
N. C. Brown, and P. Reichard.
1968.
Inhibition of ribonucleoside diphosphate reductase by hydroxyurea.
Cancer Res.
28:1559-1565[Abstract/Free Full Text].
|
| 13.
|
Manger, I. D.,
A. B. Hehl, and J. C. Boothroyd.
1998.
The surface of Toxoplasma tachyzoites is dominated by a family of glycosylphosphatidylinositol-anchored antigens related to SAG1.
Infect. Immun.
66:2237-2244[Abstract/Free Full Text].
|
| 14.
|
Mineo, J. R., and L. H. Kasper.
1994.
Attachment of Toxoplasma gondii to host cells involves major surface protein SAG-1(P30).
Exp. Parasitol.
79:11-20[CrossRef][Medline].
|
| 15.
|
Sabin, A. B., and P. K. Olitsky.
1937.
Toxoplasma and obligate intracellular parasitism.
Science
85:336-338[Free Full Text].
|
| 16.
|
Sinclair, W. K.
1967.
Hydroxyurea: effects on Chinese hamster cells grown in culture.
Cancer Res.
27:297-308[Abstract/Free Full Text].
|
| 17.
|
Yarbro, J. W.
1992.
Mechanisms of action of hydroxyurea.
Semin. Oncol.
19(3):1-10[Medline].
|
Infection and Immunity, December 2000, p. 7198-7201, Vol. 68, No. 12
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Top
Abstract
Text
