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From the
Received for publication, November 23, 1999, and in revised form, June 26, 2000
Trypanosoma cruzi, the causative
agent of Chagas' disease in humans, is an intracellular protozoan
parasite with the ability to invade a wide variety of mammalian cells
by a unique and remarkable process in cell biology that is poorly
understood. Here we present evidence suggesting a role for the host
phosphatidylinositol (PI) 3-kinases during T. cruzi
invasion. The PI 3-kinase inhibitor wortmannin marked inhibited
T. cruzi infection when macrophages were pretreated for 20 min at 37 °C before inoculation. Infection of macrophages with
T. cruzi markedly stimulated the formation of the lipid
products of the phosphatidylinositol (PI) 3-kinases, PI 3-phospate , PI
3,4-biphosphate, and PI 3,4,5-triphosphate, but not PI 4-phosphate or
PI 4,5-biphosphate. This activation was inhibited by wortmannin.
Infection with T. cruzi also stimulated a marked increase
in the in vitro lipid kinase activities that are present in
the immunoprecipitates of anti-p85 subunit of class I PI 3-kinase and
anti-phosphotyrosine. In addition, T. cruzi invasion also
activated lipid kinase activity found in immunoprecipitates of
class II and class III PI 3-kinases. These data demonstrate that
T. cruzi invasion into macrophages strongly activates
separated PI 3-kinase isoforms. Furthermore, the inhibition of the
class I and class III PI 3-kinase activities abolishes the parasite entry into macrophages. These findings suggest a prominent role for the
host PI 3-kinase activities during the T. cruzi infection process.
Trypanosoma cruzi, an intracellular protozoan parasite
that infects humans and other mammalian hosts, is the etiologic agent of Chagas' disease that is a major public health problem in Latin America (1). This parasite is now viewed as an emerging human pathogen
of HIV-1-infected individuals as it can be transmitted through blood
transfusions (2). This unicellular parasite presents three
developmental stages; epimastigote and amastigote forms correspond to
proliferative stages found in the invertebrate and vertebrate hosts,
respectively. The trypomastigote forms are infective and invade
different host cell types, first macrophages, in order to replicate
(3).
How T. cruzi trypomastigotes signal to gain entry and
survive in their host is not completely understood. However, some
evidence suggests that T. cruzi interacts with different
signaling systems of the host. It has been shown that the transforming
growth factor Several forms of evidence now indicate that phosphorylation of the D-3
position of the inositol ring of phosphoinositides, catalyzed by
phosphatidylinositol (PI)1
3-kinases, is a critical step in many cellular processes, such as
cytoskeletal rearrangement, membrane trafficking, and endosome fusion
(9, 10). Thus, tyrosine phosphorylation of proteins containing
YXXM motifs creates docking sites for Src homology 2 domains that are present on the p85 regulatory subunits of class I PI
3-kinase, p85/p110-type (9). The binding of p85 to these tyrosine-phosphorylated proteins activates the associated p110 catalytic subunit of PI 3-kinase, which catalyzes the phosphorylation of PI 4,5-P2 to PI 3,4,5-P3. The class II PI
3-kinases represent a novel group of PI 3-kinases containing a C2
domain at their C terminus, and three mammalian isoforms, namely PI
3-kinase C2 Some of the downstream elements that mediate the action of lipid
products of PI 3-kinases appear to include: 1) serine/threonine kinases
PKB and PDK-1 (9, 20) and a non-receptor tyrosine kinase Tec family,
which binds PI 3,4,5,-P3 and elicits
Ca2+-dependent signaling events (21); 2) a
family of PI 3,4,5-P3-binding proteins that contains
guanine nucleotide exchange activity for ADP-ribosylation
factors and that is potentially involved in membrane trafficking
(22, 23); and 3) a FYVE RING domain containing protein EEA-1,
which binds PI 3-P and Rab5 and appears to be required for endosome
fusion in vitro (10, 24). Thus, PI 3-kinase activity appears
to influence a multiplicity of cell functions.
A key role for host cell tyrosine phosphorylation and activation of
class I PI 3-kinase in bacteria Listeria monocytogenes (25)
and in protozoan, Cryptosporidium parvum (26) cell invasion has been shown. However, the effect of T. cruzi
invasion on the host lipid kinases has not been studied. In the present
studies, we address the question of whether T. cruzi
infection may activate host cell PI 3-kinases and if efficient
infection requires the host PI 3-kinase activity. Here we show that
T. cruzi infection causes marked increases in cellular
amounts of PI 3-P, PI 3,4-P2, and PI 3,4,5-P3,
as well as host PI 3-kinase activation. Moreover, the PI 3-kinase
inhibitor wortmannin strongly blocked T. cruzi infection.
These results suggest an important role for host PI 3-kinases in the
T. cruzi infection process.
Materials--
Anti-phosphotyrosine mouse monoclonal antibody
4G10 and anti-p85 polyclonal antibody used for immunoprecipitations
were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY).
Rabbit polyclonal anti-PI 3-kinase C2 Cell Cultures--
T. cruzi, Y strain, was maintained
through weekly passages in Swiss mice at the Department of
Ultrastructure and Cell Biology, Instituto Oswaldo Cruz. Trypomastigote
forms were obtained from the blood of these mice at the peak of
parasitemia, isolated by differential centrifugation, washed in
Dulbecco's modified Eagle's medium (DMEM), and resuspended
into the same solution before exposure to macrophage cell monolayers
(28). Mouse peritoneal macrophages were obtained from the peritoneal
cavities of Swiss mice by washing with ice-cold DMEM and by plating
them for 30 min in a cell incubator at 37 °C, 5% CO2.
The medium was then exchanged by DMEM supplemented with 10% fetal
bovine serum (DMES), and the cultures were allowed to rest for 24 h before the experiment.
Cell Invasion Assay--
To examine the effects of wortmannin on
T. cruzi infection, the macrophages were plated on 8-well
dishes. One set of plates was pretreated with dimethyl sulfoxide
(control set) and the other set with 1-20 nM wortmannin in
dimethyl sulfoxide for 20 min, and then they were washed twice with
DMES before infection. The plates were then infected with
bloodstream trypomastigotes at a 10:1 parasite:cell ratio for 30 min.
The non-interiorized parasites were removed and the macrophages were
washed in DMES, fixed in Boiun's solution, and stained with Giemsa;
the cells were counted using a Zeiss photomicroscope as described
(28).
Determination of Phosphoinositide Content in Macrophage
Cells--
To determine the effects of T. cruzi infection
on host phosphoinositides, 2 × 106 macrophages were
plated in 10-cm diameter Petri glass dishes and then incubated with
DMES containing [32P]orthophosphate (1 mCi/ml) for 3 h at 37 °C. Where appropriate, wortmannin was added before the last
20 min at a final concentration of 20 nM. Cells were then
washed twice with DMES and incubated with DMES or DMES-containing
trypomastigotes suspension (20:1 parasite:cell ratio) for 30 min. After
infection, cells were washed twice with DMES, the reaction was stopped
by adding an ice-cold solution of
CHCl3:CH3OH:12 N HCl
(200:100:0.75), and cells were recovered by scraping. Phospholipids
were then extracted and resolved by TLC as described (29).
Autoradiography of the TLC plates was carried out using x-ray film
(Kodak T-Mat) and an intensifier screen. The cassette was stored at
Cell Lysis, Immunoprecipitation, and Assay of PI 3-Kinase
Activity--
To assay activity of the PI 3-kinases in
immunoprecipitates, cells were infected or not infected (control) with
T. cruzi, treated or not treated with wortmannin as
described above, washed to remove non-interiorized parasites, lysed by
adding a detergent lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 5 mM EDTA, 100 mM NaF, 0.5 mM
Na3VO4, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, and 1 mM phenylmethylsulfonyl fluoride), and
kept on ice for 30 min. Detergent lysates were precleared by
centrifugation at 14,000 × g for 15 min at 4 °C,
and protein concentration was determined by using the Bradford method
(31). Appropriated antibodies were then added to the cleared cell
lysates standardized for total cell protein, and the lysates were
incubated overnight at 4 °C with constant mixing. Protein
A-Sepharose was added and the samples were incubated for 2 h.
Immunoprecipitated proteins were then washed three times with ice-cold
1% Nonidet P-40 in a phosphate-buffered saline, twice in a buffer
containing 10 mM Tris-HCl, pH 7.5, 100 mM NaCl,
1 mM EDTA, and once in PI 3-kinase assay buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EGTA, 10 mM MgCl2). The
immunoprecipitates were resuspended in 0.16 ml of the PI 3-kinase assay
buffer containing 200 µg of phosphoinositides as specified in figure
legends and in [ Wortmannin Inhibits T. cruzi Entry into Macrophages--
To
determine whether PI 3-kinase activity is required for T. cruzi entry into macrophages, the effects of the PI 3-kinase inhibitor wortmannin were investigated. Mouse peritoneal macrophages were treated or not treated with different concentrations of wortmannin for 30 min and then infected with T. cruzi for 30 min. As
shown in Fig. 1, wortmannin treatment
markedly inhibited the macrophage infection by T. cruzi in a
dose-dependent fashion and with a half-maximal inhibition,
IC50, occurring at 0.5 T. cruzi Infection Causes Stimulation of 3-Phosphorylated
Phosphoinositides Production in Macrophages--
We next
investigated whether T. cruzi entry into the macrophage
results in activation of PI 3-kinase(s) in vivo. Thus, the effect of T. cruzi infection on 3-phosphoinositides was
examined. [32P]Orthophosphate-labeled macrophages were
infected or not infected for 20 min with T. cruzi, and
washed to remove external parasites; phospholipids were extracted and
analyzed by TLC and HPLC.
In uninfected macrophages, [32P]PI 3,4-P2 and
[32P]PI 3,4,5-P3 were undetectable or barely
detected, and infection for 20 min caused a severalfold increase in the
amount of these two products as seen in Fig.
2A. Surprisingly, T. cruzi infection of macrophage also markedly enhanced PI 3-P
production by 6-7-fold (Fig. 2A). These increments in the
3-phosphoinositides were inhibited by 20 nM wortmannin
(Fig. 2A), a concentration that blocked the parasite entry
(Fig. 1). In contrast to 3-phosphorylated phosphoinositides, the
amounts of labeled PI 4-P and PI 4,5-P2 were unaffected by infection (Fig. 2B). Taken together, the data in Figs. 1 and
2 demonstrate that infection with T. cruzi causes a marked
increase in cellular amounts of the 3-phosphorylated phosphoinositides, and wortmannin abolishes this effect as well as invasion, suggesting a
role for PI 3-kinase(s) in the T. cruzi entrance into
macrophages.
T. cruzi Infection Increases Association of PI 3-Kinase Activity
with Tyrosine-phosphorylated Proteins--
Association of the
p85/p110-type PI 3-kinase with tyrosine-phosphorylated proteins has
been shown to promote its stimulation (9). It has been also shown that
T. cruzi infection induces phosphorylation of tyrosine in
macrophage proteins (6, 7), an event that could account for activation
of p85/p110-type PI 3-kinase. As we observed an increase of the PI
3,4,5-P3 content after T. cruzi entry into
macrophages (Fig. 2), we conducted experiments to determine whether
infection stimulates association of PI 3-kinase activity with
tyrosine-phosphorylated macrophage proteins.
As seen in Fig. 3, infection of
macrophages with T. cruzi markedly stimulated the
association of PI 3-kinase activity with tyrosine-phosphorylated
proteins immunoprecipitated with monoclonal anti-phosphotyrosine
antibody 4G10. This stimulation was also inhibited when macrophages
were treated with 20 nM wortmannin before infection. The
4G10 antibody immunoprecipitated PI 3-kinase activity from infected
macrophage phosphorylated PI, PI 4-P, and PI 4,5-P2 (data
not shown). Taken together, these data suggest that T. cruzi
entry into macrophage cells induces tyrosine phosphorylation of
proteins that recruit and activate p85/p110-type PI 3-kinase and PI
3,4,5-P3 production. These data are also consistent with other findings, showing activation of p85/p110-type PI 3-kinase by
bacterial invasion into host cells (25).
T. cruzi Infection Stimulates Class II and Class III PI
3-kinases--
It has been shown that class II PI 3-kinases
preferentially phosphorylate PI and PI 4-P, and class III enzyme can
only use PI as a substrate in vitro (11, 17-19). Thus,
these PI 3-kinase isoforms are most likely to be responsible for the
generation of a large fraction of the PI 3-P in cells. However, class
II PI 3-kinase C2
Because T. cruzi entry causes PI 3-P accumulation in
macrophage (Fig. 2), experiments were performed to examine the effects of infection on the host class II and class III PI 3-kinase activities. As seen in Fig. 4B, a 2-fold
stimulation of PI 3-kinase C2
We next conducted experiments to investigate the effects of T. cruzi infection on class III PI 3-kinase activity. As seen in Fig.
5, a 2.5-fold stimulation of class III PI
3-kinase activity was detected in immunoprecipitates from infected
macrophage cells. This stimulation was inhibited by 20 nM
wortmannin, a concentration that blocks T. cruzi entry.
Thus, these data indicate that T. cruzi interaction with
macrophage cells promotes activation of PI 3-kinase C2
The findings presented here demonstrate that infection of macrophages
with intracellular parasite T. cruzi promoted a marked activation of host p85/p110-type I PI 3-kinase, and the PI 3-kinase inhibitor wortmannin at nanomolar concentration totally blocked the
parasite entry (Fig. 1). In addition, the data also demonstrate for the
first time an activation of class II and class III PI 3-kinases
mediated by a pathogen infection. Because PI 3-kinase C2
Some of the functions regulated by PI 3-kinase activities are
cytoskeleton rearrangement, membrane trafficking, and fusion of
endocytic vacuoles (9, 10, 24). Recent evidence indeed indicates that
T. cruzi entrance into cells occurs by an unusual mechanism
that involves rearrangement of cortical actin cytoskeleton and
recruitment and fusion of host lysosomes in the invasion site (3).
Thus, the activation of different isoforms of host PI 3-kinases, as
shown in this work, could provide multiple 3-phosphoinositide products
at different intracellular locations. These kinases may be required to
promote the diverse signaling events that accompany T. cruzi
entry. Therefore, our findings are consistent with the hypothesis that
activation of different isoforms of host PI 3-kinases promoting
localized synthesis of 3-phosphoinositides is necessary to T. cruzi infection. Further work will be required to rigorously test
this hypothesis and to determine whether T. cruzi infection promotes recruitment of host PI 3-kinase isoforms to different intracellular sites. Such studies will also shed light on the mechanism
by which T. cruzi interacts with the signaling systems that
enable it to survive and replicate inside its host.
We thank Dr. Joseph V. Virbasius and
Dr. Michael P. Czech for the gifts of anti-PI 3-kinase
C2 *
This work was supported by Conselho Nacional de
Desenvolvimento Cientifico e Tecnológico, Fundacão de
Amparo à Pesquisa do Estado do Rio de Janeiro (Brazil), and
Programa de Apoio a Pesquisa Estratégica em
Saúde/Fundação Oswaldo Cruz.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.
¶
To whom correspondence should be addressed: Program in
Molecular Medicine and Dept. of Biochemistry and Molecular
Biology, University of Massachusetts Medical Center,
Worcester, MA 01605. Tel.: 508-856-6927; Fax: 508-856-4289; E-mail:
Adilson.Guilherme@umassmed.edu.
Published, JBC Papers in Press, July 25, 2000, DOI 10.1074/jbc.M909440199
2
J. V. Virbasius et al.,
unpublished observation.
The abbreviations used are:
PI, phosphatidylinositol;
DMEM, Dulbecco's modified Eagle's medium;
DMES, DMEM supplemented with fetal bovine serum;
HPLC, high performance
liquid chromatography.
Activation of Host Cell Phosphatidylinositol 3-Kinases by
Trypanosoma cruzi Infection*
,,, and¶
Instituto de Biofísica Carlos Chagas
Filho, Universidade Federal do Rio de Janeiro, Ilha do
Fundão, Rio de Janeiro 21941-900, Brazil and
§ Departamento de Ultra-estrutura e Biologia Celular,
Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro
21045-900, Brazil
ABSTRACT
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EXPERIMENTAL PROCEDURES
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INTRODUCTION
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-receptor signaling pathway is essential for T. cruzi invasion (4). Activation of a
calcium-dependent host cell pathway by T. cruzi
has also been reported (3, 5). In addition, T. cruzi
invasion has been shown to induce tyrosine phosphorylation of
macrophage proteins (6), as well as activation of the mitogen-activated protein kinase pathway (7). Thus, the blockade of tyrosine kinase and
mitogen-activated protein kinase activities in the host macrophage by
inhibitors (7, 8) ablate the infection of these cells by
T. cruzi, suggesting that activation of kinase pathways is
an important event in this process. Invasion of T. cruzi into cells also appears to trigger an unusual mechanism, which involves recruitment and fusion of host lysosomes at the invasion
site (3), suggesting that this parasite is able to interact with host
signaling systems that regulate membrane trafficking.
, PI 3-kinase C2, and PI 3-kinase C2
, have been
cloned (11-13). These PI 3-kinases prefer to phosphorylate PI and PI
4-P as substrates in vitro, but not PI 4,5-P2.
The role of class II PI 3-kinases in the cells and their regulations
are not understood, although some recent reports show activation of
class II PI 3-kinases by chemokine MCP-1 (monocyte chemoattractant
protein) in monocytes (14), by platelet aggregation (15), and by
insulin-mediated protein phosphorylation (16). Class III enzymes are
homologous to the archetypal Vps34p characterized in
Saccharomyces cerevisiae, which only produce PI 3-P (17).
Vps34p function requires its association with myristoylated
serine kinase Vps15p (18). The complex Vps15p-Vps34p is of fundamental
importance in controlling vesicular transport to the yeast vacuole
(reviewed in Ref. 19).
EXPERIMENTAL PROCEDURES
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and rabbit polyclonal
anti-class III PI 3-kinase from mouse used for
immunoprecipitation were gifts from Dr. Joseph V. Virbasius and Dr.
Michael P. Czech (University of Massachusetts Medical Center). A
cDNA for the mouse homologue of yeast VPS34 was cloned by screening
an adipocyte cDNA library (11) and subcloned in a mammalian
expression vector pCMV5 for transient expression in COS-1 cells. The
class III PI 3-kinase polyclonal antisera, against the full-length
class III PI 3-kinase and expressed in Sf9 cells as N-terminally
fused glutathione S-transferase proteins, were raised in
rabbits. These antisera were used for immunoprecipitations and Western
blots were directed at class III PI 3-kinases. A 95-kDa protein in
class III PI 3-kinase that transiently transfected COS-1 cell lysates
was recognized by this antibody but not by the preimmune serum.
Moreover, the anti-class III PI 3-kinase, and not the preimmune serum,
immunoprecipitates a Mn2+-dependent,
wortmannin-sensitive PI 3-kinase activity in untransfected or
transiently transfected COS-1 cells, indicating its
specificity.2 Protein
A-Sepharose, wortmannin, and a mixture of bovine brain phosphoinositides were from Sigma. Silica Gel G thin layer
chromatography (TLC) plates were from Merck.
[32P]Orthophosphate was from the Brazilian Institute of
Atomic Energy, Brazil and [-32P]ATP was prepared as
described previously (27). [3H]PI 4-P and
[3H]PI 4,5-P2 were from Amersham Pharmacia Biotech.
70 °C and the film developed following the manufacturer's
specifications. Radiolabeled phosphoinositides (except PI) were
recovered by scraping the appropriate spots on TLC.
[32P]Phospholipids were then deacylated and analyzed by
high performance liquid chromatography (HPLC) (30). The retention times
were compared with those of deacylated [32P]PI 3-P,
[32P]PI 3,4-P2, and [32P]PI
3,4,5-P3 standards and produced by using immunoprecipitated PI 3-kinase. Deacylated [3H]PI 4-P and
[3H]PI 4,5-P2 were also used as standards.
-32P]ATP (100 µCi at final
concentration of 0.05 mM). The reaction was incubated for
30 min at room temperature and quenched with 2 ml of ice-cold
CHCl3:CH3OH:12 N HCl
(200:100:0.75). Phospholipids were extracted, resolved by TLC, and
analyzed by HPLC as described above.
RESULTS AND DISCUSSION
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RESULTS AND DISCUSSION
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1 nM. At 10 nM, wortmannin reduced entry to about 5% of that observed
in control (dimethyl sulfoxide-treated) cells. Nonspecific phagocytosis
processes, assayed with boiled yeast, were not inhibited by wortmannin
in the same concentration range (data not shown). Thus, the mechanism of T. cruzi entry into macrophage may require PI 3-kinase
activity, as has been shown in other microorganism invasion into
mammalian cells (25, 26).
View larger version (14K):
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Fig. 1.
Wortmannin blocks the entry of T. cruzi into macrophages. Mouse peritoneal macrophages
were treated or not treated with different wortmannin concentrations
for 20 min and infected with trypomastigotes for 30 min; the
interiorized parasites were counted as described under "Experimental
Procedures." Data presented are average values from three independent
experiments ± S.E.
View larger version (20K):
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Fig. 2.
Effect of T. cruzi infection
on the amounts of cellular 3-phosphoinositides. Plated
mouse peritoneal macrophages were labeled with
[32P]orthophosphate (1 mCi/ml), treated or not treated
with 20 nM wortmannin as indicated, and then infected
(solid bars) or not infected (open bars) with
T. cruzi. Lipids were then extracted and separated by TLC,
and radiolabeled spots were analyzed by HPLC as described under
"Experimental Procedures." Effect of T. cruzi infection
on the contents of: A, PI 3-P, PI 3,4-P2, and PI
3,4,5-P3; and B, PI 4-P and PI
4,5-P2. The data presented are average values from three
independent experiments ± S.E.
View larger version (15K):
[in a new window]
Fig. 3.
T. cruzi infection stimulates the
association of PI 3-kinase activity with tyrosine-phosphorylated
proteins. Plated macrophages were treated or not treated with 20 nM wortmannin (Wort), infected or not infected
with T. cruzi as indicated, and cell lysates were prepared
as described under "Experimental Procedures." A,
tyrosine-phosphorylated protein immunoprecipitates (IP:
Anti-p-Tyr) from cell lysates were assayed for PI 3-kinase
activity using PI as lipid substrate, and products were resolved by
TLC. Spots corresponding to PI 3-P on TLC are indicated by an
arrowhead. B, the data shown in A were
quantified using a scanning densitometer. Data presented are average
values from two independent experiments.
-type is poorly sensitive to wortmannin at
concentrations that block class I and class III enzyme activities
(11).
activity in immunoprecipitates from
infected macrophage cells was found. Under these experimental
conditions, equal amounts of PI 3-kinase C2 were immunoprecipitated
from control or infected cells, as depicted in Fig. 4A. The
increased activity in PI 3-kinase C2 immunoprecipitates was specific
for PI and PI 4-P as substrate. No PI 3,4,5-P3 was formed
when PI 4,5-P2 was added to the assay (Fig. 4B).
Thus, the increased PI 3-kinase activity seen with respect to PI and PI
4-P cannot be attributed to a coprecipitating p85/p110-type PI
3-kinase. Fig. 4C also shows that immunoprecipitated PI
3-kinase C2 activity is not inhibited by 20 nM
wortmannin.
View larger version (23K):
[in a new window]
Fig. 4.
T. cruzi infection stimulates PI
3-kinase C2 activity. Macrophages were
infected (solid bars) or not infected (open bars)
with T. cruzi, and then cells were lysed and
immunoprecipitated using anti-PI 3-kinase C2 polyclonal antibody.
A, total cell lysates from control or infected cells were
incubated with anti-PI 3-kinase C2 immunoglobulin for
immunoprecipitation and subsequent immunoblotting using IP:Anti-PI
3-kinase C2 (IP:Anti-PI 3-kinase C2). The
arrow indicates the band corresponding to PI 3-kinase C2
(PI 3-K C2), 170 kDa. B, PI
3-kinase C2 activity assays were performed on immunoprecipitates
using a mixture of PI, PI 4-P, and PI 4,5-P2 as the lipid
substrates and PI 3-P, PI 3,4-P2, but not PI
3,4,5-P3 were formed in these assays. C, effect
of wortmannin on PI 3-kinase C2 activity immunoprecipitated from
infected cells. Products were resolved by TLC and analyzed by HPLC.
Data presented are average values from three independent
experiments.
and class III
enzyme activities, and this activation may contribute to the increased
PI 3-P level observed in T. cruzi-infected macrophages (Fig.
2).
View larger version (13K):
[in a new window]
Fig. 5.
T. cruzi infection stimulates
class III PI 3-kinase activity. Plated macrophages were treated or
not treated with 20 nM wortmannin (Wort),
infected or not infected with T. cruzi as indicated, and
cell lysates were prepared as described under "Experimental
Procedures." Class III PI 3-kinase immunoprecipitates from cell
lysates were assayed for PI 3-kinase activity in the presence of 3 mM MnCl2, using a mixture of PI, PI 4-P, and PI
4,5-P2 as the lipid substrates. Only PI 3-P was formed in
these assays (data not shown). Products were resolved by TLC and
analyzed by HPLC. Data presented are average values from two
independent experiments ± S.E.
is
insensitive to wortmannin at doses that block T. cruzi entry
(Fig. 1) and inhibit class I and class III kinase activities (Figs. 3
and 5), it is more likely that these latter enzymes are essential
during infection. Taken together, these results add to a growing body
of evidence suggesting a key role for the host PI 3-kinase in
intracellular pathogen infection (25, 26).
ACKNOWLEDGEMENTS
and anti-class III PI 3-kinase polyclonal antibodies.
FOOTNOTES
ABBREVIATIONS
REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1.
World Health Organization.
(1997)
Weekly Epidemiol. Rec.
1,
1-8
2.
National Institute of Allergy and Infectious Diseases.
(1996)
Council News
5,
1-16
3.
Burleigh, B. A.,
and Andrews, N. W.
(1995)
Annu. Rev. Microbiol.
49,
175-200
4.
Ming, M.,
Ewen, M. E.,
and Pereira, M. E.
(1995)
Cell
82,
287-296
5.
Moreno, S. N. J.,
Silva, J.,
Vercesi, A. E.,
and Docampo, R.
(1994)
J. Exp. Med.
180,
1535-1540
6.
Ruta, S.,
Plasman, N.,
Zaffran, Y.,
Capo, C.,
Mege, J. L.,
and Vray, B.
(1996)
Parasitol. Res.
82,
481-484
7.
Villalta, F.,
Zhang, Y.,
Bibb, K. E.,
Burns, J. M., Jr.,
and Lima, M. F.
(1998)
Biochem. Biophys. Res. Commun.
249,
247-252
8.
Vieira, M. C.,
Carvalho, T. U.,
and De Souza, W.
(1994)
Biochem. Biophys. Res. Commun.
203,
967-971
9.
Toker, A.,
and Cantley, L. C.
(1997)
Nature
387,
673-676
10.
Corvera, S.,
and Czech, M. P.
(1998)
Trends Cell Biol.
8,
442-446
11.
Virbasius, J. V.,
Guilherme, A.,
and Czech, M. P.
(1996)
J. Biol. Chem.
271,
13304-13307
12.
Arcaro, A.,
Volinia, S.,
Zvelebil, M. J.,
Stein, R.,
Watton, S. J.,
Layton, M. J.,
Gout, I.,
Ahmadi, K.,
Downward, J.,
and Waterfield, M. D.
(1998)
J. Biol. Chem.
273,
33082-33090
13.
Ono, F.,
Nakagawa, T.,
Saito, S.,
Owada, Y.,
Sakagami, H.,
Goto, K.,
Suzuki, M.,
Matsuno, S.,
and Kondo, H.
(1998)
J. Biol. Chem.
273,
7731-7736
14.
Turner, S. J.,
Domin, J.,
Waterfield, M. D.,
Ward, S. G.,
and Westwick, J.
(1998)
J. Biol. Chem.
273,
25987-25995
15.
Zhang, J.,
Banfic, H.,
Straforini, F.,
Tosi, L.,
Volinia, S.,
and Rittenhouse, S. E.
(1998)
J. Biol. Chem.
273,
14081-14084
16.
Brown, R. A.,
Domin, I.,
Arcaro, A.,
Waterfield, M. D.,
and Shepherd, P. R.
(1999)
J. Biol. Chem.
274,
14529-14532
17.
Schu, P. V.,
Takegawa, K.,
Fry, M. J.,
Stack, J. H.,
Waterfield, M. D.,
and Emr, S. D.
(1993)
Science
260,
88-91
18.
Vanhaesebroeck, B.,
and Waterfield, M. D.
(1999)
Exp. Cell Res.
253,
239-254
19.
Wurmser, A. E.,
Gary, J. D.,
and Emr, S. D.
(1999)
J. Biol. Chem.
274,
9129-9132
20.
Stokoe, D.,
Stephens, L. R.,
Copeland, T.,
Gaffney, P. R.,
Reese, C. B.,
Painter, G. F.,
Holmes, A. B.,
McCormick, F.,
and Hawkins, P. T.
(1997)
Science
277,
567-570
21.
Scharenberg, A. M.,
and Kinet, J. P.
(1998)
Cell
94,
5-8
22.
Klarlund, J.,
Guilherme, A.,
Holik, J. J.,
Virbasius, J. V.,
Chawla, A.,
and Czech, M. P.
(1997)
Science
275,
1927-1930
23.
Frank, S.,
Upender, S.,
Hansen, S. H.,
and Casanova, J. E.
(1998)
J. Biol. Chem.
273,
23-27
24.
Simonsen, A.,
Lippe, R.,
Christoforidis, S.,
Gaullier, J. M.,
Brech, A.,
Callaghan, J.,
Toh, B. H.,
Murphy, C.,
Zerial, M.,
and Stenmark, H.
(1998)
Nature
394,
494-498
25.
Ireton, K.,
Payrastre, B.,
Chap, H.,
Ogawa, W.,
Sakaue, H.,
Kasuga, M.,
and Cossart, P.
(1996)
Science
274,
780-782
26.
Forney, J. R.,
DeWald, D. B.,
Yang, S.,
Speer, A. C.,
and Healey, M. C.
(1999)
Infect. Immun.
67,
844-852
27.
Guilherme, A.,
Meyer-Fernandes, J. R.,
and Vieyra, A.
(1991)
Biochemistry
30,
5700-5706
28.
Andrews, N. W.,
and Colli, W.
(1983)
in
Genes and Antigens of Parasites: A Laboratory Manual
(Morel, C. M., ed)
, pp. 37-43, Editora Fundacao Oswaldo Cruz, Rio de Janeiro
29.
Malaquias, A. T.,
and Oliveira, M. M.
(1999)
Acta Tropica
73,
93-108
30.
Seruniam, L. A.,
Auger, K. R.,
and Cantley, L. C.
(1991)
Methods Enzymol.
198,
78-87
31.
Bradford, M. M.
(1968)
Anal. Biochem.
72,
248-254
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