From the Departments of Internal Medicine, Cell
Biology, and § Pharmacology, Yale University School of
Medicine, New Haven, Connecticut 06520 and the
Department of
Biology, University of Pennsylvania,
Philadelphia, Pennsylvania 19104
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ABSTRACT |
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The signals and the molecular machinery mediating
release of dense matrix granules from pathogenic protozoan parasites
are unknown. We compared the secretion of the endogenous dense granule marker GRA3 in Toxoplasma gondii with the release of a
stably transfected foreign reporter, Toxoplasma gondii is an obligate intracellular parasite
that survives in all nucleated cells by creating a unique membrane bounded compartment called the parasitophorous vacuole (reviewed in
Ref. 1). Like all Apicomplexan parasites, T. gondii contains three morphologically distinct secretory organelles: rhoptries, micronemes, and dense granules
(DG).1 The protein content of
these organelles, the temporal regulation of their secretion, and the
eventual localization within the infected cell of proteins secreted
from the organelles are all different. T. gondii, therefore,
represents an interesting model for analysis of the differential
control of secretion from morphologically distinct secretory compartments.
DG in T. gondii contain a homogeneous dense core and are
similar in morphology to dense matrix granules in exocrine and
neuroendocrine cells (reviewed in Ref. 2). DG exocytosis involves a
putative fusion event with either the parasite plasma membrane or the
inner membrane complex (a patchwork of closely opposed flattened
vesicles located beneath the plasma membrane). In contrast to
micronemes and rhoptries, which discharge their contents only at the
time of cell invasion (3-5), the kinetics of DG exocytosis are less well defined. The tacit assumption (based largely on their morphology) has been that DG release is also a regulated process. We and others have described a burst of DG exocytosis shortly after invasion (3, 6),
but exocytosis of DG proteins also continues during the prolonged
intracellular residence of the parasite (6-8). Spontaneous release of
DG proteins from extracellular parasites is also observed (9), and this
release may be augmented by the addition of heat-inactivated serum
(10). Thus, based on the limited information available, DG exocytosis
exhibits characteristics of both a regulated and a constitutive process.
Several common themes operate for dense matrix granule secretion in
most higher eukaryotic cells. A rise in intracellular calcium generally
triggers the final fusion event with the plasma membrane (11). An
ATP-dependent priming step precedes the
calcium-dependent event (12, 13), reflecting at least in
part ATP hydrolysis by N-ethylmaleimide-sensitive factor
(NSF) (14, 15). Additional proteins participating in regulated,
calcium-dependent exocytosis of dense core granules include
components of the 20 S vesicle docking complex (16), synaptobrevin/VAMP
(17), syntaxin, SNAP 23/25 (18-20), NSF (21), and SNAPs (22-24). The
monomeric GTPase Rab3 (25) and proteins with calcium binding domains,
most notably synaptotagmin (26), also participate.
Phylogenetically, components of the NSF/SNAP/SNARE/Rab machinery are
broadly conserved across species barriers in higher eukaryotes (27). To
date, however, the identification of these molecules in pathogenic
protozoa has been largely limited to monomeric GTPases of the Rab
family. Functional analyses of their participation in granule
exocytosis has not been reported. Experiments in the nonpathogenic
ciliate, Paramecium (28, 29), have identified Rab proteins
on secretory vesicles and a SNARE-like molecule that complements a
secretion mutant (30).
We recently reported that foreign soluble reporter proteins
(Escherichia coli We have addressed these questions by following the release of both an
endogenous DG marker (GRA3) and the secreted recombinant reporter
protein BLA in intact and streptolysin O-permeabilized extracellular
parasites. Our results demonstrate that DG markers are released
constitutively in a calcium-independent fashion, via the participation
of the NSF/SNAP/SNARE/Rab machinery. While calcium-independent
constitutive release of soluble proteins from a dense matrix granule is
unusual, the protein machinery mediating release in T. gondii is broadly conserved with that in higher eukaryotic cells.
Buffers and Reagents
The following buffers were used. Hanks' balanced salt solution
(HBSS) was supplemented with 1 mg/ml bovine serum albumin (BSA) and 10 mM Hepes buffer, pH 7.4. AISS, an intracellular buffer, contained 120 mM KCl, 20 mM NaCl, 1 mM MgCl2, 10 mM glucose, and 20 mM HEPES, pH 7.2. AISS+ buffer consisted of AISS
supplemented with 1× minimum essential medium essential and
nonessential amino acids, 1× minimum essential medium vitamins, 100 units/ml penicillin G, and 100 µg/ml streptomycin sulfate. Both
buffers contained leupeptin, aprotinin, pepstatin, chymostatin, and
phenylmethylsulfonyl fluoride at final concentrations of 5 µM, 1 µM, 5 µM, 100 µg/ml, and 1 mM, respectively. PBS/Tween 20/BSA contained 0.15 M NaCl, 0.05% Tween 20, and 0.25% BSA, pH 7.2. All
chemicals were obtained from Sigma unless stated otherwise.
Paranitrophenyl phosphate was used as a substrate for alkaline
phosphatase, and nitrocefin was used for Parasite and Cell Culture
The RH strain of T. gondii and a stable transgenic
clone of the RH strain, expressing the soluble foreign secretion
reporter BLA were described earlier (31). A stable transgenic clone of the RH strain expressing a cytosolic form of green fluorescent protein
(GFP) (34) was used to assess cytosol leakage from the parasites
permeabilized with SLO. A clone of the RH strain stably expressing the
cytosolic marker E. coli Parasites were maintained in monolayers of either Vero or HFF cells.
Extracellular parasites were harvested, washed, and counted as
described previously (9). In some experiments, extracellular parasites
were incubated in calcium- and magnesium-free PBS (dissociation buffer)
on ice for 30 min to remove network material adhered to parasites (36).
After this incubation, parasites were washed twice with ice-cold AISS
buffer and resuspended in the same buffer for secretion assays. In
several experiments, HBSS buffer was used in place of AISS.
Secretion Assays
Secretion by extracellular parasites was determined by
incubating 0.4-1 × 108 parasites/ml (total of 3 ml)
in various buffers at 37 °C. Glucose was essential for maximum
secretion (secretion was reduced by 50% less in buffer without
glucose; data not shown).
All assays were performed in prechilled Eppendorf tubes previously
coated with BSA (1 mg/ml) to prevent parasites adhesion. Before
transferring parasite suspensions from ice to 37 °C for the
secretion assay, 500 µl of parasite suspension was removed and
processed as the time 0 reading. The rest of the parasite suspension
was incubated at 37 °C in a water bath. After 15, 30, 60, and 120 min of incubation, 500 µl of the parasite suspension was removed and
centrifuged at 760 × g for 10 min at 4 °C to pellet intact parasites. The pellet was set aside, and the supernatant was
further centrifuged at 7000 × g for 10 min at 4 °C.
GRA3 in both the 7000 × g supernatant and the pellet
were determined by antigen capture sandwich ELISA (see below) and by
immunoblot, as described previously (37). BLA activity was measured as
described below.
Quantitative ELISA for GRA3 and ROP2,3
An ELISA was developed to measure GRA3 and ROP2,3 released from
extracellular parasites in secretion assays. Plates (Linbro/Titertek) were coated with 50 µl of monoclonal anti-GRA3 (T62H11) diluted 1:1000 in 0.05 M carbonate-bicarbonate buffer, pH 9.6, and
incubated overnight at 4 °C. The plates were washed four times with
PBS/Tween 20/BSA. In initial experiments, whole parasite lysates were
tested to validate the assay. A dose-response relationship was observed between the lysate from the increasing number of parasites and OD for
GRA3 (data not shown).
To measure GRA3 released by intact parasites, supernatant derived from
108 parasites/ml was diluted 1:5 with PBS/Tween 20/BSA, and
100 µl (2 × 106 parasite equivalents) was added to
each well and incubated at room temperature for 3 h. The plates
were washed six times, and 100 µl of rabbit anti-GRA3 or a control
antiserum (1:2000) was added to each well and incubated at room
temperature for 2 h. Plates were washed, incubated for an
additional 2 h with 100 µl of rabbit anti-goat IgG (Fc)-alkaline
phosphatase (Promega) (1:4000 in PBS/Tween 20/BSA), washed again, and
developed with 200 µl of p-nitrophenyl phosphate for 45 min followed by the addition of 50 µl of 3 M NaOH. Plates
were read at 550 nm in a Titertek Multiskan ELISA reader, and OD values
for each sample were calculated by subtracting the average of
triplicate wells with rabbit anti-GRA3 from the average of triplicate
wells that received normal rabbit serum.
Rhoptry protein secretion was determined by coating plates with a
1:1000 dilution of the anti-ROP2,3,4 monoclonal antibody T34A7. Rabbit
polyclonal anti-ROP2,3 (1:2000) was used as the detection antibody.
Assay conditions were as for GRA3.
Total GRA3 and ROP2,3 in the parasite pellet were determined by lysing
parasites (108/ml) in a French press cell at 10,000 p.s.i.
The parasite lysate was centrifuged at 12,500 × g for
10 min. Serial 10-fold dilutions of the 12,500 × g
supernatant were prepared in PBS/Tween 20/BSA, and cellular GRA3 and
ROP2,3 were quantitated as above.
Standard curves for GRA3 and ROP2,3 were generated by plotting net OD
versus parasite number. The percentage release of GRA3 and
ROP2,3 in the supernatant during secretion were calculated from the
standard curve.
Enzyme Assays for BLA and BLA in the parasite supernatant as well as in the pellet was
determined by the method described earlier (38) with minor modifications. Parasite supernatant (4 × 107
parasites/ml) diluted 2-fold with AISS buffer and 20 µl was added to
each well of a 96-well plate. The reaction was developed by adding 180 µl of nitrocefin mix (0.2 mM nitrocefin, 0.25 mg/ml BSA,
50 mM potassium phosphate, 0.5% Me2SO, pH 7.0)
for 30 min at 25 °C followed by the addition of 50 µl of 3 M NaOH. Plates were read at 492 nm in an ELISA reader, and
the OD values for each sample were calculated by subtracting the
average of duplicate wells with supernatant derived from RH parasites
and wells with substrate only.
In preliminary experiments, a linear relationship was observed between
BLA release and parasite number (data not shown). The enzymatic
activity of released BLA was not affected by treatment with
dithiothreitol, indicating that the oxidation-reduction state of the
enzyme did not influence BLA activity (39). BLA activity in the pellet
was substantially lower than in the supernatant, regardless of whether
parasites were lysed in TX-100 or in the French press cell. Mixing
experiments of pellet and supernatant indicated that there was an
inhibitor of BLA activity in the parasite pellet (data not shown).
Therefore, total released BLA was determined by immunoblot.
Influence of Incubation Conditions on DG Secretion in
Extracellular Parasites
Temperature, Energy, and Serum Dependence of
Secretion--
Secretion was measured during incubation of
extracellular parasites at 4 and 15 °C, or with 10 mM
sodium azide (NaN3) at 15 °C for 15 min, followed by
incubation at 37 °C for 1 h. The effect of serum on DG
secretion by extracellular parasites was determined by adding 10%
heat-inactivated fetal bovine serum to AISS buffer. The details of
other conditions tested are described in the legend to Table I.
Parasite Permeabilization with SLO and Cytosol Depletion
SLO--
Permeabilization of extracellular parasites with SLO
was done using modifications of a described protocol (40).
Extracellular parasites were washed two times with ice cold potassium
acetate buffer containing glucose (115 mM potassium
acetate, 2.5 mM MgCl2, 10 mM
glucose, and 25 mM HEPES, pH 7.2, with KOH) and then
incubated with 100 µl of 1 unit/ml of SLO for 10 min at 4 °C. The
suspension was transferred to 37 °C and incubated for 2 min, and
then parasites were pelleted at 760 × g for 5 min.
Supernatant was removed and saved to measure released BLA during the
permeabilization step. The parasite pellet was gently suspended in ice
cold potassium acetate buffer, washed two times, and resuspended to
4 × 107 parasites/ml. Permeabilized parasites were
immediately used for secretion assays at 37 °C. BLA secretion was
also monitored from control parasites, which were treated similarly at
every step without the presence of SLO.
Monitoring Permeabilization--
Parasite permeabilization was
assessed by incubating organisms with 4 µg/ml propidium iodide for 5 min at room temperature, and the percentage of parasites positive for
nuclear staining was quantitated by fluorescence microscopy.
Determining Cytosol Leakage--
Release of lactate
dehydrogenase as a cytosolic marker from parasites could not be
quantitated with accuracy, due to contamination with host cell lactate
dehydrogenase. Therefore, a stable transgenic clone of parasites
expressing a cytosolic GFP marker (34) was treated with SLO. The
percentage loss of GFP fluorescence, immediately after permeabilization
and following continued incubation at 15 °C for 1 h, was
determined by measuring the fluorescence of parasites at 490 nm and
emission of 510 nm in a fluorimeter and compared with total GFP in
nonpermeabilized parasites treated in a similar fashion in the absence
of SLO. The GFP release from SLO-permeabilized parasites was also
assessed by phase and by fluorescence microscopy.
Effects of Calcium on DG Release from Permeabilized Parasites
Extracellular parasites were washed two times with cold
potassium acetate-EGTA (5 mM) buffer and permeabilized with
SLO in the same buffer, and then various concentrations of free calcium (0-100 µM final concentration) were added in the
presence or absence of an ATP-regenerating system (ARS; 2 mM NaATP, 20 mM creatine phosphate, and 40 units/ml creatine phosphokinase). The concentrations of free
Mg2+ and Ca2+ in the buffer were calculated
using the software program of Foehr and Warchol (Ulm, Germany).
Alternatively, parasites were permeabilized with SLO in potassium
acetate buffer containing no EGTA and then preincubated with or without
MAPTA-AM (50 µM final concentration) in the presence or
the absence of ARS at 0 °C for 15 min followed by incubation at
37 °C for 30 min for secretion to occur.
Determining the Protein Machinery of DG Secretion Using
SLO-permeabilized T. gondii
The influence of N-ethylmaleimide on DG secretion was
carried out by preincubating both SLO-permeabilized as well as
nonpermeabilized parasites with or without 1 mM
N-ethylmaleimide (NEM) for 15 min at 0 °C. Excess NEM was
removed by washing parasites two times with ice-cold potassium acetate
buffer. Following NEM treatment, both nonpermeabilized and
permeabilized parasites (treated with or without NEM) were suspended in
potassium acetate buffer with or without ARS (2 mM MgATP
was used in place of Na+ATP in the ARS mixture) and
incubated at 37 °C for secretion to occur.
The effect of NSF on DG secretion was determined by incubating
SLO-permeabilized parasites with 10 µg/ml recombinant hamster NSF, or
with the catalytically inactive N domain of NSF (10 µg/ml) at 0 °C
for 1 h before switching to 37 °C for secretion to occur. Prolonged incubation of parasites at 0 °C was used due to the large
size of full-length NSF (76 kDa). These experiments were conducted only
in the presence of ARS, since NSF is unstable in the absence of ATP.
Two mM Mg2+ATP was used in place of
Na+ATP in the ARS mixture.
The involvement of DG secretion was also tested by preincubating permeabilized parasites
with a 100 µM final concentration of GTP All results were expressed as percentage of BLA release, with 100%
being the amount of BLA released by nonpermeabilized control parasites.
Statistical Analysis
Statistical analysis of the data was performed using InStat
software for MacIntosh. Student t test was used for
comparison of two groups. Results were considered significant at
p values of <0.05.
Parasites Spontaneously Secrete Dense Granule Proteins in an
Energy- and Temperature-dependent Fashion
Extracellular parasites spontaneously secreted both GRA3 and BLA
in buffer. When compared with protein in total cell lysates, 11-14%
of parasite GRA3 (Fig. 1, A
and B) and 15-18% of BLA (Fig. 2B) were secreted during a 1-h
incubation at 37 °C. Secretion was abolished at 4 °C or 15 °C
(Figs. 1A and 2A) or by the addition of 10 mM sodium azide (Fig. 1A) and was neither due to
parasite lysis or to a general secretory response of extracellular
parasites (Fig. 1A). No inhibition of BLA secretion was
observed in cycloheximide-treated parasites (Fig. 2A), a
treatment that rapidly and completely inhibits protein synthesis (41).
This latter result suggests that BLA secretion is occurring through
preformed organelles, confirming previous observations (31).
-lactamase, that localizes to
parasite dense granules. Both proteins were released constitutively in a calcium-independent fashion, as shown using both intact and streptolysin O-permeabilized parasites. N-Ethylmaleimide
and recombinant bovine Rab-guanine dissociation inhibitor inhibited
-lactamase secretion in permeabilized parasites, whereas recombinant
hamster N-ethylmaleimide-sensitive fusion protein and
bovine
-SNAP augmented release. Guanosine
5'-3-O-(thio)triphosphate, but not cAMP, augmented secretion in the presence but not in the absence of ATP. The T. gondii NSF/SNAP/SNARE/Rab machinery participates in dense granule release using parasite protein components that can interact
functionally with their mammalian homologues.
INTRODUCTION
Top
Abstract
Introduction
References
-lactamase (BLA) and alkaline
phosphatase) stably expressed in T. gondii are routed
quantitatively to DG (31), suggesting that DG are a constitutive rather
then regulated secretory organelle. Our observation raised several key
questions. Is DG release constitutive or regulated? If DG release is
regulated, what are the triggers for release? What is the protein
machinery controlling DG docking and fusion? Is the protein secretory
machinery conserved between T. gondii and higher eukaryotes?
MATERIALS AND METHODS
-lactamase (Becton
Dickinson Co., Cockeysville, MD). Streptolysin O was obtained from
Murex Diagnostic Ltd. (Dartford, United Kingdom). Recombinant bovine
Rab-GDI (stock of 2 mg/ml in HEPES-KOH, pH 8.0, 100 mM KCl,
8 mM MgCl2, 2 mM EDTA, 0.5% CHAPS
(Pierce), and 50% glycerol (v/v)) (32) was a gift from Ira Mellman
(New Haven, CT). Stocks of recombinant hamster NSF (14), the
catalytically inactive N-domain of hamster NSF (15), and recombinant
bovine
-SNAP (14) were at 0.3, 12, and 11 mg/ml, respectively, in 20 mM HEPES-KOH, pH 7.8, 200 mM KCl, 2 mM MgCl2, 10% glycerol, 1 mM ATP,
1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride. A monoclonal antibody to GRA3 (T62H11), rabbit polyclonal anti-GRA3 antiserum, an anti-ROP2,3,4 monoclonal (T34A7), and rabbit
anti-ROP2,3 antiserum described previously (33) were kind gifts from
J. F. Dubremetz (Villeneuve d'Ascq, France). Rabbit polyclonal
anti
-lactamase antiserum was obtained from 5 Prime
3 Prime,
Inc. (Boulder, CO).
-galactosidase (35) was obtained
from J. Boothroyd (Stanford, CA).
-Galactosidase
-Galactosidase activity in the supernatant as well as in the
parasite pellet from
-galactosidase-expressing parasites was determined as described previously (35).
-SNAP and Rab-GDI on DG secretion was determined
by preincubating permeabilized parasites for 15 min at 15 °C with
various concentrations of
-SNAP (0.5 to 5 µg/ml) and Rab-GDI (4 µg/ml) in the presence or absence of ARS followed by switching to
37 °C for secretion to occur.
S, ATP
S,
cAMP, and cGMP in the presence or absence of ARS at 0 °C for 15 min, after which parasites were switched to 37 °C for secretion to occur.
RESULTS
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Fig. 1.
Kinetics of GRA3 secretion from extracellular
T. gondii. A, ELISA. The percentage of total GRA3
released to the supernatant was calculated from a standard curve, as
described under "Materials and Methods." Secretion was similar in
both high potassium (AISS, ) and high sodium (HBSS,
) buffer
except at later points, where GRA3 release was significantly higher in
AISS (p
0.05) compared with HBSS. Secretion was
energy-dependent (
) and inhibited at 4 °C
(small filled triangle) and 15 °C
(×). Release of ROP2 (large filled
triangle; a marker for rhoptry secretion) and of
-galactosidase (+) (a transgenic cytoplasmic marker for parasite
lysis) were less then 1-2% over 60 min. of incubation. Results
represent mean ± S.D. from four experiments performed in
triplicate. B, parasites were incubated in AISS buffer, and
the supernatant was collected, concentrated, and analyzed by SDS-PAGE
immunoblot (top) as described under "Materials and
Methods." Supernatants are from the equivalent of 108
parasites, whereas the pellet contains the equivalent of
107 parasites. The kinetics and extent of release of GRA3,
determined by densitometric scanning (bottom
panel), were similar to those determined by ELISA in
A. Results shown are from a representative experiment
performed twice.
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Fig. 2.
Kinetics of BLA secretion from extracellular
T. gondii. A, ELISA. BLA secretion was
temperature-dependent ( , 4 °C;
, 15 °C;
,
37 °C) but not dependent upon protein synthesis over the course of
the assay (
= cycloheximide). Results represent mean ± S.D. of
two experiments done in triplicate. Since the fraction of total BLA
secreted could not be accurately determined by enzymatic assay (see
"Materials and Methods"), the amount of BLA protein released was
quantitated by immunoblot (B). B, immunoblot.
Top, detection of BLA in the supernatant and in the pellet
from 5 × 106 parasites by SDS-PAGE and immunoblot,
following incubation of parasites at 37 °C in AISS buffer.
Bottom, the percentage of BLA released was calculated by
densitometric scanning of the immunoblot. 15-18% of total parasite
BLA was secreted by extracellular parasites after 1 h at
37 °C
We therefore asked whether conditions could be identified that might trigger or inhibit DG secretion. Based on suggestions that there is a burst of DG release after parasite entry (3, 6), we reasoned that intracellular buffer conditions might augment release in comparison with extracellular buffer conditions. No difference in GRA3 or BLA release was observed when parasites were incubated in high sodium (HBSS) versus high potassium (AISS) buffer, except at later time points in the incubation for GRA3 (Fig. 1A). Secretion was not influenced by adding extracellular calcium or by chelating calcium with EGTA (Table I), although chelation of intracellular calcium with MAPTA-AM resulted in some inhibition of DG secretion. The addition of ATP, ADP, or AMP in the buffer had no effect on GRA3 or BLA release, nor was secretion altered by the addition of 10 mM glutathione at varying reduced:oxidized ratios. In contrast to one previous report (10), the addition of heat-inactivated fetal bovine serum in AISS buffer did not further enhance GRA3 or BLA release (Table I). In summary, these results suggest that DG release by extracellular T. gondii occurs constitutively.
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Dense Granule Release in Permeabilized Parasites Is ATP-dependent
To facilitate the identification of signaling molecule(s) and protein machinery involved in DG secretion, we established a permeabilized cell secretion assay with T. gondii using streptolysin O as permeabilizing agent. Under the conditions developed for permeabilization, 50-60% of parasites were permeabilized by 1 unit/ml of SLO (assessed by propidium iodide staining of the nucleus; not shown). Approximately one-quarter (see legend to Fig. 3A for calculation) of the cytosolic marker, green fluorescent protein, was released from permeabilized transgenic parasites (34) (Fig. 3A), showing that macromolecules are depleted.
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Secretion of BLA was inhibited by 49 ± 19% in SLO-permeabilized parasites as compared with untreated controls (Fig. 3B). The extent of BLA release during permeabilization was no greater than in controls incubated at 37 °C without SLO. The addition of an ARS restored secretion to 81 ± 20% of control. ARS had no effect on BLA secretion from nonpermeabilized parasites, further confirming our results (Table I). Preincubation of permeabilized parasites with an ATP depletion system (195 units/ml of hexokinase 10 mM D-glucose at 15 °C for 30 min) resulted in a further 15-20% inhibition of secretion in the absence of ATP, in comparison with permeabilized parasites not treated with hexokinase and D-glucose (data not shown). This is likely to be the base line for secretion in the absence of ATP, since only 40-60% of parasites were permeabilized by SLO. Altogether, these results show that ATP is necessary for DG secretion.
Neither Calcium nor cAMP Triggers DG Secretion from Permeabilized Parasites
An increase in intracellular calcium serves as a signal for secretion of dense matrix granules in many cells (42). In T. gondii, however, the addition of up to 100 µM calcium to SLO-permeabilized parasites had no effect on BLA secretion in either the presence or absence of ARS. Chelation of intracellular calcium with EGTA also had no effect on DG secretion (Fig. 4). MAPTA-AM, a more effective calcium chelator, inhibited BLA secretion by approximately 18% (Table II), a value somewhat lower than the inhibition by MAPTA-AM in nonpermeabilized extracellular parasites (Table I). Since cAMP can trigger granule exocytosis in the absence of calcium in some cells (43-45), we added cAMP to SLO-permeabilized parasites. As shown in Table II, cAMP did not alter BLA release. In combination with the data presented in Table I, these results further argue that DG release in T. gondii is constitutive.
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The Involvement of NSF, -SNAP, and GTP-binding Proteins in DG
Exocytosis in T. gondii
We next explored the protein machinery mediating DG release in T. gondii. In other eukaryotic systems, NSF, the SNAP-SNARE machinery, and the Rabs have been shown to play important roles in exocytosis of regulated secretory organelles (14, 17, 18, 21-25).
Involvement of NSF-- Treatment with 1 mM of the alkylating agent, NEM, resulted in 80 ± 4% inhibition of BLA secretion from nonpermeabilized parasites and 65 ± 3% inhibition of BLA secretion from permeabilized parasites in the presence of ARS (Fig. 5A). Although use of NEM at 1 mM has been previously used to inactivate NSF, NEM may have other effects. We therefore tested the effects of adding purified recombinant hamster NSF to permeabilized parasites. When parasites were incubated with recombinant hamster NSF and ARS, DG release was augmented in comparison with ARS alone (Fig. 5B). In contrast, incubation of parasites with the N domain of NSF, a fragment devoid of ATPase activity, did not augment BLA secretion from permeabilized parasites, arguing for the specificity of the NSF effect (Fig. 5B).
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Involvement of -SNAP--
Treatment of permeabilized parasites
with recombinant bovine
-SNAP resulted in stimulation of BLA
secretion. Maximum secretion occurred at 2 µg/ml of
-SNAP, in the
presence of ARS (Fig. 6A). Based on this dose-response curve, 2 µg/ml
-SNAP was used in subsequent experiments. Secretion was augmented by
-SNAP in the presence of ARS (Fig. 6B).
-SNAP alone, in the absence of
ARS, had no effect on DG secretion. These results suggest that DG
secretion is regulated by
-SNAP and that heterologous
-SNAP
proteins can interact with the SNARE/SNAP machinery to control DG
docking and/or fusion.
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Involvement of GTP-binding Proteins--
Finally, we tested the
involvement of GTP-binding proteins in DG secretion. The addition of
100 µM GTPS resulted in significant enhancement of BLA
secretion (38 ± 4%) from permeabilized parasites in the presence
of ARS (Table II), suggesting that GTP-binding proteins regulate
DG exocytosis in T. gondii. To specifically explore the
involvement of Rabs in DG secretion, SLO-permeabilized parasites
were incubated with 4 µg/ml recombinant bovine Rab-GDI. This resulted
in 11 ± 4% inhibition of DG secretion (data not shown). This
difference, while small, was reproducible and is similar to the levels
reported in other systems.
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DISCUSSION |
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The molecular machinery controlling secretion of dense core granules is broadly conserved evolutionarily. Nonetheless, until this study, there has been no direct evidence that this machinery functions in pathogenic unicellular protozoan parasites, either for constitutive or regulated secretion. In contrast to this conserved aspect of dense granule release, secretion of T. gondii dense granules is calcium-independent, reflecting the unusual nature of the protein sorting machinery within the organism (31).
In most systems, secretion from dense matrix granules is regulated by changes in intracellular calcium concentration (11), while constitutive transport between the trans-Golgi network and plasma membrane is calcium-independent (46, 47). Preliminary experiments indicated that treatment of extracellular parasites with (i) the calcium ionophore A23187, (ii) the ER CaATPase inhibitor thapsigargin, or (iii) NH4Cl, (under conditions reported to increase intracellular calcium levels in T. gondii; Ref. 48) had no effects on secretion of GRA3 and BLA (data not shown). Theoretical considerations also support the notion that increased intracellular calcium is unlikely to be the trigger for continuous exocytosis of dense granules from intracellular T. gondii, since the parasite resides in a low calcium environment and would not be capable of rapidly replenishing intracellular calcium by opening plasma membrane calcium channels (reviewed in Ref. 49). In order to examine this question further and more precisely control intracellular calcium levels, we developed a permeabilized cell system and unequivocally demonstrated that increases in intracellular calcium do not trigger DG release.
Spontaneous release of "regulated proteins" in the absence of a stimulus is generally ascribed to either low level release of dense core granules or simultaneous delivery of the regulated proteins to both regulated and constitutive secretory vesicles (50, 51). The distinction between basal and constitutive release of dense core vesicles depends on the capacity to identify triggers (typically a rise in intracellular Ca2+) for granule release in the former instance (46). The results shown above demonstrate that DG release in T. gondii is not triggered by an increase in intracellular Ca2+, precluding the use of this signal to make the distinction between basal and constitutive release. Based on our previous results with brefeldin A (31), showing quantitative delivery of GRA3 and BLA to DG, and results here showing that short term treatment of extracellular parasites with cycloheximide had no effect on DG release, it is unlikely that DG proteins are simultaneously delivered to DG and constitutive secretory vesicles. An alternative possibility is that the DG in Toxoplasma are analogous to the immature secretory granules in endocrine and exocrine cells (52-58) and that secretion occurs via budding of small constitutive secretory vesicles from the DG. Vesicle budding from immature secretory granules in mammalian cells is brefeldin A-sensitive (59, 60), arguing against this explanation for constitutive secretion of dense granule proteins in T. gondii. Moreover, neither we nor others have observed structures compatible with immature secretory granules in the organism. Taken in total, the results are most consistent with the notion that DG secretion is constitutive in T. gondii.
The discovery that hamster NSF and bovine -SNAP augment DG secretion
provides the first functional demonstration that the NSF/SNAP/SNARE
system operates in a pathogenic protozoan parasite. Although we do not
yet have direct evidence for the involvement of endogenous T. gondii components, the function of hamster and bovine proteins in
the parasite system suggests that the T. gondii homologous
will be structurally similar with their mammalian counterparts (reviewed in Ref. 27).
Numerous Rabs have been identified in Toxoplasma (61), but the specific molecules involved in regulating DG secretion are not known. Since Rab-GDI exhibits broad substrate specificity, even across species barriers (reviewed in Refs. 62 and 63), the activity of bovine protein in T. gondii does not reveal which isoform of Rab is involved. Other experiments in our laboratory suggest that both T. gondii Rab6 and Rab11 are involved in DG secretion.2 In light of the inability to demonstrate calcium-regulated secretion in Toxoplasma, however, it is interesting to note that Rab3, commonly associated with calcium-dependent exocytosis (25, 64), has not been identified in this parasite.
Treatment of permeabilized parasites with the proteolytically active light chain of tetanus toxin, which cleaves synaptobrevin/VAMP (65) and inhibits both calcium-dependent (66) and calcium-independent (67) granule secretion, had no effect on BLA release (data not shown). Unfortunately, interpretation of this experiment must await identification of T. gondii VAMP homolog, because not all VAMP/synaptobrevins are cleaved by TeTx (66).
The molecular basis for the GTPS-mediated enhancement of DG
secretion is unknown. Calcium-independent GTP
S-augmented secretion is also observed in mast cells and neutrophils (68, 69), where it is
mediated by ARF-1 activation of phospholipase D to generate phosphatidylinositol bisphosphate (70). This is the most likely explanation for the GTP
S-mediated enhancement of DG exocytosis in
T. gondii, where GTP
S is revealing the components
involved in the constitutively occurring process. Alternatively, it is possible that a parasite G-protein-coupled plasma membrane receptor could be linked to DG exocytosis. Heterotrimeric GTP-binding proteins have been identified in T. gondii, and AlF is reported to
increase the extrusion of filamentous structures from the parasite
surface (71), although in our hands BLA release was altered by NaF
alone, making interpretation of these results difficult. By identifying the mechanism for the GTP
S-augmented secretion, the possible contribution of a regulated component to DG exocytosis (3, 6) in
T. gondii may also be defined.
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ACKNOWLEDGEMENTS |
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We thank Reinhard Jahn, Norma Andrews, and members of the Joiner laboratory for useful discussions. We acknowledge the generous gift of Rab-GDI from N. Ayad and I. Mellman (New Haven, CT) and of tetanus toxin light chain from D. Bruns (New Haven, CT).
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FOOTNOTES |
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* This work was supported by Public Health Service Grant RO1 AI30060 from the National Institutes of Health and a Scholar Award in Molecular Parasitology from the Burroughs Welcome Fund (to K. A. J.).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 Cell Biology, Washington University School of Medicine, 4566 Scott Ave., Campus Box 8228, St. Louis, MO 63110.
** To whom correspondence should be addressed: Keith A. Joiner, Section of Infectious Diseases, LCI 808, Yale University School of Medicine, P.O. Box 208022, New Haven, CT 06520-8022. Tel.: 203-785-4140; Fax: 203-785-3864; E-mail: Keith.Joiner{at}yale.edu.
The abbreviations used are:
DG, dense granule(s); BLA, -lactamase; ARS, ATP-regenerating system; NEM, N-ethylmaleimide; NSF, N-ethylmaleimide-sensitive
fusion protein; SNAP, soluble NSF attachment protein; GDI, guanine
dissociation inhibitor; SNARE, SNAP receptor; GFP, green fluorescent
protein; BSA, bovine serum albumin; HBSS, Hanks' balanced salt
solution; PBS, phosphate-buffered saline; ELISA, enzyme-linked
immunosorbent assay; GTP
S, guanosine
5'-3-O-(thio)triphosphate; ATP
S, adenosine
5'-O-(thiotriphosphate); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; VAMP, vesicle-associated membrane protein; MAPTA-AM, bis-(2-amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic
acid; SLO, streptolysin o.
2 T. Stedman and K. A. Joiner, unpublished observations.
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REFERENCES |
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