From the Dental Research Institute, the
§ Canadian Institutes of Health Research Group in
Periodontal Physiology, Faculty of Dentistry, and the
Department
of Surgery, University of Toronto and the Division of Surgery, Toronto
General Hospital, Toronto, Ontario M5G 1G6, Canada
Received for publication, December 27, 2000, and in revised form, April 11, 2001
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ABSTRACT |
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The cytotoxicity of infectious agents
can be mediated by disruption of calcium signaling in target cells.
Outer membrane proteins of the spirochete Treponema
denticola, a periodontal pathogen, inhibit agonist-induced
Ca2+ release from internal stores in gingival fibroblasts,
but the mechanism is not defined. We determined here that the major
surface protein (Msp) of T. denticola perturbs calcium
signaling in human fibroblasts by uncoupling store-operated channels.
Msp localized in complexes on the cell surface. Ratio fluorimetry
showed that in cells loaded with fura-2 or fura-C18, Msp induced
cytoplasmic and near-plasma membrane Ca2+ transients,
respectively. Increased conductance was confirmed by fluorescence
quenching of fura-2-loaded cells with Mn2+ after Msp
treatment. Calcium entry was blocked with anti-Msp antibodies and
inhibited by chelating external Ca2+ with EGTA. Msp
pretreatment reduced the amplitude of [Ca2+]i
transients upon challenge with ATP or thapsigargin. In experiments
using cells loaded with mag-fura-2 to report endoplasmic reticulum
Ca2+, Msp reduced Ca2+ efflux from endoplasmic
reticulum stores when ATP was used as an agonist. Msp alone did not
induce Ca2+ release from these stores. Msp inhibited
store-operated influx of extracellular calcium following intracellular
Ca2+ depletion by thapsigargin and also promoted the
assembly of subcortical actin filaments. This actin assembly was
blocked by chelating intracellular Ca2+ with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid acetoxymethyl ester. The reduced amplitude of
agonist-induced transients and inhibition of store-operated
Ca2+ entry due to Msp were reversed by latrunculin B, an
inhibitor of actin filament assembly. Thus, Msp retards
Ca2+ release from endoplasmic reticulum stores, and it
inhibits subsequent Ca2+ influx by uncoupling
store-operated channels. Actin filament rearrangement coincident with
conformational uncoupling of store-operated calcium fluxes is a novel
mechanism by which surface proteins and toxins of pathogenic
microorganisms may damage host cells.
What if pathogenic microorganisms were able to disrupt the
regulation of intracellular calcium signals in host cells? They would
disturb a critical signaling pathway by which eukaryotic cells respond
to external stimuli (1) and would impair crucial downstream functions
by which cells maintain homeostasis. For example, calcium ion
concentration
([Ca2+]i)1
regulates the assembly and turnover of actin filaments (2), which in
turn controls cell shape, volume regulation, locomotion, and
phagocytosis. These are essential functions for maintaining tissue
integrity, for promoting wound healing, and for mediating protection
from infection.
Many human cells regulate calcium homeostasis using store-operated
calcium fluxes, a system in which Ca2+ influx through
store-operated channels (SOCs) in the plasma membrane is stimulated by
the depletion of Ca2+ in internal stores in the lumen of
the endoplasmic reticulum (ER) (3, 4). A variety of potential
cytotoxins of insects, reptiles, and microorganisms affect ion channel
function (5-7). These toxins may include the surface molecules of
opportunistic or overtly pathogenic bacteria, including some that cause
cytoskeletal perturbation or that form ion channels in the host cell
plasma membrane. Several microbial products have been reported to alter ion transport, but none is known to uncouple Ca2+ ER stores
and SOCs.
Although the mechanisms by which transient Ca2+ depletion
in the ER triggers Ca2+ influx through SOCs are not well
understood, one compelling model is based on the physical proximity
between the plasma membrane and the ER (1). This system may involve the
inositol trisphosphate receptor on the ER as a mediator of the
functional coupling of ER store depletion and Ca2+ entry
through the plasma membrane (8, 9). Notably, the assembly and
disassembly of actin filaments subjacent to the plasma membrane is a
major determinant of reversible coupling of the ER and plasma membrane
and thereby modulates the signals that regulate Ca2+ influx
through SOCs (8, 10).
Whole cells and outer membrane proteins of the oral spirochete
Treponema denticola exert profound effects on actin assembly in cultured human gingival fibroblasts and epithelial cells (11-14). These properties may contribute to bacterial pathogenicity by impeding
actin-dependent functions that are crucial for homeostasis of gingival tissues (15, 16). Since actin assembly is highly sensitive
to fluctuations in calcium concentration (2), it is notable that outer
membrane fractions of T. denticola inhibit inositol
phosphate and intracellular calcium responses of fibroblasts to
chemical agonists (14, 17). Since cortical actin and the inositol
trisphosphate receptor are evidently important in the regulation of
store-operated calcium channels in a variety of cell types (8, 18-20),
we hypothesized that the major surface protein of T. denticola, Msp,2 may be
one of the outer membrane proteins that is responsible for bacterial
perturbation of calcium flux in cells from the periodontium. We
examined the effects of Msp on calcium homeostasis in gingival fibroblasts and report that Msp mediates calcium transients and inhibits SOC activation by promoting assembly of subcortical actin filaments. These filaments in turn uncouple the ER from SOCs. Our
results provide a novel mechanism by which pathogenic bacteria may
subvert host cell signaling systems and thereby contribute to loss of
tissue homeostasis in chronic infections such as periodontitis.
Reagents--
Bovine serum albumin, Texas Red-conjugated goat
anti-rabbit antibodies, thapsigargin, ATP, ionomycin, latrunculin B,
and valinomycin were purchased from Sigma. Fura-2/AM, mag-fura-2/AM,
fura-C18, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid acetoxymethyl ester, bis-[1,3-dibutylbarbituric
acid]trimethineoxonol, and rhodamine phalloidin were obtained from
Molecular Probes, Inc. (Eugene, OR). 2-Aminoethoxydiphenyl borate
(2-APB) was obtained from Calbiochem.
Preparation of Msp--
Msp is an immunogenic surface protein of
T. denticola that complexes with a serine protease, PrtP, in
the outer sheath of the bacterium. Msp was prepared from T. denticola type strain ATCC 35405, as previously described (21,
22), with the following modifications. The starting material was ~8
g, wet weight, per 4-liter bacterial culture in late logarithmic growth
phase. After the deoxycholate and the n-octylpolyoxyethylene
extraction steps and ultracentrifugation, the enriched Msp solution was
incubated for 7 days at 37 °C until peptidase activity could no
longer be detected by the chromogenic peptides
N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine nitroanilide and
N-benzoyl-DL-arginine-p-nitroanilide (Sigma) (23). The enriched protein solution was concentrated ~30-fold by
Amicon ultrafiltration (Amicon Concentricon plus 80, Amicon Inc.,
Beverly, MA). After washing with 5 liters of 10 mM Tris (pH
8.0), five washings with double-distilled water, and
ultracentrifugation for 2 h to remove trace amounts of detergent,
the Msp was dissolved and dialyzed against double-distilled water. The
protein content was determined by Bio-Rad assay using bovine serum
albumin as a standard.
For all experiments (except dose-response experiments), a 1:100
dilution of Msp in calcium buffer was used. This preparation contained
0.41 mg of protein (dry weight) per ml. A final concentration of 30 µg of protein/ml, corresponding to ~160 nM, was used
for most challenge assays of cells. When pretreatment with Msp was specified, the Msp remained in the cell culture wells for the duration
of the experiment unless mentioned. In some experiments, Msp was boiled
for 10 min to denature proteins or heated at 60 °C for 30 min. In
other experiments, the cells were pretreated with a mixture of Msp and
anti-Msp antibodies at a 2:3 ratio (protein concentration) that had
been preincubated for 60 min.
Gel Electrophoresis and Immunoblotting--
SDS-polyacrylamide
gel electrophoresis was performed as previously described in 10% (w/v)
polyacrylamide gels with a current of 200 V (24, 25). Proteins or
peptides were solubilized in sample buffer either at room temperature
or heated at 100 °C for 5 min prior to electrophoresis and staining
with Coomassie Brilliant Blue. For immunoblotting, proteins were
transferred electrophoretically to nitrocellulose membranes (26).
Following transfer, membranes were blocked with 5% bovine serum
albumin in Tris-buffered saline (20 mM Tris, 0.5 M NaCl, pH 7.6) and probed with rabbit polyclonal antibodies raised against Msp of ATCC 35405, kindly provided by B. C. McBride and P. Hannam (27). After incubation with horseradish peroxidase-conjugated secondary antibodies at appropriate dilutions, enhanced chemiluminescence (Amersham Pharmacia Biotech) was used to
detect the signal.
Cell Culture--
Human gingival fibroblasts were derived from
primary explant cultures as described (28). Cells from passages 6-15
were grown as monolayers in T-75 flasks in Intracellular Calcium--
For measurement of whole cell
intracellular calcium ion concentration
([Ca2+]i), cells on coverslips were loaded with 3 µM fura-2/AM (a Ca2+-sensitive fluorescent
dye) for 20 min at 37 °C (17). For measurement of near-plasma
membrane Ca2+ concentration, cells attached to coverslips
were briefly permeabilized with saponin and incubated with 2 µM fura-C18, pentapotassium salt (29) at 25 °C for 10 min followed by three washes with PBS. For estimation of
[Ca2+]ER, cells on coverslips were incubated
with mag-fura-2/AM (4 µM) (30) for 150 min at 37 °C,
in
After incubation with fura-2/AM, inspection of cells by fluorescence
microscopy demonstrated no vesicular compartmentalization of fura-2,
suggesting that the dye loading method permitted measurement of
cytosolic [Ca2+]i. Visual inspection of
fura-C18-loaded cells and mag-fura-2-loaded cells showed fluorescent
labeling of the plasma membrane and intracellular organelles,
respectively. Estimates of whole cell [Ca2+]i and
near plasma membrane [Ca2+]i were calculated
according to the equation of Grynkiewicz (32) from the emitted
fluorescence measurements, using a Nikon Diaphot II inverted microscope
optically interfaced to a Deltascan 4000, dual beam, epifluorescence
spectrofluorimeter and analysis system (Photon Technology Int., London,
Ontario, Canada), as described previously (17).
Msp Perturbation of Calcium Fluxes--
To examine the effect of
Msp on internal release of Ca2+, ATP (100 µM;
Sigma) and thapsigargin (1 µM; Sigma) were used as
agonists and added to resting cells in calcium-free buffer with EGTA
(17). Cells that were pretreated with Msp for 40 min at 25 °C were
compared with untreated cells. To compare the function of SOCs in these cells, the influx of extracellular calcium following depletion of
intracellular stores was also studied. Fibroblasts were exposed to 1 µM thapsigargin in 1 mM EGTA buffer for 30 min to deplete internal Ca2+ and to chelate residual
extracellular Ca2+. CaCl2 (2 mM)
was added, and [Ca2+]i increases due to calcium
influx were measured. These experiments were repeated in cells
pretreated with 1 µM latrunculin B (30 min, 25 °C), an
inhibitor of actin polymerization.
In some experiments, the influx pathway was examined after the addition
of Msp or water vehicle in cells loaded with fura-2. We measured the
fluorescence of single cells at the isosbestic point (356 nm) in the
presence of MnCl2 (1 mM) in the standard Ca2+-containing buffer (33). For indirect assessment of
general cation permeability of the plasma membrane to monovalent
cations, we measured membrane potential using flow cytometry and the
bisoxonol dye bis-[1,3-dibutylbarbituric acid]trimethineoxonol (3) as described (34).
Immunocytochemistry and Confocal Microscopy--
To examine the
location of Msp binding to cells, indirect immunofluorescence
microscopy was performed on Msp-pretreated cells using polyclonal
anti-Msp-antibodies as the primary antibody. Cells grown on coverslips
were fixed and permeabilized with methanol at
Laser-scanning confocal microscopy (Leica CLSM) was used to examine
actin filament assembly induced by Msp. The cells were fixed in 3%
paraformaldehyde for 15 min, permeabilized in 0.2% Triton X-100 for 5 min, and stained with rhodamine phalloidin (35). The excitation filter
used was 530/20 nm, and emitted fluorescence was collected through an
emission filter (620/40 nm). Cells were imaged with a × 63 oil
immersion lens, 1.4 NA, and transverse optical sections were obtained
in 1-µm steps from the level of cell attachment at the substratum to
the dorsal surface of the cell (33). In some experiments, the
fluorescence intensity of rhodamine phalloidin staining was measured
with a fluorescence spectrophotometer (Leitz MVP; Wetzlar, Germany)
(28) in square sampling zones (25 µm2) immediately
beneath the cell membrane.
Cell Viability--
Propidium iodide staining was used to
determine if Msp was toxic to cells during the duration of the
experiments. Flow cytometry analyses were performed as described (36).
Briefly, cells were trypsinized and suspended in PBS, pelleted,
resuspended in PBS to a cell concentration of 1 × 106/ml, and stained with propidium iodide (10 µl/ml;
Calbiochem). After a 5-min incubation, cells were analyzed by flow cytometry.
Data Analysis--
Means and S.E. values were
calculated for [Ca2+]i measurements. Calcium
measurements were restricted to (i) base-line [Ca2+]i; (ii) percentage change of the transient
[Ca2+]i above base-line; (iii) net change in
[Ca2+]i above base line; and (iv) time to peak
[Ca2+]i. For continuous variable data, means and
S.E. of [Ca2+]i were computed, and, when
appropriate, comparisons between two groups were made with the unpaired
Student's t test with statistical significance set at
p < 0.05.
Preparation of Msp--
The Msp preparation was highly enriched by
sequential detergent extraction and autoproteolysis of T. denticola extracts. We isolated a fraction that contained
primarily Msp oligomers, which migrated as a single ~190-kDa band in
SDS-polyacrylamide gel electrophoresis (21) (Fig.
1A). Boiled samples
demonstrated a 53-kDa polypeptide that is known to be the monomer (27,
37). The 95-kDa chymotrypsin-like protease that usually associates with
Msp in the outer membrane was not detected in the gels; nor was its
peptidase activity detected. Immunoblots of the preparation using
anti-Msp antibodies showed almost exclusive staining of the prominent
190-kDa oligomer and the 53-kDa monomer in native and heat-denatured
preparations, respectively (Fig. 1B), confirming that these
bands contained the authentic Msp from T. denticola.
Previous studies demonstrated that Msp binds to fibronectin,
fibrinogen, and laminin and associates with epithelial cell surfaces and cytoplasmic membrane proteins (22, 37). When the Msp preparation was incubated with fibroblasts, immunostaining showed that Msp associated with the fibroblast surface (Fig. 1C). After a
30-min incubation, intense Msp staining was clustered in one area of the cell, indicating a capping phenomenon and possible aggregation of
ligand-bound receptors.
Msp Induces Ca2+ Transients--
Single cell ratio
fluorimetry was used to test whether the association of Msp with the
fibroblast plasma membrane caused perturbation of calcium homeostasis.
For all experiments, [Ca2+]i was stable in
untreated cells, even over prolonged time course experiments (>120
min). In calcium buffer, exposure of cells to Msp induced a robust
Ca2+ transient that often returned to a level ~15-30%
higher than base-line within 90 s of the peak (Fig.
2, A-C); subsequent smaller transients could also be detected over a period of 30 min in some cells
(Fig. 2B, inset). Treatment of cells with
increasing concentrations of Msp (1, 10, 30 µg/ml) showed a
dose-response relationship with the amplitude of the calcium peak (Fig.
2H). In control cells treated with the vehicle (distilled
H2O added to cell culture buffer), there was no
Ca2+ response (Fig. 2D), indicating that the
Msp-induced Ca2+ responses were not simply due to physical
disturbance or an anisosmotic change. Further, only cells with bright
fura-2 staining in the cytoplasm were included in analyses to overcome
the possibility of measuring dead or dying cells (see data on cell
death below). In cells challenged with Msp that was preincubated with
anti-Msp antibodies, there was no Ca2+ response detected
upon the addition of Msp (Fig. 2E), indicating that the
Msp-induced transient was due to a specific interaction between Msp and
the cells. Boiling the Msp eliminated the Ca2+ response
(Fig. 2F); the amplitude of the Ca2+ transient
was much lower than controls when the Msp was heated to 60 °C for 30 min (Fig. 2G). Thus, the ability of Msp to interact with
cells and to generate Ca2+ responses evidently depends on
the undenatured protein complex. Finally, cells that were incubated
repeatedly with fresh doses of Msp showed progressively diminished
calcium responses (Fig. 2I).
To determine if Msp could induce Ca2+ transients subjacent
to the cell membrane, cells were loaded with fura-C18. In these
experiments Msp evoked large amplitude calcium transients,
demonstrating a sharp elevation of [Ca2+]i near
the plasma membrane (Fig. 2J). These transients occurred
more rapidly than the cytoplasmic calcium signals following Msp
treatment (n = 10 experiments; p < 0.05), indicating that Msp may stimulate Ca2+ influx
through plasma membrane-permeable channels. We determined the source of
Ca2+ for the observed rise in
[Ca2+]i. Fura-2-loaded cells were incubated in
Ca2+-free buffer containing EGTA. The cells were rapidly
switched to the EGTA buffer prior to the addition of Msp to avoid
depletion of intracellular stores. Under these conditions, Msp
treatment failed to evoke a defined Ca2+ transient (Fig.
2K), indicating that most of the Ca2+ for the
robust intracellular response was probably derived from influx through
plasma membrane-permeable channels. We examined the influx pathway
further by performing quenching experiments in which the extracellular
bathing buffer was supplemented with 1 mM MnCl2
and the fluorescence of fura-2 was excited at 356 nm (the isosbestic or
calcium-insensitive point) (33). When single emission photon counts
were measured, cells demonstrated a drop in fluorescence ~90 s after
Msp was added to the culture medium, probably due to influx of
Mn2+ and fluorescence quenching of fura-2 (Fig.
2L). These results indicate that the Msp-induced
Ca2+ transient is due to entry from the extracellular medium.
To assess whether Msp causes a general increase in cation permeability,
we measured the membrane potential with the bisoxonol dye
bis-[1,3-dibutylbarbituric acid]trimethineoxonol and flow cytometry.
Measurement of bis-[1,3-dibutylbarbituric acid]trimethineoxonol fluorescence after vehicle or Msp treatment showed only a 2% reduction of fluorescence, while KCl depolarization in the presence of
valinomycin (3 µM) produced large reductions (~25%
reductions after each of the sequential additions of 50 mM
KCl; 5000 cells/run). Experiments conducted with calcium buffer and
ionomycin (2 µM) treatments indicated that cells
preincubated with Msp or vehicle responded with an equivalent increase
in [Ca2+]i (p > 0.2;
n = 5 cells each). These data indicated that the cell
membranes of Msp-treated cells were intact. Consistent with these
observations, the basal [Ca2+]i was unchanged in
Msp-treated and vehicle-treated cells (~150 nM).
We determined if Msp is cytotoxic to fibroblasts in the short term
(i.e. under the conditions used for these experiments) because of a rise in intracellular Ca2+ (38). Propidium
iodide staining experiments were done as described under
"Experimental Procedures." Flow cytometry analysis showed that
there were ~15% propidium iodide-permeable cells after treatment with Msp (30 µg/ml, 25 °C) for 40 min compared with ~9% for
control cells (n = 5; p < 0.05).
Therefore, most of the cells that were pretreated with Msp remained
viable under the conditions of the experiments; those that were
permeable to propidium iodide also exhibited less fura-2 staining and
were not included in the measurements of [Ca2+]i.
In contrast, after 24 h of exposure to Msp (10 µg/ml, 37 °C),
there were ~66% propidium iodide-permeable cells after treatment
(n = 5; p < 0.05), which is consistent
with both the concentration and time course for the cytotoxicity of Msp
complex reported previously (39). An alternative viability assay based on a replating and cell attachment method (40) showed no difference in
viability between control and Msp-challenged cells over a 60-min incubation (data not shown).
Msp Inhibits Ca2+ Release from Internal
Stores--
Outer membrane extracts of T. denticola inhibit
agonist-induced increases of [Ca2+]i in human
gingival fibroblasts (17). We used two agonists capable of generating
internal calcium release to study the effect of Msp pretreatment on
calcium release from intracellular stores. ATP (200 µM)
or thapsigargin (1 µM) was incubated with cells in
calcium-free buffer containing 1 mM EGTA, a protocol that
ensures that the induced calcium transients were attributable solely to
release from internal calcium stores. Prior to the addition of
agonists, the calcium-free buffer was quickly switched with the
calcium-containing buffer to minimize calcium depletion from the cells.
Indeed, measurement of the resting [Ca2+]i values
showed no significant reduction after EGTA treatment under these
conditions ([Ca2+]i ~150 nM). In
cells treated with ATP, there was a nearly 2-fold increase of
[Ca2+]i above basal levels (Fig.
3A; p < 0.05). In contrast, pretreatment of cells with Msp (40 min; 25 °C)
followed by stimulation with ATP showed a 70% reduction of the
amplitude of ATP-induced calcium transients. In an identical
experimental design, the mean amplitude of the calcium transient
induced by thapsigargin was 80% less in the Msp-treated cells than
controls (Fig. 3B; p < 0.05). We ran
analogous experiments using 2-APB, an inhibitor thought to act by
interference with InsP3 receptors of the ER (9); 2-APB (75 µM) reduced the thapsigargin-releasable Ca2+
transients by ~50% (maximum [Ca2+]i change (in
nM) as follows: control cells, 127.5 ± 20.9;
2-APB-pretreated cells, 66.7 ± 6.7; p < 0.05;
n = 5 per treatment). These experiments seemed to
indicate that Msp interferes with the process of Ca2+
release from internal stores. The effect of Msp on ATP-induced Ca2+ transients was progressively reversed during increased
time periods following wash-out of free Msp from the assay buffer (Fig.
3C). Notably, the cells were partially protected from the
effect of Msp on both ATP- and thapsigargin-induced Ca2+
transients by pretreatment with the subcortical actin filament assembly
inhibitor latrunculin B (1 µM; Fig. 3, A and
B); the reversal was greater for the thapsigargin- than the
ATP-challenged cells. Thus, the effect of Msp on the release of
Ca2+ from ER stores in response to these agonists is
probably functionally linked, in part, to its effects on the
cytoskeleton and SOCs (see below).
For estimation of Ca2+ concentration in the ER stores,
cells were loaded with mag-fura-2 according to the methods of Hofer
et al. (30), and the mag-fura-2 emission ratio was
calculated. Fluorescence microscopic examination of these cells showed
that the dye was compartmentalized in discrete vesicular zones that closely resembled previously published images (30). Digitonin experiments as well as stimulation with ATP showed that the mag-fura-2 loading protocol was indeed reporting on calcium stores with properties similar to that of the endoplasmic reticulum. Msp produced no abrupt
change in the mag-fura-2 ratio (Fig. 3D), but it did cause a
slight decline in base-line measurements over the time course of the
experiment. Pretreatment of cells with Msp (25 °C, 40 min) followed
by incubation with ATP (200 µM) blocked the ATP-induced reduction of the mag-fura-2 ratio (Fig. 3E). In contrast,
treatment with ATP alone induced a sharp reduction of the mag-fura-2
ratio followed by a rapid recovery to base-line (Fig. 3F),
indicating that ATP induced calcium efflux from ER stores. Traces of
the mag-fura-2 ratio of single cells (Fig. 3, E and
F) and summary analyses (Fig. 3G) showed that
pretreatment with Msp lowered the basal mag-fura-2 ratio and the rate
or amount of Ca2+ release from the ER stores upon ATP
challenge. Collectively, these data seemed to suggest that Msp may
either block calcium release or affect the kinetics of depletion and
replenishment cycles of internal Ca2+ stores.
Alternatively, we considered that the combined effects of Msp and ER
agonists may have so completely depleted intracellular stores that
there was no more calcium to be released after ATP or thapsigargin
treatment. Accordingly, we performed experiments in which cells were
rapidly switched over to an EGTA (1 mM), calcium-free buffer prior to Msp or vehicle treatment and then treated with ionomycin (2 µM). In contrast to the ATP- or
thapsigargin-induced response (Fig. 3, A and B;
also see Fig. 4, C and
D), the ionomycin-triggered Ca2+ transients were
not different in control and Msp-pretreated cells. Indeed, both the
amplitude (Fig. 4, A and B; summary analyses: vehicle = 263 ± 22 nM; Msp = 250 ± 25 nM; p > 0.2; n = 4 per
treatment) and the kinetics of the transients were similar in vehicle-
and Msp-pretreated fibroblasts. These results indicate that the
inhibitory effect of Msp on the release of Ca2+ from the
stores was not due to depletion of the stores prior to adding the
agonists. Rather, Msp appears to specifically reduce the rate and/or
the amount of agonist-induced Ca2+ release, per
se. To determine whether Msp affected the total amount of the
released Ca2+ or the kinetics of the release, we further
analyzed the thapsigargin-induced [Ca2+]i
transients. To this end, the cells were incubated in the presence of
EGTA (1 mM) and then challenged by thapsigargin. Finally,
to assess the amount of releasable Ca2+ after thapsigargin
treatment, ionomycin (2 mM) was added. The total
Ca2+ released by thapsigargin, determined by measuring the
area under the curve of [Ca2+]i traces, was only
marginally lower for the Msp-pretreated cells (vehicle = 9.13 ± 0.25 × 10 Msp Inhibits Store-operated Calcium Influx--
We determined if
Msp may inhibit the refilling of internal Ca2+ stores by
blocking the influx of extracellular calcium following depletion of
these stores. Cells were pretreated with thapsigargin, and the medium
was augmented with EGTA (1 mM) to deplete the stores and
prevent refilling. As shown above, after sufficient time, the ER stores
were similarly depleted in control and Msp-pretreated cells. After the
addition of CaCl2 (2 mM),
[Ca2+]i rose to a peak and then returned to a
somewhat higher base-line (Fig.
5A). The maximum influx of
Ca2+ was diminished >2-fold (p < 0.05) in
Msp-treated cells (Fig. 5, B and C). This finding
suggests that Msp may have inhibited the influx of extracellular
calcium through SOCs and uncoupled the feedback connection between
internal Ca2+ release and store-operated calcium entry.
Msp Induces Actin Reorganization in Human Gingival
Fibroblasts--
Since T. denticola causes actin
rearrangement in fibroblasts (11), we determined if Msp may inhibit
activation of SOCs by promoting actin assembly (5, 7). Cells were
pretreated with latrunculin B (1 µM) to prevent
subcortical actin assembly and incubated with vehicle or Msp, and then
intracellular calcium was depleted with thapsigargin and EGTA as
described for cells in Fig. 5A. Under these conditions,
internal Ca2+ stores of Msp-pretreated and control cells
were comparably depleted. The amplitude of acute Ca2+
transients in response to Msp (30 µg/ml) was unaffected by
latrunculin B (maximum [Ca2+]i change (in
nM): control cells, 258.8 ± 50.0; latrunculin B-pretreated cells, 221.7 ± 15.9; n = 4 per
treatment). Yet, under the same conditions, Msp barely inhibited
Ca2+ depletion-induced Ca2+ influx (Fig. 5,
D-F) when compared with its strong inhibition in cells that
were not treated with latrunculin B (Fig. 5C). Since these
data indicated that the mechanism by which Msp uncouples SOCs is
related to its effects on actin assembly, we used confocal microscopy
to examine the effects of Msp on the distribution of actin filaments
(Fig. 5, G-I). After a 5-min treatment with Msp (30 µg/ml), there was an almost complete loss of
rhodamine-phalloidin-stained actin filaments in stress fibers and a
greatly increased staining in the subcortical region. After treatment
of cells with Msp for 45 min, the increased density of actin filaments
subjacent to the plasma membrane was maintained. These data suggested
that the early Msp-induced calcium transients may have promoted the assembly of subcortical actin filaments, which in turn uncoupled the
SOCs from the intracellular stores. Accordingly, we examined if
chelation of the Msp-induced calcium transient would prevent the
formation of subcortical actin filaments. Cells were preincubated (or
not) with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (3 µM) for 15 min
prior to Msp or vehicle treatment for 30 min. Fixed cells were stained
with rhodamine phalloidin, and subcortical actin filaments were
measured by quantitative fluorescence photometry. Consistent with the
images obtained by confocal microscopy, Msp induced substantial
increases of actin filament staining in subcortical zones (vehicle = 52 ± 8 fluorescence units; Msp = 129 ± 24 fluorescence units; p < 0.02; n = 10 cells/treatment). In contrast, preincubation with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester abrogated the Msp-induced actin
assembly (vehicle = 40 ± 5 fluorescence units; Msp = 47 ± 5 fluorescence units; p > 0.2;
n = 10 cells/treatment).
The central finding in this investigation is that the major
surface protein of the spirochete T. denticola promotes
subcortical actin assembly that subsequently perturbs calcium signaling
in human fibroblasts. Since calcium signals are tightly and
reciprocally linked to regulation of the actin cytoskeleton (2),
microbial perturbation of this system has profound effects on cell
regulation. Not only is the activity of actin-binding and -severing
proteins determined by local concentrations of calcium, but the
assembly of subcortical actin filaments evidently affects the coupling of two key components of store-operated calcium flux: depletion/refill cycles of ER store Ca2+ and Ca2+ entry through
SOCs (5-7). Since store-operated flux is a major pathway by which
intracellular Ca2+ concentration is regulated in response
to extracellular agonists, it occurred to us that pathogenic
microoganisms may perturb host cells by affecting their store-operated
calcium pathways. In contrast to the exotoxins or surface proteins of
bacteria that stimulate host cell calcium signaling, our previous data
have shown that protein(s) in the T. denticola outer
membrane inhibit calcium signals in gingival fibroblasts (17). In this
study, we examined the mechanism by which the major outer sheath
protein of T. denticola, Msp, perturbs calcium signaling. We
have identified two potentially pathological responses: (i) an early,
acute rise in [Ca2+]i due to increased
conductance across the plasma membrane and (ii) a delayed perturbation
of store-operated calcium flux. The latter includes two unprecedented
effects, alteration of ER Ca2+ release kinetics and
inhibition of Ca2+ entry through putative SOCs. We have
also provided substantial support for the hypothesis that
Ca2+ ER stores and SOCs are conformationally coupled (5-7)
by using the gingival fibroblast as a target cell and Msp as a
biologically relevant antagonist isolated from a pathogenic bacterium.
Msp bound and clustered into complexes on the cell surface, perhaps due
to receptor aggregation after ligand occupancy. Both the bacterium
T. denticola and Msp can bind to extracellular matrix proteins (37, 41), which could provide the initial adhesion of Msp to
gingival fibroblasts. Indeed, abundant cell surface-associated fibronectin (23) could have bound Msp and effectively lowered its
bioavailability for the induction of Ca2+ transients in our
experiments. Nevertheless, when sufficient amounts of Msp were added
(~160 nM), there was a robust intracellular Ca2+ response.
Msp failed to induce Ca2+ transients in cells incubated in
Ca2+-free buffer and failed to stimulate Ca2+
release from ER stores. As Msp increased manganese conductance, it is
evident that the Msp-induced calcium transients probably arose from the
influx of external Ca2+. Indeed, we found that the elapsed
time following exposure of the cells to Msp and both the ensuing peak
in cytoplasmic Ca2+ and the rapid quenching of fluorescence
by Mn2+ was coincidentally ~90 s. Acute Ca2+
influx may be due to activation of ligand-gated channels (42) or from
the translocation of some of the bound Msp into the plasma membrane to
increase plasma membrane conductance to cations. This latter notion is
consistent with earlier evidence showing that Msp can form transient,
ion-permeable channels in HeLa cells (22, 39) and is reminiscent of
recent reports on the formation of cation-permeable channels by
complexes of Longer term experiments (~30 min) showed that Msp reduced ATP- and
thapsigargin- induced [Ca2+]i transients and
blocked subsequent Ca2+ entry following thapsigargin
treatment, indicating that Msp uncouples physiologic store-operated
calcium flux. These findings were not due to prior store depletion,
since ionomycin induced immediate and equivalent calcium release in
control and Msp-pretreated cells in calcium-free buffer. Further
experiments with ionomycin showed that thapsigargin-sensitive
Ca2+ stores were comparably depleted in control and
Msp-pretreated cells. Yet, the amplitude of thapsigargin-induced
calcium transients was clearly decreased, and the kinetics of
Ca2+ release were clearly altered in the Msp-pretreated
cells. The mechanisms accounting for this unprecedented effect of a
bacterial toxin are not entirely clear. They may involve perturbation
of Ca2+ release and/or reuptake into ER or other internal
Ca2+ stores and may be affected by de novo actin
filament assembly near the ER and other calcium-containing organelles.
Notably, the reduction of thapsigargin- and ATP- induced
Ca2+ transients by Msp was reversed in latrunculin
B-pretreated cells. Thus, the altered kinetics of Ca2+
release and/or replenishment of ER stores following incubation of
Msp-pretreated cells with agonists may be caused, in part, by actin
filament reorganization. Evidently, the key element in the perturbation
of store-operated calcium flux by Msp is that it induces a
calcium-dependent assembly of subcortical actin filaments in gingival fibroblasts, suggesting that Msp may uncouple the ER and
Ca2+ channels in the plasma membrane.
There is growing experimental support for conformational coupling as a
principal regulatory mechanism that links Ca2+ store
depletion and Ca2+ influx through SOCs (1). Experimental
strategies using drugs that affect actin assembly, translocation of
actin to the cell periphery, and the association of actin and
actin-binding proteins with the plasma membrane support the hypothesis
that de novo assembly of an actin filament network subjacent
to the plasma membrane impairs conformational coupling (5, 7). We have
shown previously that T. denticola induces rearrangement of
actin (11, 12, 15), with a proportional shift in actin filaments from
the stress fiber-rich ventral interface to the dorsal area of the cell
(14). Here, we observed that the Msp-induced calcium transient is
required for subcortical actin filament assembly, and this actin
network is formed contemporaneously with the block of store-operated
calcium flux. However, in cells pretreated with latrunculin B to
inhibit actin filament assembly, both Ca2+ release from
thapsigargin-sensitive stores and Ca2+ entry following
Ca2+ store depletion were hardly affected upon challenge
with Msp. We interpret these findings to indicate that the Msp of
T. denticola perturbs store-operated calcium flux by
uncoupling SOCs, and we provide corroborating evidence for actin
assembly-dependent conformational coupling of SOCs in an
additional cell type, the human gingival fibroblast.
The binding of many pathogenic bacterial species or their toxins to
host cells can elicit intracellular calcium signals that are often
associated with substantial cytoskeletal rearrangement (44-48). Some
bacterial outer membrane porins can even translocate to the plasma
membrane of target cells and induce apoptosis secondary to the rapid
influx of Ca2+ (49, 50). For another example, cholera toxin
increases Ca2+ current through calcium release-activated
calcium channels in mast cells (11). Therefore, pathogens may induce
cytotoxicity by causing abnormally elevated cytoplasmic
Ca2+ levels in target cells, which is one of the acute
effects of T. denticola outer membrane proteins (17),
specifically Msp. Although presently unique, our central finding is
that the major surface protein of the spirochete T. denticola inhibits calcium flux by uncoupling store-operated
calcium channels. Conceivably, this may well become a more general
theme of pathogenic host-parasite interactions. Indeed, our data
suggest a general mechanism by which pathogens can block one of the key
cellular signaling pathways for responses to natural agonists such as
growth factors, extracellular matrix proteins, cytokines, and a variety
of other mediators of intercellular communication networks.
Pharmacological intervention to block this pathway of bacterial
pathogenicity may provide a novel approach to inhibit the cytotoxic
effects of some species of spirochetes and other pathogens that cause
chronic disease.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimal essential medium
with 10% fetal bovine serum and antibiotics. Cells from these cultures were harvested and replated onto glass coverslips 2 days before each
experiment, as described previously (17). The cells were grown to
confluence prior to all experiments except when sparse cultures were
used as indicated.
-minimal essential medium containing fetal bovine serum (10%)
(31). The calcium-free buffer consisted of a bicarbonate-free medium
containing 150 mM NaCl, 5 mM KCl, 10 mM D-glucose, 1 mM
MgSO4, 1 mM Na2HPO4,
and 20 mM HEPES at pH 7.4 with an osmolarity of 291 mosM. For experiments requiring external calcium, 1 mM CaC12 was added to the buffer; for
experiments requiring chelation of external Ca2+, 2 mM EGTA was added. Fmax and
Fmin were determined using ionomycin and EGTA as
described previously (17).
20 °C for 10 min,
blocked with 1:1000 mouse serum in PBS for 10 min, incubated with
primary antibody (1:100 dilution) for 1 h at room temperature,
washed three times with PBS containing 0.2% bovine serum albumin, and
incubated with Texas Red-conjugated goat-anti-rabbit antibodies
(1:100). The coverslips were washed with PBS and mounted with an
antifade mounting medium (ICN, Montreal, Canada). The fibroblasts were
examined using a × 40, 1.3 NA oil immersion objective under
epifluorescence optics (Leica CLSM, Heidelberg, Germany).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (37K):
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Fig. 1.
Enriched Msp complex and immunolocalization
of Msp on fibroblasts. A, Msp was prepared as described
in "Experimental Procedures" and Refs. 21 and 22, subjected
to SDS-polyacrylamide gel electrophoresis, and stained with Coomassie
Brilliant Blue. Lane 1, boiled
preparation; lane 2, unheated Msp preparation;
lane M, molecular mass standards. B,
immunoblot. Lane 1, boiled preparation;
lane 2, unheated Msp preparation. C,
indirect immunofluorescence microscopy of cells incubated with Msp (30 µg/ml, 25 °C, 30 min). 1, secondary antibody control
labeled only with Texas Red-conjugated goat anti-rabbit antibody shows
a small amount of nonspecific staining and autofluorescence.
2, similar results were obtained with primary anti-Msp
control but with no secondary goat anti-rabbit conjugate. Cells
incubated with Msp for 1 min (3) or 30 min (4)
and stained with both primary anti-Msp and secondary goat anti-rabbit
conjugate show specific fluorescence staining along cell membranes.
Note the clustering of Msp to one pole of cell at 30 min postincubation
with Msp.
View larger version (25K):
[in a new window]
Fig. 2.
Msp induces Ca2+ transients in
fibroblasts. A-C, cells were treated with Msp as
indicated (1 µg/ml, 10 µg/l, 30 µg/ml) in 1 mM
Ca2+ buffer, and intracellular calcium
([Ca2+]i) was measured in fura-2-loaded cells by
ratio fluorimetry. There was a ~90-s delay after incubation,
presumably due to Msp binding to cell surface fibronectin. B
(inset), an example of prolonged post-transient fluctuations
of [Ca2+]i observed in many cells treated with
Msp at 10 and 30 µg/ml. D, cells were treated with
distilled H2O added to cell culture medium. E,
cells were treated with Msp after incubation with anti-Msp antibody
(2:3 protein ratio). F, cells were treated with boiled Msp
(30 µg/ml, 100 °C, 30 min). G, cells were treated with
heated Msp (30 µg/ml, 60 °C, 30 min). H, summary of
increases of [Ca2+]i above unstimulated control
values (data from n = 9 experiments; means ± S.E.; values in nM). Data show increased
[Ca2+]i above base-line after the addition of
distilled H2O to cell culture medium, Msp following
anti-Msp antibody, boiled Msp, heated Msp, or Msp at the indicated
concentrations. All experiments were performed in calcium buffer (1 mM). I, cells incubated repeatedly with fresh
doses of Msp (30 µg/ml) but without wash-out showed no response.
J, cells loaded with the membrane-linked calcium reporter
dye fura-C18 and treated with Msp (30 µg/ml) produced large amplitude
rise of submembrane [Ca2+]i. A similar result was
obtained in n = 5 cells. K, Msp failed to
evoke Ca2+ transients in cells incubated in calcium-free
buffer augmented with EGTA (2 mM); similar results were
seen in n = 8 cells. L, typical traces of
photon counts of single cells showing a sharp decline in the
fluorescence of fura-2 excited at 356 nm ~2 min after the addition of
Msp in buffer containing manganese chloride (1 mM). The
loss of fluorescence after the addition of Msp was contemporaneous with
Msp-induced calcium transients (~90 s; see Fig. 1C). The
data are representative of five independent experiments. The
inset in L shows Msp-treated cells in
medium without manganese chloride.
View larger version (26K):
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Fig. 3.
Msp inhibits ATP- and thapsigargin-induced
Ca2+ release from internal stores. Shown is the
increase of [Ca2+]i in single cells that were
untreated (control) or pretreated with Msp (30 µg/ml, 25 °C, 40 min) or Msp and latrunculin B (1 µM, 25 °C, 40 min)
and then incubated with 200 µM ATP (A) or 1 µM thapsigargin (B) in calcium-deficient
EGTA-containing medium. The calcium-deficient media were switched
immediately before experiments to reduce intracellular calcium
depletion. The calcium-deficient media did not significantly reduce
basal [Ca2+]i over the time course of these
experiments. Data are mean ± S.E. from n = 8 experiments showing maximum [Ca2+]i change (in
nM) induced by ATP or thapsigargin treatment. Pretreatment
of cells with latrunculin B reverses the decrease in
[Ca2+]i change caused by Msp. C,
summary of data (mean ± S.E.) from n = 4-8
experiments per group showing that the effects of Msp on ATP-induced
Ca2+ transients were progressively reversed during
increasing time periods following wash-out of free Msp from the assay
buffer. D-G, Mag-fura-2 ratio data for estimation of
[Ca2+]ER. Msp has no acute effect on base-
line mag-fura-2 ratio (D). After the addition of ATP (200 µM), Msp pretreatment (30 µg/ml, 25 °C, 40 min)
blocks ATP-induced reduction of mag-fura-2 ratio (E). In
control cells with vehicle, the mag-fura-2 ratio is transiently reduced
and then returns to base-line after refilling of ER stores
(F). Summary data showing basal
[Ca2+]i and peak ATP-induced change of mag-fura-2
ratio (G; means ± S.E.; data are from n = 9 experiments). Note that pretreatment with Msp reduces the basal
mag-fura-2 ratio by ~25%.
6 nM·s;
Msp = 7.75 ± 0.19 × 10
6
nM·s; p < 0.005). Yet, Msp pretreatment
clearly reduced the amplitude of the Ca2+ transient and
slowed down the rate of Ca2+ release and return of the
transient to base-line values (Fig. 4, C and D).
Moreover, the thapsigargin-sensitive Ca2+ stores of the
Msp-pretreated and control cells were comparably depleted, as shown by
the similar residual Ca2+ release upon the addition of
ionomycin (Fig. 4E). Taken together, this series of
experiments indicates that Msp interferes with the process of
Ca2+ release from ER stores, seen as the lower amplitude
and altered kinetics of [Ca2+]i transients,
despite its minimal effect on the amount of Ca2+ released
over time.
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Fig. 4.
Msp alters ER store Ca2+ release
kinetics. Shown are representative calcium traces after the
incubation of fura-2-loaded vehicle- or Msp-pretreated cells (30 µg/ml, 25 °C, 40 min) followed by the addition of ionomycin or
thapsigargin. Typical calcium signals evoked by ionomycin (2 µM) are similar in vehicle-treated control cells
(A) and Msp-pretreated cells (B). Shown are
typical calcium signals evoked after thapsigargin (1 µM)-EGTA, showing different kinetics for
[Ca2+]i transients in control (C) and
Msp-pretreated cells (D). The amplitude of the
[Ca2+]i transient is diminished, and the
Ca2+ release is prolonged in the Msp-pretreated cells.
E, summary data (mean ± S.E.) from n = 4-8 experiments per group showing that internal stores of both control
and Msp-pretreated cells are comparably depleted of Ca2+
after thapsigargin (1 µM)-EGTA treatment, by measuring
subsequent ionomycin (2 µM)-releasable Ca2+
transients.
View larger version (35K):
[in a new window]
Fig. 5.
Msp inhibits store-operated Ca2+
influx in fibroblasts. Shown are representative calcium traces
after the incubation of fura-2-loaded cells with 1 µM
thapsigargin in calcium-deficient medium for 30 min, followed by the
addition of 2 mM extracellular calcium. Shown are typical
calcium signals evoked by the addition of 2 mM
extracellular calcium in vehicle-pretreated control cells
(A) and Msp-pretreated cells (30 µg/ml, 25 °C, 40 min)
(B). C, summary data from n = 9 experiments showing basal [Ca2+]i and peak
[Ca2+]i after the addition of extracellular
calcium (means ± S.E. of [Ca2+]i in
nM above base line). D and E, typical
calcium signals for latrunculin B-pretreated cells with and without Msp
challenge. Cells were pretreated with 1 µM latrunculin
followed by the protocols described in Fig. 5, A and
B, respectively. F, summary data from
n = 5 experiments comparing Msp-challenged and control
cells that had been pretreated with latrunculin B (n = 5 independent experiments; means ± S.E. of
[Ca2+]i in nM). G-I,
confocal microscopy of human gingival fibroblasts stained with
rhodamine-phalloidin showing assembly of subcortical actin induced by
Msp. G, vehicle-treated control cells. H,
fibroblasts pretreated with Msp for 5 min. Note that rhodamine
phalloidin staining for actin filaments in stress fibers is prominent
in control cells (G; arrows) but is lost in
Msp-treated cells, while staining near the cortex is greatly
intensified (arrowheads; H). The increased
density of actin filaments in subcortical regions was confirmed by
quantitative fluorescence spectrophotometry (see "Results").
Pretreatment of cells with Msp for 45 min induces almost complete
depolymerization of stress fibers and the formation of a dense
subcortical actin filament network (I).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-latrotoxin oligomers (43). However, our flow cytometry
measurements with a bisoxonol dye showed no significant loss of
membrane potential, indicating that there was no general increase of
cation conductance (e.g. sodium). Thus, it appears that Msp
represents a novel type of bacterial cytotoxin whose immediate
pathogenic effect is the specific promotion of increased calcium conductance.
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ACKNOWLEDGEMENTS |
---|
We thank David A. Grove, Pam Arora, and Wilson Lee for technical assistance. We thank Pauline Hannam and Barry C. McBride for supplying the anti-Msp polyclonal antibodies and for helpful advice about the isolation of enriched Msp. We thank Milton Charlton and Philip Sherman for insightful criticism of the draft manuscript.
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FOOTNOTES |
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* This work was supported by Canadian Institutes of Health Research (CIHR) Grant MOP-5619, a maintenance grant, and a group grant.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U66256.
¶ Supported by a CIHR fellowship.
** To whom correspondence should be addressed. Tel.: 416-979-4917 (ext. 1-4456); Fax: 416-979-4936; E-mail: richard.ellen@utoronto.ca.
Published, JBC Papers in Press, April 18, 2001, DOI 10.1074/jbc.M011735200
2 The amino acid sequence of Msp protein can be accessed through NCBI protein data base under NCBI accession number AAB47939 (51).
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ABBREVIATIONS |
---|
The abbreviations used are: [Ca2+]i and [Ca2+]ER, intracellular and ER calcium ion concentration, respectively; ER, endoplasmic reticulum; SOC, store-operated channel; Msp, major surface protein; 2-APB, 2-aminoethoxydiphenyl borate.
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