Vol. 91, No. 2, 1979 November
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BIOCHEMICAL
Pages 629-636
28, 1979
CALCIUM-INDEPENDENT
AND DEPENDENT STEPS IN ACTION OF CLOSTRIDIIJM PERFRINGENS ENTEROTOXIN ON HELA AND VERO CELLS Morihiro
Research
Received
Matsuda
and Nakaba
Institute for Microbial Yamada-kami, Suita,
October
12,
Sugimoto
Diseases, Osaka University Osaka, Japan 565
1979
SDMMARY. Purified enterotoxin (20-200 rig/ml) of Clostridium perfringens rapidly induced bleb and balloon formation on HeLa and Vero cells in the presence, but not the absence, of Ca2+. The action of the toxin involved two, sequential, temperature-dependent steps:lhe first was Ca*+-' independent and included binding of toxin and the bound toxin after 30-60 set could no longer be removed by washing. The second step was Ca*+-dependent and eventually led to bleb and balloon formation. On adding Ca2+ to cells pretreated with toxin in Ca2+-free medium, bleb The ionophore A23187 mimicked the and balloon formation started immediately. action of toxin. The effects of sucrose (0.2 M), trypsin-treatment of the cells and various pretreatments of the toxin on the action of enterotoxin were studied. Clostridium produces
perfringens,
an enterotoxin
enterotoxin bolism
is
(5,
believed
that
known
rapidly
isolation
12,
(1,
by direct
about
35,000
have
facilitated
precise
simple
defined
conditions.
toxin
depresses
amino
responsible
toxin
interaction
with
as a highly
purified
studies
Thus recently
acid
transport
it
specifically
to Vero
thesis,
and causing
detachment
and death
This fied
paper
mechanism reports
enterotoxin
two steps
in its
of action
studies
cells,
membranes
(8,
protein
of the cells
and is 9, 10).
with
cells
(4,
toxin
a mol. 9, 14,
action
wt. 15, 16)
under
to show that
hepatocytes
inhibiting
(4)
and that
macromolecular (15).
the
But little
synis
known
of the enterotoxin.
on the requirements
of -C. perfringens
systems
rat
The
2, 3, 4) and meta-
model
of the
poisoning
of diarrhea.
has been possible
in cultured
binds
(1,
culture
on the mechanism
toxin
the exact
of transport
13) and the use of tissue
of food
the symptoms
7, 8) in various
labeled
about
common causes for
alterations
damage
of the
(11,
is
to induce
6) and tissue to act
Recent
one of the most
for
on HeLa and Vero
the action cells
of the puri-
and demonstration
of
action.
Abbreviations: PBS hosphate-buffered Ca*+(0.9 mM) and Mg$21; (0.5 mM) (17);
saline (17); PBS(+), PBS containing PBS(-), PBS free from Ca2+ and Mg2+. 0006-291X/79/220629-08$01.00/0 629
Copyright All rights
@ I979 by Academic Press. Inc. of reproduction in anyform reserved.
Vol. 91, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
was prepared from sporulated cultures of a MATERIALS AND METHODS. Enterotoxin obtained through Dr. G. Sakaguchi. It was strain NCTC 8239 of C. perfringens purified as describe; by Sakaguchi et a1.(12). Toxin was diluted in PBS (17) free from Ca2+ and Mg2+(PBS(-)) and added to the cells at a final concentration of 20-200 rig/ml medium. Rabbit antitoxin serum was prepared as described by Sakaguchi et a1.(12). Ionophore A23187(A grade) was purchased from Calbiochem., and cholesterol(guaranteed reagent) from Nakarai Chemicals, Ltd, Kyoto. HeLa-S3 cells and Vero (African green monkey kidney) cells were provided by Dr. Y. Sakaue of this Institute and by Dr. R. Murata of the National Institute of Health, Tokyo, respectively. The growth medium was 95% Eagle's minimum essential medium and 5% calf serum(Biken). Cells were grown at 37'C under a watersaturated atmosphere of 5% CO2 in air. Stock cultures were maintained as monolayers in flasks(5 x 11 cm) containing 15 ml of medium. Subculture every 4-6 days was performed by detaching the cells with 0.25% trypsin-0.02% EDTA mixture (1:l by volume) after they had become confluent. For experiments, cells were removed from the stock flask with trypsin-EDTA mixture and seeded into 60-mm diameter plastic dishes(Falcon) at densities of 3 x lo4 and 1 x 105 cells per dish. With these inocula toxin was added 4 and 2 days later, respectively. Just before adding toxin, the medium was replaced by fresh growth medium or the cells were washed three times with PBS(-) and then an appropriate buffer was added (final volume, 5 ml per dish). RESULTS AND DISCUSSION Requirement of calcium cells: ml)
In growth induced
at 0-4°C.
medium
bleb Similar
bleb
Further
analyses
toxin
toxin
toxin
(Fig.
only
had a rapid
below
0.9 uM Ca2+ the
These acid
effect
pretreated
cellular
transport
slow
of
took
toxin(200 with
PBS(+)
in PBS(-)(final responses in cultured
did
1 hour
did
not have
not
produce
concentrations: are
faster
hepatocytes
2+
(4),
120 min by 40 or 100 ng of toxin/ml.
630
which
but
but
for no Ca2+
concen-
and with
within
120 min.
such morphological 200 rig/ml;
was not
medium.
even when
at lower
such effect
sensitive
in PBS
90 uM Ca 2+
at 9 uM Ca*+)
toxin,
and more
cells
1C and 2B),
cells
in
was essential
to 1 mM. In PBS containing
longer(about rig/ml)
media
Mg
not
with
as in growth
in PBS containing
HeLa and Vero
but
treated
on these
as well
(Fig.
at 37"C,
simpler
acted
the cells
min at 200 ng/
ileum
not Mg 2+ , in PBS(+)
, but
on both
cells
of various
mM)(PBS(+)(17))
swelling
lo-15
in rat
the toxin
1 and 2 A-D);
effect
of Ca2+ its
of cells
that
of Mg2+ was increased
tration
tions
2+
rapidly(in
observed
the effects
and found
Ca
on HeLa and Vero
on HeLa and Vero
has been
We tested
that
induced
rig/ml)
formation
formation
medium
of enterotoxin
toxin(20-200
showed
of the
Enterotoxin
the action
mM) and Mg2+(0.5
the concentration the
the
10).
Ca2+(0.9
the action the
(8,
of the growth
containing
for
and balloon
the enterotoxin place
ions
to toxin depressed
alteraCa 2+, 0.9 than within
uM).
amino
Vol. 91, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fig. 1. Phase contrast micrographs of HeLa cells cultures) treated at (2-da 37'C. (A) Control without toxin in PBS (-)(free from Ca 3+ and Hg2+), 15 min; (B) with toxin (200 rig/ml), in PBS(-) 15 min; (C) with toxin (200 "g/ml), in PBS containing Mg2+(0.5 mM) but no Cai +, 15 min; (D) with toxin (200 rig/ml), in PBS containing Ca2+(0.9 mM) but no Mg2+, 15 min; (E) with toxin (200 &ml) in PBS(+) containing 0.2 M sucrose, 20 min and (F) with ionophore A23187 (2 pg/ ml) in PBS(+), 35 min. The scale indic ates 100 urn.
631
Vol. 91, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fig. 2. Phase contrast micrographs of Vero cells (4-da cultures) treated at 37'C. (A) Control without toxin, in PBS containing Mg 2Y (0.5 mM) but no Ca2+, 15 min; (B) with toxin (200 rig/ml) in the same buffer as for (A), 15 min; (C) control without toxin in PBS containing Ca2+(0.9 a&f) but no Mg2+, 15 min; (D) with toxin (200 rig/ml) in the same buffer as for (C), 15 min; (E) pretreated with toxin (200 rig/ml) in PBS(-) for 20 min, then washed and incubated in PBS(+), 5 min and (F) trypsin-EDTA treated cells in suspension, in PBS(+), toxin (200 rig/ml), 15 min. The scale indicates 100 urn.
632
Vol. 91, No. 2, 1979
Dissociation
of two steps
Ca*+was recently Vero
BIOCHEMICAL
essential McDonel
cells
in the action
for
the action
et a1.(15)
in Ca*+-
and Mg*+ -free
the cells
PBS(PBS(-)).
balloons.
But when Ca*+(f* inal they
Under
that
pretreatment
resulted
in bleb
and balloon
Ca*+ but
without
toxin of
Therefore,
the
and a second
bleb
that
and balloon
is
Rapidity
of response
toxin(40
rig/ml)
showed
that
30-60
set
ical
times
from
This
cannot
toxin
pg toxin
adding 3A),
cells
equivalent),
of toxin
at 0-4°C
but
that
60 set, could
with
and then
toxin
wash-
rescu-
them after morpholog-
at 37°C after
prevent
the
Results
(0 set)
washing
even washing not
not
at 37°C in PBS(+).
on the ultimate observed
to to
did
the cells
the
from
HeLa cells.
60 or 120 set)
had no effect
for
present
them at 37°C in PBS(+).
(Fig.
toxin
of Ca 2+
with the action
20 min PBS(-) of
3C and D). on 30-60
proceeds
be rescued
of the toxin
Figure
ultimately
and incubated
after
or with-
of the enterotoxin:
and leads
We incubated
immediately
both with
toxin
or
in medium with
the binding
the
30,
with
formation.
in the action
(0,
form blebs
in the absence
obtained
with
with
shows that
enterotoxin
pretreated
periods
not
incubation
Ca 2+ -dependent
in Ca *+-and
to the cells,
and includes
preincubation
antitoxin(10 (Fig.
two steps
3B) and Vero
containing
were
and incubated
the
did
and balloon
when washed
PBS(-)
to
Thus neither
rig/ml)
2+ and Ca were
results
is
short
with
added
on subsequent
of HeLa (Fig.
After
toxin
for
toxin(200
the toxin
to enterotoxin:
the cells
in PBS(+).
the
of cells
the action
alterations
that Cells
with
preincubation
with
Ca 2+ -independent step
However,
enterotoxin
solution.
the cells
when toxin
alterations
at 37'C
washing
ed them all
formation
Similar
formation.
show such morphological
ed them three
cells
must be at least
step
cells,
20 min with
to show bleb
as observed
an initial
salt
cells:
binding.
0.9 mM) was then
of Vero
above.
I 125 II-labeled
of
conditions
began
on HeLa and Vero
as described
balanced
toxin
these
incubation.
there
Hank's for
immediately
2E shows
the beginning
binding
at 37°C for
Mg*+-free
out washing,
of enterotoxin
of the toxin
observed
Ca*+ nor Mg 2+ must be necessary We incubated
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
set
exposure
to an irreversible
to the stage
by washing.
633
toxin,
from which
the action
of the
the intoxicated
cells
Vol. 91, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fii. 3. Phase contrast micrographs of H eLa cells (2-day cultures) treated at or (D) 60 set after 37 C. Cells washed, (A) immediately. (B #) and (C) 30 sec. adding toxin (40 rig/ml) in PBS(-), with PBS(-) (A and B) or with PBS(-) containing antitoxin (C and D), and subseq uently incubated in PBS(+), 20 min. Cells in PBS(+) for 15 min with toxin ( 200 rig/ml) that had been pretreated with (E) 20 mM cysteine at 2S°C for 60 min and (F) with cholesterol (200 rig/ml) at 37“C for 60 min. The scale indicates 100 pm.
634
BIOCHEMICAL
Vol. 91, No. 2, 1979
Effect
of ionophore
ethanol
and 25 pl
concentration sence,
of the solution
the ionophore formation
induced
by the
alterations.
molar possibly
with
there
of various
treatments
immediately attached
inhibited
the action
Heating or antitoxin
(37°C
activated enterotoxin
rapidly,
toxins,
action
leading
alterations
ionophore
induced
of Ca2+ ions.
molecular
alterations.
mechanism
for
of the action
635
in 40 min at 37'C).
with
0.2% formalin(18)
activity.
Unlike
thiol-
2. p erfringens
60 min)(Fig.
3E) but
enterotoxin
proceeds
trypsin-resistant
Ca2+ is
required
receptors for
The osmotically similar
the enterotoxin
of the membranes findings
1E) and partially
3F).
and the
that
suspended
of sucrose
B-toxin,
with
and that
of the above
of the entero-
The presence
cytotoxic
(25'C
by the enterotoxin
On the basis
the ionophore,
to toxin
it
of -C. perfringens
in permeability
C6. On a
Cells
formed
60 min)(Fig.
of Ca 2+ suggest
that
as sensitive
(Fig.
were
its
an interaction
to morphological
by change
2F).
10 min or pretreating
cells
and
and balloon
cells
enterotoxin:
cells
vesicles
the action
sensitive
in the presence
effect
of the
37°C for
bleb
than
were
(Fig.
by 20 mM cysteine
through
of the
of the
inactivated
ab-
cells.
such as -C. perfringens
show that
probably
on the surface
on exact
60 min)
(ZOO rig/ml,
The above data
toxic
for
its
bleb
of interaction
treatment
(fine
in
from
glial
more effective
as monolayers
toxin
in
such morphological
similar
N18 and rat
sensitive
the response
induced
not produce
mechanism
trypsin-EDTA
was inactivated
not by cholesterol
670 times
at 80°C for
cytolytic
clone
not
indistinguishable
produced
on the action
of the
the toxin
Cl300
on the
after
delayed
did
2+ , but
at 0-4"C,
cells
the ionophore
to the dish
M in PBS(+))
alone
was dissolved
in 5 ml of PBS (final
of Ca
but not
is some efficient
receptors
as cells
surface
(0.5%)
roughly
specific
in PBS(+)
quent
is
to cells
1F) and Vero
the toxin,
the toxin
because
Effects
very
at 37"C,
Ethanol
A23187
In the presence
on mouse neuroblastoma
basis
toxin
0.5%).
on HeLa (Fig. toxin.
cells:
was added
(2 pg/ml)
Unlike
formation
on HeLa and Vero
of ethanol,
balloon
(0.2
A23187
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
studies
of enterotoxin.
the
subse-
sensitive effect
of the
exerts
its
and consequent
cyto-
influx
are now in progress
Vol. 91, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL
ACKNOWLEDGEMENT. We thank Drs. G. Sakaguchi Osaka Prefecture for providing the bacterial tion of purified enterotoxin.
RESEARCH COMMUNICATIONS
and T. Uemura of University of strain and for advice on prepara-
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
McDonel, J. L. (1974) Infect. Immun. 10, 1156-1162. McDonel, J. L., and Asano, T. (1975) Infect. Immun. 11, 526-529. McDonel, J. L., and Duncam, C. L. (1977) J. Infect. Dis. 136, 661-666. M. W. (1978) Biochem. Biophys. Res. Commun. Geiger, O., and Pariza, 82, 378-383. McDonel, J. L., and Duncan, C. L. (1975) Infect. Immun. 12, 274-280. McDonel, J. L., and Duncan, C. L. (1976) Infect. Immun. 15, 999-1001. McDonel, J. L., and Duncan, C. L. (1975) Infect. Immun. 12, 1214-1218. McDonel, J. L., Chang, L. W., Pounds, J. G., and Duncan, C. L. (1978) Lab. Invest. 39, 210-218. McDonel, J. L. (1979) Am. J. Clin. Nutr. 23, 210-218. McClane, B. A., and McDonel, 3. L. (1979) J. Cell Physiol. 99, 191-200. Stark, R. L., and Duncan, C. L. (1972) Infect. Immun. 6, 662-673. Sakaguchi, G., Uemura, T., and Riemann, H. P. (1973) Appl. Microbial. 26, 762-767. Enders, G. Jr., and Duncan, C. L. (1977) Infect. Immun. 17, 425-429. Gyobu, Y., and Kodama, H. (1978) J. Food Hyg. Sot. Jpn. 19, 294-298 (in Japanese). McDonel, J. L., and McClane, B. A. (1979) Biochem. Biophys. Res. Commun. 87, 497-504. Dis. 131, 58-63. Keusch, G. T., and Donta, S. T. (1975) J. Infect. Dulbecco, R., and Vogt, M. (1954) J. Exp. Med. 99, 167-182. Matsuda, M., and Yoneda, M. (1976) Biochem. Biophys. Res. Commun. 68, 668-674.
636
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BIOCHEMICAL
Pages 629-636
28, 1979
CALCIUM-INDEPENDENT
AND DEPENDENT STEPS IN ACTION OF CLOSTRIDIIJM PERFRINGENS ENTEROTOXIN ON HELA AND VERO CELLS Morihiro
Research
Received
Matsuda
and Nakaba
Institute for Microbial Yamada-kami, Suita,
October
12,
Sugimoto
Diseases, Osaka University Osaka, Japan 565
1979
SDMMARY. Purified enterotoxin (20-200 rig/ml) of Clostridium perfringens rapidly induced bleb and balloon formation on HeLa and Vero cells in the presence, but not the absence, of Ca2+. The action of the toxin involved two, sequential, temperature-dependent steps:lhe first was Ca*+-' independent and included binding of toxin and the bound toxin after 30-60 set could no longer be removed by washing. The second step was Ca*+-dependent and eventually led to bleb and balloon formation. On adding Ca2+ to cells pretreated with toxin in Ca2+-free medium, bleb The ionophore A23187 mimicked the and balloon formation started immediately. action of toxin. The effects of sucrose (0.2 M), trypsin-treatment of the cells and various pretreatments of the toxin on the action of enterotoxin were studied. Clostridium produces
perfringens,
an enterotoxin
enterotoxin bolism
is
(5,
believed
that
known
rapidly
isolation
12,
(1,
by direct
about
35,000
have
facilitated
precise
simple
defined
conditions.
toxin
depresses
amino
responsible
toxin
interaction
with
as a highly
purified
studies
Thus recently
acid
transport
it
specifically
to Vero
thesis,
and causing
detachment
and death
This fied
paper
mechanism reports
enterotoxin
two steps
in its
of action
studies
cells,
membranes
(8,
protein
of the cells
and is 9, 10).
with
cells
(4,
toxin
a mol. 9, 14,
action
wt. 15, 16)
under
to show that
hepatocytes
inhibiting
(4)
and that
macromolecular (15).
the
But little
synis
known
of the enterotoxin.
on the requirements
of -C. perfringens
systems
rat
The
2, 3, 4) and meta-
model
of the
poisoning
of diarrhea.
has been possible
in cultured
binds
(1,
culture
on the mechanism
toxin
the exact
of transport
13) and the use of tissue
of food
the symptoms
7, 8) in various
labeled
about
common causes for
alterations
damage
of the
(11,
is
to induce
6) and tissue to act
Recent
one of the most
for
on HeLa and Vero
the action cells
of the puri-
and demonstration
of
action.
Abbreviations: PBS hosphate-buffered Ca*+(0.9 mM) and Mg$21; (0.5 mM) (17);
saline (17); PBS(+), PBS containing PBS(-), PBS free from Ca2+ and Mg2+. 0006-291X/79/220629-08$01.00/0 629
Copyright All rights
@ I979 by Academic Press. Inc. of reproduction in anyform reserved.
Vol. 91, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
was prepared from sporulated cultures of a MATERIALS AND METHODS. Enterotoxin obtained through Dr. G. Sakaguchi. It was strain NCTC 8239 of C. perfringens purified as describe; by Sakaguchi et a1.(12). Toxin was diluted in PBS (17) free from Ca2+ and Mg2+(PBS(-)) and added to the cells at a final concentration of 20-200 rig/ml medium. Rabbit antitoxin serum was prepared as described by Sakaguchi et a1.(12). Ionophore A23187(A grade) was purchased from Calbiochem., and cholesterol(guaranteed reagent) from Nakarai Chemicals, Ltd, Kyoto. HeLa-S3 cells and Vero (African green monkey kidney) cells were provided by Dr. Y. Sakaue of this Institute and by Dr. R. Murata of the National Institute of Health, Tokyo, respectively. The growth medium was 95% Eagle's minimum essential medium and 5% calf serum(Biken). Cells were grown at 37'C under a watersaturated atmosphere of 5% CO2 in air. Stock cultures were maintained as monolayers in flasks(5 x 11 cm) containing 15 ml of medium. Subculture every 4-6 days was performed by detaching the cells with 0.25% trypsin-0.02% EDTA mixture (1:l by volume) after they had become confluent. For experiments, cells were removed from the stock flask with trypsin-EDTA mixture and seeded into 60-mm diameter plastic dishes(Falcon) at densities of 3 x lo4 and 1 x 105 cells per dish. With these inocula toxin was added 4 and 2 days later, respectively. Just before adding toxin, the medium was replaced by fresh growth medium or the cells were washed three times with PBS(-) and then an appropriate buffer was added (final volume, 5 ml per dish). RESULTS AND DISCUSSION Requirement of calcium cells: ml)
In growth induced
at 0-4°C.
medium
bleb Similar
bleb
Further
analyses
toxin
toxin
toxin
(Fig.
only
had a rapid
below
0.9 uM Ca2+ the
These acid
effect
pretreated
cellular
transport
slow
of
took
toxin(200 with
PBS(+)
in PBS(-)(final responses in cultured
did
1 hour
did
not have
not
produce
concentrations: are
faster
hepatocytes
2+
(4),
120 min by 40 or 100 ng of toxin/ml.
630
which
but
but
for no Ca2+
concen-
and with
within
120 min.
such morphological 200 rig/ml;
was not
medium.
even when
at lower
such effect
sensitive
in PBS
90 uM Ca 2+
at 9 uM Ca*+)
toxin,
and more
cells
1C and 2B),
cells
in
was essential
to 1 mM. In PBS containing
longer(about rig/ml)
media
Mg
not
with
as in growth
in PBS containing
HeLa and Vero
but
treated
on these
as well
(Fig.
at 37"C,
simpler
acted
the cells
min at 200 ng/
ileum
not Mg 2+ , in PBS(+)
, but
on both
cells
of various
mM)(PBS(+)(17))
swelling
lo-15
in rat
the toxin
1 and 2 A-D);
effect
of Ca2+ its
of cells
that
of Mg2+ was increased
tration
tions
2+
rapidly(in
observed
the effects
and found
Ca
on HeLa and Vero
on HeLa and Vero
has been
We tested
that
induced
rig/ml)
formation
formation
medium
of enterotoxin
toxin(20-200
showed
of the
Enterotoxin
the action
mM) and Mg2+(0.5
the concentration the
the
10).
Ca2+(0.9
the action the
(8,
of the growth
containing
for
and balloon
the enterotoxin place
ions
to toxin depressed
alteraCa 2+, 0.9 than within
uM).
amino
Vol. 91, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fig. 1. Phase contrast micrographs of HeLa cells cultures) treated at (2-da 37'C. (A) Control without toxin in PBS (-)(free from Ca 3+ and Hg2+), 15 min; (B) with toxin (200 rig/ml), in PBS(-) 15 min; (C) with toxin (200 "g/ml), in PBS containing Mg2+(0.5 mM) but no Cai +, 15 min; (D) with toxin (200 rig/ml), in PBS containing Ca2+(0.9 mM) but no Mg2+, 15 min; (E) with toxin (200 &ml) in PBS(+) containing 0.2 M sucrose, 20 min and (F) with ionophore A23187 (2 pg/ ml) in PBS(+), 35 min. The scale indic ates 100 urn.
631
Vol. 91, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fig. 2. Phase contrast micrographs of Vero cells (4-da cultures) treated at 37'C. (A) Control without toxin, in PBS containing Mg 2Y (0.5 mM) but no Ca2+, 15 min; (B) with toxin (200 rig/ml) in the same buffer as for (A), 15 min; (C) control without toxin in PBS containing Ca2+(0.9 a&f) but no Mg2+, 15 min; (D) with toxin (200 rig/ml) in the same buffer as for (C), 15 min; (E) pretreated with toxin (200 rig/ml) in PBS(-) for 20 min, then washed and incubated in PBS(+), 5 min and (F) trypsin-EDTA treated cells in suspension, in PBS(+), toxin (200 rig/ml), 15 min. The scale indicates 100 urn.
632
Vol. 91, No. 2, 1979
Dissociation
of two steps
Ca*+was recently Vero
BIOCHEMICAL
essential McDonel
cells
in the action
for
the action
et a1.(15)
in Ca*+-
and Mg*+ -free
the cells
PBS(PBS(-)).
balloons.
But when Ca*+(f* inal they
Under
that
pretreatment
resulted
in bleb
and balloon
Ca*+ but
without
toxin of
Therefore,
the
and a second
bleb
that
and balloon
is
Rapidity
of response
toxin(40
rig/ml)
showed
that
30-60
set
ical
times
from
This
cannot
toxin
pg toxin
adding 3A),
cells
equivalent),
of toxin
at 0-4°C
but
that
60 set, could
with
and then
toxin
wash-
rescu-
them after morpholog-
at 37°C after
prevent
the
Results
(0 set)
washing
even washing not
not
at 37°C in PBS(+).
on the ultimate observed
to to
did
the cells
the
from
HeLa cells.
60 or 120 set)
had no effect
for
present
them at 37°C in PBS(+).
(Fig.
toxin
of Ca 2+
with the action
20 min PBS(-) of
3C and D). on 30-60
proceeds
be rescued
of the toxin
Figure
ultimately
and incubated
after
or with-
of the enterotoxin:
and leads
We incubated
immediately
both with
toxin
or
in medium with
the binding
the
30,
with
formation.
in the action
(0,
form blebs
in the absence
obtained
with
with
shows that
enterotoxin
pretreated
periods
not
incubation
Ca 2+ -dependent
in Ca *+-and
to the cells,
and includes
preincubation
antitoxin(10 (Fig.
two steps
3B) and Vero
containing
were
and incubated
the
did
and balloon
when washed
PBS(-)
to
Thus neither
rig/ml)
2+ and Ca were
results
is
short
with
added
on subsequent
of HeLa (Fig.
After
toxin
for
toxin(200
the toxin
to enterotoxin:
the cells
in PBS(+).
the
of cells
the action
alterations
that Cells
with
preincubation
with
Ca 2+ -independent step
However,
enterotoxin
solution.
the cells
when toxin
alterations
at 37'C
washing
ed them all
formation
Similar
formation.
show such morphological
ed them three
cells
must be at least
step
cells,
20 min with
to show bleb
as observed
an initial
salt
cells:
binding.
0.9 mM) was then
of Vero
above.
I 125 II-labeled
of
conditions
began
on HeLa and Vero
as described
balanced
toxin
these
incubation.
there
Hank's for
immediately
2E shows
the beginning
binding
at 37°C for
Mg*+-free
out washing,
of enterotoxin
of the toxin
observed
Ca*+ nor Mg 2+ must be necessary We incubated
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
set
exposure
to an irreversible
to the stage
by washing.
633
toxin,
from which
the action
of the
the intoxicated
cells
Vol. 91, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fii. 3. Phase contrast micrographs of H eLa cells (2-day cultures) treated at or (D) 60 set after 37 C. Cells washed, (A) immediately. (B #) and (C) 30 sec. adding toxin (40 rig/ml) in PBS(-), with PBS(-) (A and B) or with PBS(-) containing antitoxin (C and D), and subseq uently incubated in PBS(+), 20 min. Cells in PBS(+) for 15 min with toxin ( 200 rig/ml) that had been pretreated with (E) 20 mM cysteine at 2S°C for 60 min and (F) with cholesterol (200 rig/ml) at 37“C for 60 min. The scale indicates 100 pm.
634
BIOCHEMICAL
Vol. 91, No. 2, 1979
Effect
of ionophore
ethanol
and 25 pl
concentration sence,
of the solution
the ionophore formation
induced
by the
alterations.
molar possibly
with
there
of various
treatments
immediately attached
inhibited
the action
Heating or antitoxin
(37°C
activated enterotoxin
rapidly,
toxins,
action
leading
alterations
ionophore
induced
of Ca2+ ions.
molecular
alterations.
mechanism
for
of the action
635
in 40 min at 37'C).
with
0.2% formalin(18)
activity.
Unlike
thiol-
2. p erfringens
60 min)(Fig.
3E) but
enterotoxin
proceeds
trypsin-resistant
Ca2+ is
required
receptors for
The osmotically similar
the enterotoxin
of the membranes findings
1E) and partially
3F).
and the
that
suspended
of sucrose
B-toxin,
with
and that
of the above
of the entero-
The presence
cytotoxic
(25'C
by the enterotoxin
On the basis
the ionophore,
to toxin
it
of -C. perfringens
in permeability
C6. On a
Cells
formed
60 min)(Fig.
of Ca 2+ suggest
that
as sensitive
(Fig.
were
its
an interaction
to morphological
by change
2F).
10 min or pretreating
cells
and
and balloon
cells
enterotoxin:
cells
vesicles
the action
sensitive
in the presence
effect
of the
37°C for
bleb
than
were
(Fig.
by 20 mM cysteine
through
of the
of the
inactivated
ab-
cells.
such as -C. perfringens
show that
probably
on the surface
on exact
60 min)
(ZOO rig/ml,
The above data
toxic
for
its
bleb
of interaction
treatment
(fine
in
from
glial
more effective
as monolayers
toxin
in
such morphological
similar
N18 and rat
sensitive
the response
induced
not produce
mechanism
trypsin-EDTA
was inactivated
not by cholesterol
670 times
at 80°C for
cytolytic
clone
not
indistinguishable
produced
on the action
of the
the toxin
Cl300
on the
after
delayed
did
2+ , but
at 0-4"C,
cells
the ionophore
to the dish
M in PBS(+))
alone
was dissolved
in 5 ml of PBS (final
of Ca
but not
is some efficient
receptors
as cells
surface
(0.5%)
roughly
specific
in PBS(+)
quent
is
to cells
1F) and Vero
the toxin,
the toxin
because
Effects
very
at 37"C,
Ethanol
A23187
In the presence
on mouse neuroblastoma
basis
toxin
0.5%).
on HeLa (Fig. toxin.
cells:
was added
(2 pg/ml)
Unlike
formation
on HeLa and Vero
of ethanol,
balloon
(0.2
A23187
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
studies
of enterotoxin.
the
subse-
sensitive effect
of the
exerts
its
and consequent
cyto-
influx
are now in progress
Vol. 91, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL
ACKNOWLEDGEMENT. We thank Drs. G. Sakaguchi Osaka Prefecture for providing the bacterial tion of purified enterotoxin.
RESEARCH COMMUNICATIONS
and T. Uemura of University of strain and for advice on prepara-
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
McDonel, J. L. (1974) Infect. Immun. 10, 1156-1162. McDonel, J. L., and Asano, T. (1975) Infect. Immun. 11, 526-529. McDonel, J. L., and Duncam, C. L. (1977) J. Infect. Dis. 136, 661-666. M. W. (1978) Biochem. Biophys. Res. Commun. Geiger, O., and Pariza, 82, 378-383. McDonel, J. L., and Duncan, C. L. (1975) Infect. Immun. 12, 274-280. McDonel, J. L., and Duncan, C. L. (1976) Infect. Immun. 15, 999-1001. McDonel, J. L., and Duncan, C. L. (1975) Infect. Immun. 12, 1214-1218. McDonel, J. L., Chang, L. W., Pounds, J. G., and Duncan, C. L. (1978) Lab. Invest. 39, 210-218. McDonel, J. L. (1979) Am. J. Clin. Nutr. 23, 210-218. McClane, B. A., and McDonel, 3. L. (1979) J. Cell Physiol. 99, 191-200. Stark, R. L., and Duncan, C. L. (1972) Infect. Immun. 6, 662-673. Sakaguchi, G., Uemura, T., and Riemann, H. P. (1973) Appl. Microbial. 26, 762-767. Enders, G. Jr., and Duncan, C. L. (1977) Infect. Immun. 17, 425-429. Gyobu, Y., and Kodama, H. (1978) J. Food Hyg. Sot. Jpn. 19, 294-298 (in Japanese). McDonel, J. L., and McClane, B. A. (1979) Biochem. Biophys. Res. Commun. 87, 497-504. Dis. 131, 58-63. Keusch, G. T., and Donta, S. T. (1975) J. Infect. Dulbecco, R., and Vogt, M. (1954) J. Exp. Med. 99, 167-182. Matsuda, M., and Yoneda, M. (1976) Biochem. Biophys. Res. Commun. 68, 668-674.
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