Low pH-induced Formation of Ion Channels by Clostridium
difficile Toxin B in Target Cells*
Holger
Barth
,
Gunther
Pfeifer
,
Fred
Hofmann
,
Elke
Maier§,
Roland
Benz§, and
Klaus
Aktories
¶
From the
Institut für Experimentelle und
Klinische Pharmakologie und Toxikologie der
Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany,
and § Lehrstuhl für Biotechnologie,
Theodor-Boveri-Institut (Biozentrum) der Universität
Würzburg, Am Hubland, D-97074 Würzburg, Germany
Received for publication, October 17, 2000, and in revised form, December 20, 2000
 |
ABSTRACT |
Clostridium difficile toxin B (269 kDa), which is one of the causative agents of antibiotic-associated
diarrhea and pseudomembranous colitis, inactivates Rho GTPases by
glucosylation. Here we studied the uptake and membrane interaction of
the toxin with eukaryotic target cells. Bafilomycin A1, which prevents
acidification of endosomal compartments, blocked the cellular uptake of
toxin B in Chinese hamster ovary cells cells. Extracellular
acidification (pH
5.2) induced uptake of toxin B into the
cytosol even in the presence of bafilomycin A1. Toxin B increased
86Rb+ release when preloaded Chinese hamster
ovary cells were exposed to low pH (pH
5.6) for 5 min. Release
of 86Rb+ depended on the concentration of toxin
B and on the pH of the extracellular medium. An antibody directed
against the holotoxin prevented channel formation, whereas an antibody
against the N-terminal enzyme domain was without effect. The
N-terminally truncated toxin B fragment consisting of amino acids
547-2366 increased 86Rb+ efflux when cells
were exposed to low pH. Toxin B also induced pH-dependent
channel formation in artificial lipid bilayer membranes. Clostridium sordellii lethal toxin, another member of the
family of large clostridial cytotoxins, also induced increased
86Rb+ release at low pH. The results
suggest that large clostridial cytotoxins including C. difficile toxin B and C. sordellii lethal toxin
undergo structural changes at low pH of endosomes that are accompanied
by membrane insertion and channel formation.
 |
INTRODUCTION |
The large clostridial cytotoxins from Clostridium
difficile toxin A (308 kDa) and toxin B (269 kDa) are major
virulence factors of antibiotic-associated diarrhea and the causative
agents of pseudomembranous colitis (1-4). Both toxins are
O-glucosyltransferases that modify the small GTPases of the
Rho family (Rho, Rac, and Cdc42) by monoglucosylation at threonine 37 and threonine 35, respectively (5, 6). The glucosylation blocks the
biological functions of Rho GTPases, which are molecular switches in a
large array of signal processes including regulation of the actin
cytoskeleton (7, 8). Accordingly, some of the best-documented effects of large clostridial cytotoxins are the toxin-induced redistribution of
actin filaments, morphological changes, and rounding-up of cells.
Structure-function analyses of large clostridial cytotoxins suggest a
tripartite organization of the toxins similar to that known for
diphtheria toxin. According to this model, the catalytic domain is
located at the N terminus (9), whereas the C-terminal part of the
toxins is believed to bind to the cellular receptor (10-12). In the
middle of the molecule is a rather small hydrophobic region possibly
involved in the translocation of the toxin into the cytoplasm. Because
the substrates of the toxins are intracellularly located, translocation
of the toxins across the cell membrane is a prerequisite for their
actions on Rho GTPases. However, little is known about the site and
mechanism of translocation of the toxins. Two major traffic pathways
for bacterial exotoxins have been described. One group of toxins
including cholera toxin (13), shiga toxins (14), and Pseudomonas
aeruginosa exotoxin A (15) appears to be internalized after
binding to specific receptors and then follows a retrograde pathway
back to the endoplasmic reticulum, where the membrane
translocation occurs. Another group of toxins appears to enter the
cytosol from the low pH compartment of endosomes. Prototypes of this
group are diphtheria toxin (16), anthrax toxin (17), and
Clostridium botulinum C2 toxin (18). It is believed that
endosomal acidification leads to a conformational change of these
toxins, thereby allowing insertion into the endosomal membrane and
eventually allowing translocation across the endosomal membrane into
the cytosol. Some previous reports suggested that toxin B also escapes
from the endosome into the cytoplasm (19). Toxins that enter the
cytosol from endosomes were shown to induce ion-permeable channels in
artificial membranes and in cell membranes when exposed to acidic pH
(20, 21). Therefore, we investigated whether acidification also
triggers the formation of channels by large clostridial cytotoxins.
Here we report that under acidic conditions, C. difficile
toxin B and Clostridium sordellii lethal toxin induce
membrane permeabilization in eukaryotic cells as well as channel
formation in artificial lipid bilayer membranes.
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EXPERIMENTAL PROCEDURES |
Materials--
Cell culture medium was from Biochrom (Berlin,
Germany), and fetal calf serum was from PAN Systems (Aidenbach,
Germany). Cell culture materials were obtained from Falcon (Heidelberg,
Germany). Thrombin was from Sigma (Deisenhofen, Germany). Rubidium-86
(specific activity, 2 mCi/mg) was from PerkinElmer Life Sciences
(Boston, MA). Bafilomycin A1 was from Calbiochem (Bad Soden, Germany). Toxin B from C. difficile VPI 10463 and C. sordellii lethal toxin were purified as described elsewhere (22).
A polyclonal antibody against toxin B (anti-Tox B) was produced in a
rabbit. The toxin B fragment CDB1-546 (catalytic domain of
C. diffcile toxin B (amino acids 1-546)) was cloned,
expressed, and purified as described previously (22), and a monoclonal
antibody (anti-CDB1-546) was produced against this fragment.
Cloning and Expression of Toxin B Fragment
CDB547-2366--
CDB547-2366 (enzymatic
inactive fragment of C. diffcile toxin B (amino acids
547-2366)) was amplified from genomic DNA from C. difficile VPI
10463 by polymerase chain reaction with Taq polymerase
(Roche Diagnostics, Mannheim, Germany) and the oligonucleotide primers CDB547-BamHI (5'-GGGGATCCGATAATCTTGATTTTTCTCAAAAT-3') and
CDB3N-EcoRI (5'-GAATTCCTATTCACTAATCACTAATTG-3').
Amplification was done by denaturing at 94 °C for 10 s, primer
annealing at 55 °C for 30 s, and extension at 68 °C for 6 min and repeated for 25 cycles. The resulting polymerase chain reaction
product (2 µl) was cloned into pCR2.1 vector (Invitrogen, NV Leek,
The Netherlands) according to the manufacturer's instructions. For
expression of CDB547-2366, the gene was excised with
BamHI/EcoRI (NEB Biolabs, Schwalbach, Germany)
and cloned in BamHI/EcoRI-digested pGEX2T plasmid
(Amersham Pharmacia Biotech, Uppsala, Sweden) containing a double
glycine linker. Proteins were expressed in Escherichia
coli-TG1 as recombinant glutathione
S-transferase-fusion proteins and purified by affinity chromatography with glutathione-Sepharose 4B according to the manufacturer's instructions. Glutathione S-transferase was
cleaved off by thrombin, and proteins were analyzed by
SDS-polyacrylamide gel electrophoresis according to the method of
Laemmli (23).
Cell Culture and Toxin Translocation
Assay--
CHO1-K1, HT-29,
Caco-2, and Vero cells were cultivated in tissue culture flasks at
37 °C and 5% CO2 in Ham's F-12/Dulbecco's modified
Eagle's medium (DMEM) (1:1) containing 5% heat-inactivated (30 min,
56 °C) fetal calf serum, 2 mM L-glutamate,
100 units/ml penicillin, and 100 µg/ml streptomycin. Rat basophilic
leukemia (RBL) cells were cultivated in Eagle's minimal essential
medium plus Earle's salts containing 15% (v/v) heat-inactivated fetal calf serum, 4 mM glutamine, 100 units/ml penicillin, and
100 µg/ml streptomycin. All cells were routinely trypsinized and
reseeded twice a week. For the toxin B translocation assay, CHO cells
were preincubated for 15 min at 37 °C with 100 nM
bafilomycin A1 in serum-free medium (Ham's F-12/DMEM), toxin B (100 ng/ml) was subsequently added, and cells were incubated for an
additional 2 h at 4 °C in serum-free medium. Cells were washed
with cold medium and incubated for 5 min in serum-free medium (Ham's
F-12/DMEM) at pH 7.5, pH 5.2, or pH 4.5, respectively. Subsequently,
cells were incubated in complete medium, pH 7.5, at 37 °C in the
presence of bafilomycin A1, and after 1.5 h, phase-contrast
pictures were taken.
86Rb+ Efflux Measurements--
For
86Rb+ efflux experiments, cells were plated in
complete medium (Ham's F-12/DMEM containing 5% fetal calf serum) at a
density of ~2 × 105 cells/well in 24-well culture
plates. At 8 h after plating, fresh medium containing
86Rb+ (1 µ Ci/ml) was added, and cells were
incubated for an additional 14 h. Cells were chilled at 4 °C,
and fresh medium (4 °C; Ham's F-12/DMEM without serum) containing
toxin A or toxin B was added. Toxins were allowed to bind for 2 h
at 4 °C and then washed two times with cold medium to remove unbound
toxin. To initiate membrane insertion of the toxins, cells were treated
with warm medium (37 °C; Ham's F-12/DMEM without serum, pH
4.0-7.5) for 5 min at 37 °C. Cells were further incubated at
4 °C, and after various incubation times, aliquots of the medium
were removed, and 86Rb+ release was determined
by liquid scintillation counting in a 1209 Rackbeta beta counter from
LKB Wallac (Gräfeling, Germany).
Black Lipid Bilayer Experiments--
The methods used for black
lipid bilayer experiments have been described previously in detail
(24). Membranes were formed from a 1% solution of asolectin or
diphytanoyl phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) in
n-decane.
 |
RESULTS |
Translocation of Cell-bound Toxin B into the
Cytoplasm--
Treatment of CHO cells with bafilomycin A1, which
blocks the vacuolar H+-ATPase pump, prevented the cytotoxic
effect of toxin B. To test the effect of pH on toxin uptake, toxin B
was allowed to bind to cells at 4 °C. After 2 h, cells were
incubated for 5 min at 37 °C with bafilomycin-containing medium of
pH 7.5, pH 5.2, or pH 4.5, respectively. Thereafter, the acidified
media were replaced by neutral medium containing bafilomycin, and cells
were incubated for an additional 1.5 h at 37 °C. The cells were
then analyzed for cytopathic effects induced by toxin B by
phase-contrast microscopy. As shown in Fig.
1, cells rounded up when the pH was
shifted for 5 min to pH 5.2 or pH 4.5 but not at pH 7.5. The results
indicate that at low pH, the toxin crosses the cell membrane directly
even in the presence of bafilomycin. The data are in line with the recent report that toxin B escapes from an acidic endosomal compartment into the cytoplasm (19).

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Fig. 1.
Influence of extracellular pH on toxin B
uptake into bafilomycin A1-treated CHO cells. CHO cells were
preincubated for 15 min at 37 °C with 100 nM bafilomycin
A1, followed by a 2-h incubation with toxin B (100 ng/ml) in serum-free
medium. After washing, cells were incubated for 5 min in serum-free
medium (37 °C + bafilomycin A1) at pH 7.5, pH 5.2, or pH 4.5, respectively. Cells were further incubated at 37 °C in complete
neutral medium (pH 7.5) containing bafilomycin A1. After 1.5 h,
phase-contrast pictures were taken. In parallel, cells were incubated
with 100 ng/ml toxin B (Tox B) or without any drug
(con).
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Toxin B-induced 86Rb+ Release from CHO
Cells--
The conditions allowing direct uptake of toxin B across the
cytoplasmic membrane were used to test whether extracellular
acidification leads to channel formation and membrane permeabilization
by toxin B. To this end, the efflux of 86Rb+
from preloaded cells was studied. At first,
86Rb+-preloaded CHO cells were incubated
without and with toxin B and heat-inactivated toxin B, respectively. An
increase in 86Rb+ release was measured when
toxin B-treated cells were incubated in an acidified medium of pH 5.2 (Fig. 2A). The increased
86Rb+ release depended not only on low pH but
also required short-term (5 min) incubation at 37 °C. It is
noteworthy that under these conditions, cells did not round up,
indicating no intoxication by toxin B. Acidification of medium in the
absence of toxin B and in the presence of heat-inactivated toxin or
binding of toxin B to cells without subsequent acidification did not
increase 86Rb+ release. This result indicates
that toxin B was inserted into the membrane of eukaryotic cells
and increased membrane permeability after exposure of cells to acidic
pH. In Vero cells, toxin B caused a similar increase in efflux of
86Rb+ at low pH (Fig. 2B). This
finding indicates that channel formation by toxin is not restricted to
CHO cells. However, we were not able to detect an increase in
86Rb+ release in HT-29 and Caco-2 cells.
Additional studies on toxin B-induced 86Rb+
release were performed with the CHO-K1 cell line.

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Fig. 2.
Toxin B-mediated release of
86Rb+ from CHO and Vero cells after
acidification. A, CHO cells were preloaded with
86Rb+ (14 h, 1 µCi/ml) and subsequently
incubated for 2 h at 4 °C with toxin B (100 ng/ml) or with
heat-inactivated toxin B (100 ng/ml) in fresh medium without serum. For
control, cells were incubated without toxin. Cells were washed to
remove unbound toxin, and prewarmed medium (37 °C; pH 7.5 or pH 5.2, respectively) was added. Cells were incubated at 37 °C for 5 min and
then at 4 °C for an additional 55 min. The complete medium (500 µl) was removed, and 86Rb+ release was
determined by liquid scintillation counting. Values are given as
mean ± S.D. (n = 3). B, Vero cells
preloaded with 86Rb+ were incubated with toxin
B (100 ng/ml) or, for control, without toxin B. Cells were shifted for
5 min to pH 5.2 and further incubated at 4 °C for 25 min. The medium
was assayed for 86Rb+. Values are given as
mean ± S.D. (n = 3).
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Characterization of Toxin B-induced 86Rb+
Release from CHO Cells--
At first, the influence of pH on the
kinetics of the 86Rb+ release from toxin
B-treated CHO cells was studied. When the medium of toxin B-treated
cells was shifted to pH 5.2 for 5 min at 37 °C and cells were
subsequently incubated on ice at pH 5.2, the increase in
86Rb+ release occurred mainly within 20-30
min. A low additional increase in 86Rb+ release
occurred in a second phase within 90 min (Fig.
3). A similar release of
86Rb+ was observed when toxin B-treated cells
were kept for 5 min in a medium of pH 5.2 and then incubated at 4 °C
at pH 7.5. This finding suggests that membrane permeabilization by
toxin B is achieved by a brief acidic pulse. Once formation of channels
has occurred, the channels remained open to release
86Rb+ even at physiological pH. To further
characterize the formation of channels, we studied the pH dependence of
the toxin effects in more detail.
86Rb+-preloaded CHO cells were incubated with
toxin B and shifted to pH 7.5, pH 5.6, pH 5.2, or pH 5.0, respectively.
At the lowest pH (5.0), the largest increase in release of
86Rb+ occurred. At pH 5.6, 86Rb+ efflux was much less pronounced than that
at pH 5.2 or pH 5.0, and at pH 7.5, no increased release was measured
(Fig. 4A). Next, 86Rb+-preloaded CHO cells were incubated with
increasing concentrations of toxin B (25, 100, and 500 ng/ml,
respectively). Cells were then shifted to pH 5.2, and released
86Rb+ was determined.
86Rb+ efflux depended on the concentration of
the toxin. Treatment of cells with 100 and 500 ng/ml toxin B,
respectively, did not result in significant differences in
86Rb+ release (Fig. 4B), most likely
indicating saturation of the toxin binding capacity of CHO cells. This
finding suggests that the specific binding of toxin B to its cell
membrane receptor is a prerequisite for subsequent channel formation.
Moreover, the data indicate that channel formation is not caused by an
unspecific integration of toxin B into cell membranes.

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Fig. 3.
Effect of pH on toxin B-mediated
86Rb+ efflux.
86Rb+-preloaded CHO cells were incubated for
2 h at 4 °C with toxin B (100 ng/ml) or, for control, without
toxin. Cells were washed to remove unbound toxin, and prewarmed medium
(pH 5.2) was added. Cells were incubated at 37 °C for 5 min and
subsequently at 4 °C at pH 5.2 (control, ; toxin B, ) or pH
7.5 (control, ; toxin B, ), respectively. After an additional 5, 10, 15, 25, 55, and 85 min, 100 µl of the medium were removed, and
86Rb+ release was determined by scintillation
counting.
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Fig. 4.
pH and concentration dependence of toxin
B-mediated 86Rb+ efflux.
86Rb+-preloaded CHO cells were incubated for
2 h at 4 °C with toxin B (100 ng/ml) or, for control, without
toxin. Cells were washed to remove unbound toxin, and prewarmed medium
(pH 7.5, ; pH 5.6, ; pH 5.2, ; pH 5.0, ) was added. Cells
were incubated at 37 °C for 5 min and then incubated at 4 °C.
After 10, 20, 30, and 60 min, aliquots (100 µl) of the medium were
removed, and released 86Rb+ was determined
(A). 86Rb+-preloaded CHO cells were
incubated for 2 h at 4 °C with toxin B (25, 100, and 500 ng/ml,
respectively) or, for control, without toxin B. Cells were washed, and
prewarmed medium (pH 5.2) was added. Cells were incubated at 37 °C
for 5 min and then incubated at 4 °C. After 10 ( ), 20 ( ), 30 ( ), and 60 min ( ), aliquots (100 µl) of the medium were
removed, and 86Rb+ was determined
(B).
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Influence of Anti-toxin B Antibodies on Toxin B-induced
86Rb+ Release--
To confirm that receptor
binding of toxin B is necessary for subsequent membrane
permeabilization, toxin B was incubated with an antibody directed
toward the complete toxin, which recognizes mainly the receptor binding
domain of toxin B (25), or with an antibody against its N-terminal
catalytic domain, respectively. CHO cells were then incubated with the
respective toxin/antibody mixture. The antibody against the holotoxin
inhibited binding of toxin B to CHO cells and prevented the cytotoxic
effects (Fig. 5A). In
contrast, the antibody against the catalytic domain did not inhibit
toxin B-induced rounding of the cells (Fig. 5A).
Accordingly, the latter antibody did not affect toxin B-induced
86Rb+ release from CHO cells (Fig.
5B). Incubation of toxin B with the antibody against the
holotoxin prevented toxin-induced membrane permeabilization. However,
this antibody was without effect when added to cells after binding of
toxin B (Fig. 5B). This observation suggests that only toxin
B that was already bound to the cell membrane receptor formed channels
at low pH.

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Fig. 5.
Influence of anti-toxin B antibodies on
cytotoxic toxin B effects and pH-induced release of
86Rb+ from CHO cells. Toxin B was
preincubated with an antibody against the holotoxin (anti-Tox B) or
with an antibody against its catalytic domain
(anti-CDB1-546) for 30 min at 4 °C. For control, toxin
B was preincubated with phosphate-buffered saline. A, CHO
cells were incubated with the respective pretreated toxin B (100 ng/ml)
at 37 °C. The morphology of the cells after 3 h is shown.
B, 86Rb+-preloaded CHO cells were
incubated for 2 h at 4 °C in serum-free medium with the
respective pretreated toxin B (100 ng/ml), and pH shift assay (pH 7.5 versus pH 5.2) was performed. After 30 min at 4 °C, the
medium was removed, and 86Rb+ release was
measured. Values are given as mean ± S.D. (n = 3). C, cells were treated as described above. Additionally,
toxin B (100 ng/ml) was bound to CHO cells for 2 h at 4 °C,
anti-Tox B was added, and cells were incubated for 1 h. After pH
shift (pH 7.5 and pH 5.2, respectively), the cells were incubated for
30 min at 4 °C, and the medium was removed and assayed for
86Rb+. Values are given as mean ± S.D.
(n = 3).
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Effect of Toxin B Fragment CDB 547-2366 on
86Rb+ Release from CHO Cells--
To determine
the part of toxin B that mediates membrane permeabilization of cells
after acidification, we cloned and expressed a toxin B fragment. CDB
547-2366 consists of residues 547-2366, i.e.
of the putative receptor binding and membrane insertion domains, and
lacks the catalytic domain (Fig.
6A). The proteins were allowed
to bind to 86Rb+-preloaded CHO cells, and cells
were subsequently exposed to pH 7.5 or pH 4.5, respectively. The
release of 86Rb+ was determined. Fig. 6 shows
that the release of 86Rb+ from preloaded CHO
cells was increased when cells were treated with CDB
547-2366 and then exposed to acidic medium. It is
noteworthy that the concentration of toxin B fragment
CDB547-2366 (4 µg/ml) was higher than that of the
full-length toxin B (200 ng/ml), which was applied in parallel.

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Fig. 6.
Effect of toxin B fragment
CDB547-2366 on 86Rb+
release from CHO cells. A, SDS-polyacrylamide gel
electrophoresis of recombinant CDB547-2366.
CDB547-2366 was expressed in E. coli and
purified as described. After cleavage with thrombin, 1 µg of protein
was analyzed by 7% SDS-polyacrylamide gel electrophoresis and
Coomassie blue staining. B, release of
86Rb+ from CHO cells after treatment of cells
with toxin B and CDB547-2366, respectively, and exposure
to low pH medium. 86Rb+-preloaded (1 µCi/ml)
cells were incubated for 2 h at 4 °C with toxin B (200 ng/ml)
or with CDB547-2366 (4 µg/ml) in fresh medium without
serum. For control, cells were incubated without toxin. Cells were
washed, and prewarmed medium (37 °C; pH 7.5 or pH 4.5, respectively)
was added. Cells were incubated at 37 °C for 5 min and then
incubated at 4 °C for an additional 55 min. The medium (500 µl)
was removed and assayed for 86Rb+. Values
are given as mean ± S.D. (n = 3).
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Effect of C. sordellii Lethal Toxin on
86Rb+ Release from Cells--
Lethal toxin of
C. sordellii was tested for an influence on
86Rb+ release from cells after exposure to low
pH medium. Therefore, the toxin was allowed to bind to preloaded CHO
cells, and cells were subsequently shifted to pH 7.5 or pH 4.5, respectively, for 5 min at 37 °C and incubated for an additional 55 min at 4 °C. As shown in Fig. 7,
treatment of cells with lethal toxin of C. sordellii and
subsequent exposure to low pH medium increased the efflux of
86Rb+ from cells. Similar results were obtained
when RBL cells were used.

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Fig. 7.
pH-induced release of
86Rb+ from CHO cells after treatment with
lethal toxin (LT).
86Rb+-preloaded CHO cells were incubated for
2 h at 4 °C in serum-free medium with lethal toxin (400 ng/ml).
Cells were washed to remove unbound toxin, and prewarmed medium
(37 °C; pH 7.5 or pH 4.5) was added. Cells were incubated at
37 °C for 5 min and then incubated at 4 °C for an additional 55 min. The medium (500 µl) was removed to determine released
86Rb+. Values are given as mean ± S.D.
(n = 3).
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Toxin B Leads to Channel Formation in Artificial Lipid Bilayer
Membranes under Conditions of Low pH--
The toxin B-mediated efflux
of 86Rb+ from cells indicated that toxin B
formed channels, which were at least permeable to ions. Therefore, we
studied whether ion-permeable channels are also formed in lipid bilayer
membranes. In a first set of experiments, we studied the effect of
toxin B on membranes formed from different lipids such as
phosphatidylcholine and asolectin at pH 6. Under these
conditions, we observed only rare current fluctuations in single-channel recordings (see Fig.
8A). The fluctuations were very rapid as Fig. 8A demonstrates and had only a short
lifetime on the order of seconds. Their single-channel conductance was ~0.2-1.5 nS in 1 M KCl. Enhanced single-channel activity
was observed, however, when the pH was lowered to pH 5, and many more
channels were recorded under the conditions of the lowered pH (see Fig. 8B). The current fluctuations were again very rapid, and it
was difficult to provide a precise value for the single-channel
conductance. Histograms of the fluctuations that could be resolved
suggest that the pH did not influence the size of the channels as
compared with pH 6. The low pH seemed to increase simply the
channel-forming activity. Measurement with other salts such as LiCl
suggested that a variety of ions were permeable through the toxin B
channel, but it seemed to have generally a higher permeability for
cations than for anions.

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Fig. 8.
Channel formation of toxin B in artificial
lipid bilayer membranes. A, single-channel recording of
an asolectin/n-decane membrane in the presence of toxin B from C. difficile. 10 min after the formation of the membrane, 80 ng/ml
toxin B was added to the aqueous phase on one side of the membrane. The
aqueous phase contained 1 M KCl and 10 mM MES,
pH 6. B, an experiment similar to that described in
A. The left side of the record demonstrates the
small channel-forming activity of toxin B at pH 6. About 30 min after
membrane formation, the pH was lowered to pH 5 (right side,
arrow). Shortly after the decrease of the pH, the membrane
conductance increased due to the formation of many channels,
which had a small lifetime. The applied membrane potential was 50 mV;
temperature = 20 °C.
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DISCUSSION |
Bacterial toxins that modify intracellular substrates must
translocate across a lipid bilayer membrane to reach their targets (26). After endocytosis, two pathways are described that are used by
bacterial toxins to reach their cytosolic substrates. One possible
route for toxin uptake (e.g. cholera toxin (13)) is the
retrograde transport from endosomal compartments via the Golgi
apparatus to the endoplasmic reticulum, from which the toxins translocate into the cytoplasm. Other toxins (e.g.
diphtheria toxin (16) or the binary anthrax toxin (17)) translocate
directly from the acidic endosomal compartment into the cytosol.
Inhibition of the cytotoxic action of toxin B by bafilomycin, as
reported recently and shown in this report, indicates that toxin B also requires an acidic endosome for cellular uptake (19). In line with this
notion is the finding that the lowering of the extracellular pH allows
uptake of the toxin even in the presence of bafilomycin. Thus far, our
knowledge of the events that cause translocation of toxin B is very
limited. In the present study, we report that toxin B is capable of
inducing ion-permeable channels, a process that might reflect molecular
mechanisms closely related to translocation of the toxin into the
cytosol. For detection of channel formation, the release of
86Rb+ from preloaded cells was measured, and
conditions were chosen that mimic the endosomal compartment,
e.g. cell-bound toxin B was exposed to an acidic buffer
(pH < 5.6) for a short time (5 min) at 37 °C to induce channel
formation by the toxin. Similarly, we observed a strong induction of
channel formation in lipid bilayer membranes when the pH was lowered to
pH 5 (see Fig. 8). At pH 6, only a very small channel-forming activity
was observed. It is noteworthy that pH-dependent channel
formation has been reported for several protein toxins that are taken
up from endosomes (18, 21). It is believed that the low pH present in
endosomal compartments causes conformational changes of the toxin,
which are accompanied by surface exposition of the otherwise
intramolecularly located hydrophobic region of the toxins. The
hydrophobic regions are inserted into the bilayer membrane to
form channels and/or to allow protein translocation. Recently, Qa'Dan
et al. (19) analyzed the pH-induced conformational changes
occurring in toxin B. Using various fluorescence methods, it was shown
that the hydrophobicity of toxin B was increased at pH
5.0 but
not at pH
5.5 and concluded that toxin B undergoes pH-induced
structural changes that finally allow membrane insertion and
translocation of the toxin from the acidic endosome into the cytosol.
Thus far, the structural basis of the pH-dependent channels
is not clear. Our studies show that the N-terminal catalytic domain (toxin B fragment consisting of amino acids 1-546) is not necessary for channel formation. In contrast, a N-terminal truncated toxin B
fragment that consisted of amino acid residues 547-2366 was capable of
channel formation. However, because the C-terminal fragment was only
active at concentrations higher than that of the holotoxin, it
is suggested that the N terminus might also play a minor role in
binding/insertion or stabilization of the holotoxin. Nevertheless, the
results are in line with the view that the C terminus and the middle
part of large clostridial toxins are involved in receptor binding and
translocation. This hypothesis is supported by the findings that an
antibody directed against the holotoxin, which recognizes mainly the
receptor binding domain of toxin B (25), inhibited channel formation
and intoxication, whereas an antibody against the N-terminal catalytic
domain of toxin B was not able to prevent the increase in Rb efflux. In experiments with artificial black lipid bilayer membranes, membrane permeabilization by toxin B was detected at pH 6.0 and increased under
more acidic conditions (pH 4.5). Here, the antibody against the
holotoxin had no effect on toxin B-induced membrane permeabilization. This indicates that the antibody did not affect membrane insertion of
toxin B but might inhibit the toxin binding to the cell surface. However, it remains to be clarified whether insertion into artificial membranes and insertion into cell membranes follow identical mechanisms.
Moreover, the functional dissociation of channel formation and
cytopathic effects induced by the toxin is corroborated by the finding
that despite increased 86Rb+ efflux, the cells
still showed the control morphology when experiments were performed at
4 °C and only the acidic shift was done at 37 °C.
An increased 86Rb+ efflux was measured when
CHO, Vero, or RBL cells were studied. These cell lines show high
sensitivity toward toxin B and appear to have a sufficient amount of
cell membrane receptor molecules. In contrast, we were not able to
detect toxin B-induced channel formation in some other cell lines
(HT-29 and Caco-2). In agreement with the possible destruction of the
barrier function of the cytoplasmic membrane of CHO, Vero, or RBL
cells, we were able to identify channel formation in artificial lipid bilayer membranes. Channels were formed with the two lipids tested. This result seems to represent a contradiction to the obvious receptor-mediated permeabilization of cells. However, it has to be kept
in mind that other toxins, such as C2 toxin (20) or the repeat in
toxin toxins (27-29), form channels in lipid bilayer membranes
without the need for receptors, whereas they all need a receptor for
biological activity. An important role of the toxin receptor in events
finally resulting in channel formation is supported by the
concentration effect studies at high concentration of toxin, which show that limitation of channel formation is most likely due to
reaching the binding capacity of cells. In contrast to toxin B, toxin A
from C. difficile showed no detectable channel-forming activity in the 86Rb+ release assay with CHO
cells. This might be due to the relative insensitivity of CHO cells to
toxin A and to their low content of toxin A receptor molecules. Toxin
A, which is also termed enterotoxin, is known to exhibit much less
cytotoxic effect on many cultured cells than toxin B. Similar to toxin
B, C. sordellii lethal toxin caused channel formation in CHO
and RBL cells, which are especially sensitive to the lethal toxin,
suggesting that channel formation is a general activity of large
clostridial cytotoxins.
 |
ACKNOWLEDGEMENTS |
We thank Katrin Thoma and Otilia Wunderlich
for expert technical assistance. We thank Dr. Ingo Just for the
polyclonal anti-toxin B antibody and for discussion of the results.
 |
FOOTNOTES |
*
This work was supported by the Deutsche
Forschungsgemeinschaft (Sonderforschungsbereich 388 and 487) and by the
Fonds of the Chemische Industrie.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Institut für
Pharmakologie und Toxikologie, Hermann-Herder-Str. 5, D-79104 Freiburg, Germany. Tel.: 49-761-2035301; Fax: 49-761-2035311; E-mail:
aktories@uni-freiburg.de.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M009445200
 |
ABBREVIATIONS |
The abbreviations used are:
CHO, Chinese hamster
ovary;
DMEM, Dulbecco's modified Eagle's medium;
RBL, rat basophilic
leukemia.
 |
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