Although the hemolytic activity of AC toxin has a lag of >1 h,
intoxication starts immediately. Because of this difference, we sought
a surrogate or precursor lesion that leads to hemolysis, and potassium
efflux has been observed from erythrocytes treated with other
pore-forming toxins. AC toxin elicits an increase in K+
efflux from sheep erythrocytes and Jurkat cells, a human T-cell leukemia line, that begins within minutes of toxin addition. The toxin
concentration dependence along with the analysis of the time course
suggest that toxin monomers are sufficient to elicit release of
K+ and to deliver the catalytic domain to the cell
interior. Hemolysis, on the other hand, is a highly cooperative event
that likely requires a subsequent oligomerization of these individual
units. Although induction of K+ efflux shares some
structural and environmental requirements with both intoxication and
hemolysis, it can occur under conditions in which intoxication is
reduced or prevented. The data presented here suggest that the
transmembrane pathway by which K+ is released is separate
and distinct from the structure required for intoxication but may be
related to, or a precursor of, that which is ultimately responsible for
hemolysis.
 |
INTRODUCTION |
Adenylate cyclase (AC)1
toxin is an important virulence factor for Bordetella
pertussis and probably Bordetella bronchiseptica and
Bordetella parapertussis (1-4). It is an acylated, 177-kDa protein that can deliver its catalytic domain to the interior of intact
target cells (5-8). Inside the cell AC toxin binds endogenous
calmodulin, resulting in a 1000-fold increase in its enzymatic activity
and production of supraphysiologic levels of intracellular cAMP from
host ATP (9). This process is referred to as intoxication. Although
intoxication is believed to be the primary contribution of AC toxin to
Bordetella virulence, this protein is also lytic for sheep
erythrocytes and, thus, responsible for the hemolytic phenotype of
phase I B. pertussis (10, 11). Removal of the N-terminal
catalytic domain (amino acids 1-400) does not affect hemolysis or pore
formation in artificial lipid bilayers (12, 13). Both of these
activities are attributed to the C-terminal end of AC toxin that is
homologous to Escherichia coli hemolysin and other members
of the RTX family of bacterial toxins (13-15). Intoxication and
hemolysis require post-translational acylation of the toxin at one or
two internal lysine residues, which converts the inactive protoxin to
the active form (8, 16). Acylation appears to occur within the
bacterium and is dependent on the product of a separate gene,
cyaC (17, 18). A similar acylation is required for
activation of E. coli hemolysin and probably other members
of the RTX family (15, 19, 20).
As with other hemolysins, AC toxin has been shown to produce
transmembrane ion conductance in artificial lipid bilayers, and the
properties of the responsible structure have been investigated (13,
21). We have shown that pore formation is
calcium-dependent, as are intoxication and hemolysis, and
have demonstrated that this activity has a cubic or higher
concentration dependence, suggesting that an oligomer is the active
species (21). Benz et al. (13) studied the single channel
properties of the pore formed by AC toxin and found it to be
cation-selective and considerably smaller than that of E. coli hemolysin. In keeping with that observation, the hemolytic
activity of AC toxin is modest, relative to E. coli hemolysin or other RTX hemolysins (13, 14). The time course is
prolonged (hours rather than minutes), and the concentration required
to elicit >90% hemolysis is high, in the 10 µg/ml range (22). By
using the method of osmotic solute exclusion, Ehrmann et al.
(22) estimated the pore created by AC toxin to be <0.6 nm, which is
significantly smaller than the 2-3 nm estimated for E. coli
hemolysin (23).
A major focus of our work has been to determine the sequence of events
involved in AC toxin's interaction with target cells and the
relationships among these activities. The onset of intoxication is
rapid and maximal in 30-60 min at toxin concentrations of about 1 µg/ml (22, 24). However, the hemolytic activity of AC toxin demonstrates a lag of >1 h even at toxin concentrations of 10 µg/ml
(22). The significant difference in both time course and concentration
dependence for intoxication and hemolysis suggested to us that these
two processes occur by different mechanisms. In order to address this,
we hypothesized that there might be an earlier event in the course of
toxin-cell interaction that is an antecedent of hemolysis. Potassium
efflux has been used previously to measure pore formation by toxins and
other proteins that traverse cell membranes (25-32). This efflux of
K+ has been shown to be rapid in onset and to precede cell
swelling and eventual lysis. Potassium is present inside the cell at
concentrations greater than 100 mM and can be measured
simply and directly in the culture medium by flame photometry. By using
this method, we have found that AC toxin-treated sheep erythrocytes
release K+ with an onset comparable to that of
intoxication. The toxin concentration dependence of K+
efflux is a first order process, suggesting that a monomer is responsible. Whereas intoxication has a similar concentration dependence, hemolysis shows a cubic or higher power dependence, suggesting that an oligomer is necessary. Potassium efflux can be
dissociated from intoxication because it occurs under conditions in
which intoxication is reduced or prevented. These results suggest that
K+ efflux is an early event in toxin-cell interaction that
can be dissociated from intoxication. This process may reflect the
initial insertion of the toxin monomer into the target cell membrane in a process culminating in toxin oligomerization and hemolysis.
 |
EXPERIMENTAL PROCEDURES |
Production and Purification of Adenylate Cyclase
Toxin--
Production of recombinant AC toxin was done as described
previously (33) with minor modifications. E. coli BL21 cells
(Stratagene, La Jolla, CA) containing plasmid pT7CACT1 for expression
of wild type AC toxin, or plasmid pACT7 for production of
CyaC
AC toxin, were grown to an optical density of 0.2 at
600 nm at 37 °C in 2× YT medium (1.6% Bacto-tryptone, 1%
Bacto-yeast, 85 mM NaCl) containing 150 µg/ml ampicillin.
Isopropyl-
-D-thiogalactopyranoside (1 mM)
was added to the cultures which were incubated an additional 4 h.
Cultures were centrifuged, and the resulting pellet was stored at
70 °C overnight. Pellets were resuspended in 50 mM
Tris, pH 7.5, sonicated, and extracted with 8 M urea.
Urea-extracted AC toxin was purified on a calmodulin affinity column as
described previously (34). B. pertussis strain BPLME58IE
(11) was grown, and the toxin was purified as described previously
(34). AC toxin was stored at
70 °C in 8 M urea, 10 mM Tricine, 0.5 mM EDTA, 0.5 mM
EGTA, pH 8.0.
Adenylate Cyclase Enzymatic Activity--
AC activity was
measured by the conversion of [32P]ATP to
[32P]cAMP as described previously (35, 36). Briefly, each
assay tube contained 60 mM Tricine, 10 mM
MgCl2, 2 mM ATP, with 2 × 105
cpm of [
-32P]ATP and 1 µM calmodulin at
pH 8.0. All buffers and samples were equilibrated to 0-2 °C or
37 °C as indicated. The reaction was initiated with the addition of
[
-32P]ATP and carried out in an ice bath at 0-2 °C
or at 37 °C for 10, 20, and 30 min. This reaction was terminated by
the addition of a solution containing 1% SDS, 20 mM ATP,
and 6.24 mM cAMP (with 1.5 × 104 to
2.0 × 104 cpm of [3H]cAMP). Cyclic AMP
was separated from substrate ATP by the double column method of Salomon
et al. (37), and enzymatic activity was determined by
measuring [32P]cAMP in a scintillation counter (Beckman
Instruments, Palo Alto, CA).
Sheep and Human Erythrocytes--
Blood was drawn into a flask
containing Alsever's Solution and immediately placed on ice. Within 30 min of obtaining it, the blood was centrifuged, and the serum and buffy
coat were removed. Erythrocytes were washed 3 times in Hanks' balanced
salt solution, pH 7.4 (HBSS), and the hematocrit was adjusted to 40%
(approximately 1010 RBC/ml). Erythrocytes were used within
2 days.
Intoxication Assay--
AC toxin activity was measured by
incubating 1 ml of erythrocytes at a 40% hematocrit with AC toxin at
37 °C unless otherwise indicated. Cells were then centrifuged, the
pellet washed 3 times in HBSS, and 10% trichloroacetic acid added to
lyse the cells and precipitate the hemoglobin. The supernatant (400 µl) containing the cellular cAMP was removed and extracted 3 times
with H2O-saturated ether to remove residual trichloroacetic
acid. HCl was added to yield a final concentration of 0.1 N, and cAMP was measured by an automated radioimmunoassay
(38).
Hemolytic Activity and K+ Efflux--
Hemolytic
activity and K+ efflux were measured by incubating 1 ml of
erythrocytes at a 40% hematocrit with AC toxin for the indicated
amount of time. Cells were centrifuged, and the supernatant was used to
measure hemolysis as described previously (22), and K+
efflux, using an Instrumentation Laboratory model IL943 flame photometer. In experiments comparing K+ efflux with
hemolysis, control cells were lysed to determine the total
K+ present; AC toxin-induced K+ efflux is
reported as a percent of total using Equation 1.
|
(Eq. 1)
|
In all other situations, K+ efflux was expressed as
mmol/liter, with K+ efflux from control cells typically
being 5.2-6.7 mmol/liter. For K+ efflux in Jurkat cells,
2 × 107 cells/500 µl were incubated with AC toxin
for 30 min at 37 °C. Cells were centrifuged and the supernatant
collected to measure K+ efflux.
Trypsin Treatment of Cell-associated Adenylate Cyclase
Toxin--
After initial incubation of erythrocytes with AC toxin,
cells were washed 3 times in HBSS, treated with 1 mg/ml trypsin (Sigma) for 20 min at 0-2 °C, and the reaction stopped with 2 mg/ml lima bean trypsin inhibitor (Sigma). Control cells were treated with trypsin
inhibitor first and then trypsin. Where indicated, cells were washed 2 times, resuspended to 40% hematocrit, and incubated an additional 30 min at 37 °C.
Renaturation of AC Toxin in the Presence of Calcium--
AC
toxin, which is stored in 8 M urea, 0.5 mM
EDTA, 0.5 mM EGTA was renatured by a >15-fold dilution of
toxin solution into HBSS. The urea concentration present after this
dilution was <500 mM and free calcium concentration was
1.1 mM.
Data Analysis--
The time courses of intoxication,
K+ efflux, and hemolysis shown in Fig. 1 were fitted by
Equation 2.
|
(Eq. 2)
|
where t is time,
is the time constant of the rise
of effect,
is the amplitude of the effect, and n is a
parameter related to the stoichiometry of the molecular complex
involved in this process. A value of n = 1 corresponds
to a simple exponential rise expected for a first order process. Larger
values of n are expected when several molecules must combine
to form a functional complex.
Nonlinear curve fitting was carried out using the program Origin
(Microcal Software). The resulting fits are shown as solid lines, and
the corresponding parameters are given in the figure legends. An
analogous procedure was used to fit the Hill equation to the data of
Fig. 3.
 |
RESULTS |
Temporal Relationships among Intoxication, Hemolysis, and
K+Efflux--
The difference in onset of intoxication and
hemolysis has made it difficult to determine the relationship between
these two functional activities of AC toxin and suggests that they
occur by different mechanisms. In studies using erythrocytes, addition of other pore-forming toxins has been shown to cause a rapid release of
K+ which was followed by cell swelling and eventual lysis.
That is to say, efflux of K+ can be used as a diagnostic
tool to measure increases in membrane permeability and is thought to
represent the initial manifestation of the transmembrane pore that is
responsible for ultimate osmotic lysis (25-32). In the present study,
we hypothesized that K+ efflux would occur in erythrocytes
treated with AC toxin and that it would precede hemolysis. The timing
of K+ efflux from AC toxin-treated sheep erythrocytes in
relation to intoxication or hemolysis is illustrated in Fig.
1, A and B,
respectively. Both intoxication and K+ efflux (Fig.
1A) exhibit virtually immediate onset with first order
kinetics. In comparison, hemolysis is delayed and negligible in
magnitude for periods as long as 90 min even at a high toxin concentration (20 µg/ml) (Fig. 1B). These data clearly
suggest that K+ efflux is an indicator of toxin-mediated
membrane perturbation, which precedes hemolysis and is associated
temporally with intoxication. As shown in Fig.
2, AC toxin also elicits K+
release from Jurkat cells, a human T-cell leukemia line that is not
lysed by AC toxin. This observation indicates that the process
resulting in K+ efflux is not restricted to non-nucleated
cells or cells such as erythrocytes that are lysed by AC toxin. In
light of these observations, it was important to characterize in depth
K+ efflux as a consequence of AC toxin interaction with
target cells. In order to determine relevance of this phenomenon to
hemolysis, sheep erythrocytes were chosen for use in subsequent
experiments as the model system.

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Fig. 1.
Time course of intoxication, K+
efflux, and hemolysis in sheep erythrocytes. AC toxin was
incubated with erythrocytes at 37 °C. At the time indicated, cells
were spun, and the supernatant was removed to measure K+
efflux and hemolysis. The pellet was washed and intracellular cAMP
measured as described under "Experimental Procedures." Solid
lines show the fit of Equation 2 to the data. A, AC
toxin, 10 µg/ml. Parameters for the fit of the intoxication data were
= 331 ± 30, = 17 ± 5 min, n = 1.4 ± 0.3 and for the K+ efflux data were = 6.2 ± 0.5, = 106 ± 16 min, n = 0.8 ± 0.02. B, AC toxin, 20 µg/ml. Parameters for the fit of
the hemolysis data were = 66 ± 0.5, = 1.2 ± 0.02 h, n = 14 ± 0.4 and for K+
efflux data were = 97 ± 6, = 1.9 ± 0.5 h,
n = 1.0 ± 0.1. Each point is the mean of
duplicate determinations. This graph is representative of
three separate experiments.
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Fig. 2.
AC toxin causes an increase in K+
efflux in Jurkat cells. AC toxin at indicated concentrations was
added to 2 × 107 Jurkat cells in 500 µl and
incubated for 30 min at 37 °C. Cells were spun, and the supernatant
was used to measure K+ efflux. Each point is the mean of
duplicate determinations. This graph is representative of
three separate experiments.
|
|
Concentration Dependences of Intoxication, Hemolysis, and
K+ Efflux--
To examine more closely the relationship
between K+ release and hemolysis, we evaluated the
concentrations of toxin required for these two activities and compared
them to that necessary for intoxication. The concentration dependences
of intoxication and K+ efflux at 30 min and hemolysis at
4.5 h are shown in Fig. 3. The
differences between intoxication or K+ efflux and hemolysis
are striking. A quantitative assessment of these differences was
obtained by fitting the data to the Hill equation, shown as solid
lines. Intoxication and K+ efflux demonstrate Hill
coefficients near unity (n = 1.39 ± 0.10 and
0.82 ± 0.04 respectively), whereas that for hemolysis is markedly higher (n = 4.99 ± 0.71), suggesting
cooperativity as observed previously (21). These data along with the
analysis of the time course strongly suggest that toxin monomers are
sufficient to elicit release of K+ and the delivery of the
catalytic domain to the cell interior. Hemolysis, on the other hand, is
a highly cooperative event that likely requires a subsequent
oligomerization of these individual units.

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Fig. 3.
Concentration dependence for intoxication,
K+ efflux, and hemolysis in sheep erythrocytes. AC
toxin at indicated concentrations was added to 1010 sheep
erythrocytes and incubated at 37 °C. Incubation time for cAMP
accumulation and K+ efflux was 30 min; hemolysis was
4.5 h. Each point is the mean of duplicate determinations.
Solid lines show the fit of the Hill equation with maximal
effect fixed at 100%. The best fitting parameters were as follows.
Intoxication: K50 = 0.78 ± 0.06, nH = 1.4 ± 0.1; K+ efflux:
K50 = 6.9 ± 0.6, nH = 0.8 ± 0.04; hemolysis: K50 = 12 ± 0.5, nH = 5.0 ± 0.7. This graph
is representative of seven separate experiments.
|
|
Structural and Functional Requirements for Intoxication, Hemolysis,
and K+ Efflux--
In light of the discrepancy between the
stoichiometry for K+ efflux and hemolysis, processes that
were expected to be closely linked, we began an investigation to
compare and contrast K+ efflux with intoxication and
hemolysis. Both intoxication and hemolysis require acylation of an
internal lysine residue in a process mediated by CyaC (8, 16, 17). The
data in Fig. 4 demonstrate that
non-acylated protoxin (CyaC
) does not elicit
K+ efflux even at toxin concentrations of 20 µg/ml. Thus,
acylation is necessary for all three activities.

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Fig. 4.
Acylation of AC toxin by CyaC and toxin-bound
calcium are necessary to elicit release of K+ from sheep
erythrocytes. Sheep RBC were washed in 10 mM HEPES,
140 mM NaCl, 5 mM KCl, 0.1% glucose, 2 mM EDTA, 3 mM MgCl2, pH 7.4. Where
indicated, calcium was added to washed cells to achieve a free
Ca2+ concentration of 1.2 mM. AC toxin (10 µg/ml), AC toxin (10 µg/ml) renaturated in the presence of
Ca2+ (AC toxin RN+Ca2+), or CyaC
AC toxin (20 µg/ml) were added to sheep erythrocytes and incubated
for 30 min at 37 °C. Cells were spun, and the supernatant was used
to measure efflux of K+. Each point is the mean of
duplicate determinations. This graph is representative of
two separate experiments. , 2 mM EDTA; , 1.2 mM Ca2+.
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|
Calcium Requirement--
The amino acid sequence of AC toxin
includes approximately 41 copies of a nonameric, glycine, and aspartic
acid-containing motif that is involved in calcium binding in other
bacterial proteins (6, 14, 15, 39). The functional importance of this
domain is illustrated by the observation that extracellular calcium is necessary for intoxication and hemolysis (34, 35, 40-43). Fig. 4
demonstrates that K+ efflux is similarly
calcium-dependent. Furthermore, Rose et al. (43)
and Rogel et al. (10) have observed that toxin prepared in
the presence of calcium, but assayed in the presence of the calcium
chelator EGTA, is able to induce hemolysis but not intoxication. Rose
et al. (43) suggested that AC toxin contains approximately 45 low affinity binding sites for calcium and another 3-5 high affinity sites (43). Calcium bound to these few high affinity sites has
been postulated to be responsible for the hemolytic activity of AC
toxin when assayed in the presence of calcium chelators. As shown in
Fig. 4, when AC toxin is exposed to calcium before addition to sheep
RBC in the presence of EDTA, K+ efflux was 71% that
induced by AC toxin assayed with sheep erythrocytes in the presence of
calcium. In contrast, AC toxin exposed to calcium but incubated with
RBC in EDTA, increased intracellular cAMP <1% that observed in RBC
assayed in the presence of calcium (data not shown). These latter
observations are comparable with those reported previously (10, 43) and
suggest that calcium occupancy of both high and low affinity sites is
necessary for intoxication, whereas occupancy of the high affinity
sites alone is sufficient for both K+ release and
hemolysis.
Calmodulin Effect--
Differences in calcium requirement
notwithstanding, similarities in time course and concentration
dependence of intoxication and K+ efflux raised the
question of how these two activities are related. It has been shown
previously that exposure of AC toxin to calmodulin prior to its
addition to erythrocytes markedly inhibits intoxication (10, 44, 45)
but does not influence hemolysis (10). The effects of calmodulin on the
ability of AC toxin to produce K+ efflux and intoxication
are shown in Fig. 5. Extracellular
calmodulin did inhibit intoxication by >90%, consistent with previous
data (10, 44, 45). In contrast, K+ efflux was not affected
by the presence of calmodulin (Fig. 5). This observation not only
dissociates intoxication from K+ efflux but suggests that
intoxication-associated increases in cAMP are not responsible for
K+ efflux. To demonstrate further that cAMP accumulation
does not induce K+ efflux, we used a mutant AC toxin
(BPLME58IE), which contains a single amino acid substitution at amino
acid 58, that eliminates catalytic activity (11, 46). This mutant toxin
caused K+ efflux to an extent similar to that of wild type
toxin (data not shown). Thus the ability of AC toxin to increase
intracellular cAMP is not required to cause K+ efflux.

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Fig. 5.
Calmodulin inhibits the intoxication of sheep
erythrocytes by AC toxin, without affecting its ability to increase
K+ efflux. AC toxin (10 µg/ml) was incubated with
calmodulin (CaM, 6 µM) for 20 min at room
temperature. Sheep erythrocytes were added and incubated for 1 h
at 37 °C. Cyclic AMP accumulation and K+ efflux were
measured as described under "Experimental Procedures." Each point
is the mean of duplicate determinations. This graph is
representative of two separate experiments. , intoxication; ,
K+ efflux.
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|
Temperature Dependences of Intoxication and K+
Efflux--
Translocation of the catalytic domain across the cell
membrane is highly temperature-dependent, occurring only
above 20 °C (47). The temperature dependences of K+
efflux and intoxication at 0-2 and 37 °C are illustrated in Fig. 6. Incubation of target erythrocytes at
0-2 °C has only a modest effect on the initial rate of
K+ efflux and results in no cumulative difference from
incubation at 37 °C by 1.5 h. On the other hand, delivery of
the catalytic domain, as measured by accumulation of intracellular
cAMP, is markedly impaired at the reduced temperature (Fig. 6 and Table I). The reduction in cAMP accumulation at
0-2 °C far exceeds the expected temperature-dependent
decrease in enzymatic activity (Table I). As shown in Table I, in
vitro enzymatic activity at 0-2 °C is 6.4-6.6% that at
37 °C, whereas intracellular cAMP accumulation ranges from only 0.14 to 0.48% that observed at 37 °C over 30-90 min. Thus, reduced
temperature impairs delivery of the catalytic domain to the cell's
interior, thereby inhibiting intoxication, with only a small effect on
K+ efflux.

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Fig. 6.
Intoxication, but not K+ efflux,
is markedly reduced at 0-2 °C. Sheep erythrocytes at 0-2 or
37 °C were incubated with AC toxin (10 µg/ml) for the indicated
times. Cyclic AMP accumulation and K+ efflux were measured
as described under "Experimental Procedures." Each point is the
mean of duplicate determinations. This graph is
representative of three separate experiments. , intoxication,
37 °C; , intoxication, 0 °C; , K+ efflux,
37 °C; , K+ efflux, 0 °C.
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|
The temperature dependence of delivery of the catalytic domain was
confirmed by additional studies illustrated in Table
II. These experiments were based on the
concept that if the catalytic domain of AC toxin has not entered the
cytoplasm at 0-2 °C, it will remain susceptible to degradation by
trypsin added to the extracellular medium. On the other hand, if the
catalytic domain has been delivered to the cytoplasm it will be
protected from tryptic digestion (18, 42, 47). Erythrocytes exposed to AC toxin at 37 °C for 30 min accumulate 464 nmol of
cAMP/1010 cells, as compared with 1 nmol of
cAMP/1010 cells at 0-2 °C. Cells exposed to AC toxin at
0-2 °C for 30 min, then treated with trypsin and allowed an
additional incubation at 37 °C, accumulated 6.5 nmol of
cAMP/1010 RBC. This value represents just 1.2% of the
intracellular cAMP produced in cells incubated at 37 °C for the
initial 30 min, treated with trypsin, and incubated an additional 30 min at 37 °C (544 nmol cAMP/1010 RBC). This indicates
that at least 98.8% of the AC toxin had remained on the surface of
cells held at 0-2 °C and was removed by trypsin treatment of those
cells. These data suggest that the catalytic domain is not inserted in
the membrane of the target cell at 0-2 °C, a condition under which
K+ efflux is occurring (Fig. 6), and provide further
evidence for K+ efflux under conditions that do not support
intoxication. From the data presented thus far we conclude the
following: 1) the ability of wild type toxin to elicit K+
efflux is independent of its ability to increase intracellular cAMP
levels; 2) K+ efflux is not mediated by a defect in the
cell membrane resulting from delivery of the catalytic domain, and; 3)
an endogenous, cAMP-activated channel is not responsible for
K+ efflux.
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Table II
The catalytic domain of AC toxin is susceptible to trypsin treatment
when incubated with sheep erythrocytes at 0-2 °C
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|
Temperature Dependences of K+ Efflux and
Hemolysis--
To explore further the relationship between
K+ efflux and release of hemoglobin, we investigated the
temperature dependence of hemolysis as compared with K+
release. As shown in Fig. 7, hemolysis
was markedly delayed at 0-2 °C as compared with 37 °C. As noted
earlier, K+ efflux was only modestly affected by a
reduction in temperature, indicating that K+ efflux can be
dissociated from hemolysis by a change in temperature. This effect of
temperature might be expected if K+ efflux was the result
of insertion of a toxin monomer, whereas hemoglobin release required
oligomerization of toxin molecules resulting from lateral diffusion
within the plasma membrane. This interpretation is consistent with the
stoichiometric data showing different concentration dependences for the
two activities. The data suggest that additional steps, including
oligomerization, are required for progression from the initial event
responsible for K+ efflux to hemoglobin release.

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Fig. 7.
Effect of reduced temperature on
K+ efflux and hemolysis of sheep erythrocytes. Sheep
erythrocytes at 0-2 °C or 37 °C were incubated with AC toxin at
20 µg/ml for the indicated time. Cells were spun, and the supernatant
was used to measure K+ efflux and hemolysis. Each point is
the mean of duplicate determinations. This graph is
representative of three separate experiments. , K+
efflux, 37 °C; , K+ efflux, 0 °C; , hemolysis,
37 °C; , hemolysis, 0 °C.
|
|
Effects on Human Erythrocytes--
For reasons that are unknown,
human erythrocytes are resistant to hemolysis by AC toxin but can
become intoxicated, albeit at a comparatively reduced level (10). The
data in Fig. 8 show that treatment of
human RBC with AC toxin causes cAMP accumulation to levels
approximately 30% those achieved in sheep RBC. In marked contrast, no
K+ efflux occurred in human RBC treated with AC toxin for
1 h at 37 °C, even at a concentration of toxin that was 3-fold
greater than that typically used for study of K+ efflux
(Fig. 8). This observation further demonstrates that K+
efflux and intoxication are independent events, each able to occur in
the absence of the other. Furthermore, these data suggest that the
transmembrane pathway by which K+ is released is separate
from the structure required for intoxication but may be related to, or
a precursor of, that which is ultimately responsible for hemolysis.

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Fig. 8.
Human erythrocytes are resistant to
K+ efflux produced by AC toxin. Human erythrocytes
were exposed to AC toxin at the concentrations indicated for 60 min at
37 °C. Intracellular cAMP accumulation and K+ efflux was
measured as described. Each point is the mean of duplicate
determinations. This graph is representative of three
separate experiments.
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|
 |
DISCUSSION |
Although efflux of potassium from target cells has been described
previously as a marker of pore formation in cell membranes by a variety
of toxins (25-32), its demonstration here in response to AC toxin has
special significance in the investigation of the complex sequence of
events by which this toxin acts. In the past, the delayed onset and
variability of the hemolytic response precluded its direct comparison
with intoxication, which is rapid and robust, suggesting that different
mechanisms may be responsible for these two activities. We show here
that K+ efflux occurs at a rate similar to intoxication and
at toxin concentrations similar to those necessary to increase
intracellular cAMP levels. Intoxication and hemolysis are both
acylation- and calcium-dependent (8, 16, 34, 35, 40-43);
K+ efflux is similarly dependent on both the acylation and
calcium concentration. AC toxin produced in B. pertussis is
acylated exclusively at lysine 983, whereas recombinant toxin produced
in E. coli is acylated at lysine 860 and lysine 983. This
double acylation of recombinant toxin has been shown to result in
reduced hemolytic potency compared with toxin produced in B. pertussis (16). We found that the same is also true for
K+ efflux, namely AC toxin produced in B. pertussis is more potent than the recombinant toxin (data not
shown). Whereas the previous report suggested that the additional
acylation at lysine 860 might impair oligomerization (16), the power
dependence derived from both the time course and the concentration
response reveals that monomers are likely responsible for
K+ efflux and intoxication. Since the stoichiometry
strongly suggests hemolysis being elicited by an oligomer, the
mechanistic basis for the difference between native and recombinant
toxin remains to be determined.
To confirm that K+ efflux, thought to be a precursor of
hemolysis, is not somehow dependent on intoxication, we used several methods to determine that K+ efflux and intoxication are
separate events. Both K+ efflux and hemolysis can occur
when AC toxin is exposed to calcium, then added to erythrocytes in the
presence of a molar excess of EDTA. There is, however, no intoxication
under these conditions, indicating the requirement for extracellular
calcium at the time of toxin interaction with the erythrocyte. This
suggests that while the calcium requirements for K+ efflux
and hemolysis are similar, that for intoxication is different.
Inhibition of intoxication by exogenous calmodulin is a peculiar
phenomenon because it is target cell-specific (44, 45, 48). In several
cell types, addition of calmodulin to AC toxin prior to addition to
cells has little or no effect on intoxication, whereas in others an
inhibitory effect is striking. The working hypothesis is that
calmodulin bound to the calmodulin-binding site in the catalytic domain
can interfere with its delivery to the cell interior. Why this would be
cell-specific remains unknown, although membrane composition and
transmembrane potential may be involved (21, 24). Nevertheless,
previous data showed that exposure of AC toxin to calmodulin prior to
its addition to sheep RBC inhibited intoxication, whereas it had no
effect on hemolysis (10). This observation served as a way to determine
if K+ efflux was associated with intoxication or hemolysis.
In fact, we observed that K+ efflux from sheep RBC is not
affected by prior addition of calmodulin to AC toxin, indicating again
that K+ efflux is dissociable from intoxication.
Previous studies showed that toxin insertion, measured by the presence
of AC toxin tightly bound to sheep erythrocytes, occurs at temperatures
between 4 and 36 °C, but that translocation of the catalytic domain
across the red cell membrane required temperatures above 20 °C (47).
Our results confirm those reported earlier that delivery of the
catalytic domain does not occur at 0-2 °C. Surprisingly, we found
that K+ efflux was only slightly affected at the reduced
temperature when compared with that at 37 °C. This observation
suggests that K+ efflux may represent the initial
interaction of a part of the toxin molecule with the cell membrane and
establishes that K+ efflux is not dependent on the delivery
of the catalytic domain and/or the resultant cAMP accumulation. In
addition, a mutant AC toxin containing a single amino acid
substitution, which inhibits its enzymatic activity at least 1000-fold,
elicited an efflux in K+ similar to that of wild type
toxin.
Whereas these observations support the suggestion that intoxication and
K+ efflux are fully dissociable, they do link
K+ efflux and hemolysis. Although the concentration
dependence of K+ efflux suggests that a monomer is
responsible for efflux, data presented here and elsewhere (21) indicate
that an oligomer is necessary for hemolysis. In addition, temperature
had only a modest effect on K+ efflux, although its effect
on hemolysis was dramatic. This suggests that the insertion of
monomeric AC toxin allows K+ release, which is followed by
a time-, temperature-, and concentration-dependent oligomerization leading to the formation of a larger transmembrane pore
required for hemolysis. A diagrammatic representation of these data is
presented in Fig. 9.

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Fig. 9.
Schematic representation of proposed activity
of AC toxin. *, in the presence of 1.2 mM calcium; **,
at 0-2 °C in the presence of 1.2 mM calcium or at 0-2
or 37 °C when AC toxin is exposed to calcium and then incubated with
RBC in the presence of EDTA, hence, with only the high affinity calcium
sites filled. , amino acids 1-400, catalytic domain; , amino
acids 401-1706.
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Human erythrocytes are resistant to hemolysis by AC toxin even though
the toxin does bind and they do become intoxicated (10). In the context
of evaluating K+ efflux, we have confirmed these earlier
results, and we demonstrate that AC toxin does not elicit
K+ efflux from human RBC. In sheep erythrocytes, we have
shown by several different approaches that we could inhibit the
intoxication by AC toxin without affecting its capability to elicit
K+ efflux. The ability of AC toxin to intoxicate human red
blood cells without causing an increase in K+ efflux shows
that the converse is also true, namely that the delivery of the
catalytic domain is not dependent upon the toxin-cell interaction by
which K+ efflux occurs. Although it is possible that the
lack of K+ efflux from human red blood cells is due to a
difference in membrane composition from sheep erythrocytes, these data
establish that K+ efflux and intoxication can be mutually
exclusive events. Given these observations, one must consider that
concurrent intoxication and K+ efflux might even reflect a
mixed population of AC toxin molecules exhibiting one or the other
activity.
Taken together these data strongly support the concept that AC toxin
binds to the erythrocyte membrane as a monomer. This interaction with
the membrane can result in K+ efflux and/or the insertion
of the catalytic domain resulting in increases in intracellular cAMP.
These two pathways are distinct and separable, each able to occur in
the absence of the other. Insertion of the monomeric AC toxin allowing
K+ efflux is a precursor of toxin oligomerization which is
necessary for hemolysis. Because of its relative temperature
insensitivity, K+ efflux may result from partial insertion
of the toxin molecule in the membrane. This step may be followed by a
temperature-sensitive, more complete insertion of AC toxin that results
in delivery of the catalytic domain to the interior of the cell. On the
other hand, K+ efflux and intoxication could be the result
of a mixed population of AC toxin molecules, each able to achieve only
a single function. Nevertheless, the monomers involved in
K+ efflux then coalesce to form a larger, oligomeric pore
responsible for hemolysis. These observations provide a new perspective
on the functions of this novel toxin, and studies are underway to characterize further its mechanisms of action.
We thank Peter Sebo for the constructs used
for expression of AC toxin; Dede Haverstick for assistance with the
Jurkat cells; Dr. Robert Carey and Nancy Howell for the use and
assistance with their flame photometer; and Starr Palmore for help with
the manuscript.