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Applied and Environmental Microbiology, February 1999, p. 457-464, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Toxicity, Binding, and Permeability Analyses of Four
Bacillus thuringiensis Cry1 
-Endotoxins Using Brush
Border Membrane Vesicles of Spodoptera exigua
and Spodoptera frugiperda
Ke
Luo,1
David
Banks,1 and
Michael J.
Adang1,2,*
Department of
Entomology,1 and
Department of
Biochemistry and Molecular Biology,2
University of Georgia, Athens, Georgia 30602
Received 8 July 1998/Accepted 1 November 1998
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ABSTRACT |
The binding and pore formation properties of four Bacillus
thuringiensis Cry1 toxins were analyzed by using brush border
membrane vesicles from Spodoptera exigua and
Spodoptera frugiperda, and the results were compared to the
results of toxicity bioassays. Cry1Fa was highly toxic and Cry1Ac was
nontoxic to S. exigua and S. frugiperda larvae, while Cry1Ca was highly toxic to S. exigua and weakly toxic to S. frugiperda. In
contrast, Cry1Bb was active against S. frugiperda but
only marginally active against S. exigua. Bioassays
performed with iodinated Cry1Bb, Cry1Fa, and Cry1Ca showed that the
effects of iodination on toxin activity were different. The toxicities
of I-labeled Cry1Bb and Cry1Fa against Spodoptera species
were significantly less than the toxicities of the unlabeled toxins,
while Cry1Ca retained its insecticidal activity when it was labeled
with 125I. Binding assays showed that iodination prevented
Cry1Fa from binding to Spodoptera brush border membrane
vesicles. 125I-labeled Cry1Ac, Cry1Bb, and Cry1Ca bound
with high-affinities to brush border membrane vesicles from
S. exigua and S. frugiperda. Competition binding experiments performed with heterologous toxins revealed two major binding sites. Cry1Ac and Cry1Fa have a common binding site, and Cry1Bb, Cry1C, and Cry1Fa have a second common binding site. No obvious relationship between dissociation of bound
toxins from brush border membrane vesicles and toxicity was detected.
Cry1 toxins were also tested for the ability to alter the permeability
of membrane vesicles, as measured by a light scattering assay. Cry1
proteins toxic to Spodoptera larvae permeabilized brush
border membrane vesicles, but the extent of permeabilization did not
necessarily correlate with in vivo toxicity.
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INTRODUCTION |
The Cry1 
-endotoxins of
Bacillus thuringiensis are a family of 130- to 140-kDa
proteins that exhibit specific toxicities to numerous lepidopteran
insects (1, 19). Several Cry1 -endotoxins, including
Cry1Bb, Cry1Ca, and Cry1Fa, are toxic to important agricultural pests
belonging to the genus Spodoptera (3, 7, 46).
Biopesticides based on the Cry1Ca and Cry1Fa toxins are now available
for control of Spodoptera exigua and Plutella
xylostella in the field (29, 37). The most recent
application of Cry proteins for the control of Spodoptera
larvae is the development of transgenic crops that express a synthetic
cry1Ca gene (36). Evolution of insect resistance to Cry proteins will undoubtedly challenge the long-term use of these
toxins. Field results obtained with P. xylostella and
laboratory results obtained with S. exigua and
Spodoptera littoralis have shown that pests evolve
resistance to the Cry1Ca and Cry1Fa toxins (30, 31, 39).
B. thuringiensis Cry proteins intoxicate insects via the
larval midgut (16, 21). Toxin binding to midgut brush border sites is a key determinant of toxicity to insects (40). For example, loss of binding sites, as measured by reduced toxin binding, is associated with acquired resistance in Plodia
interpunctella and P. xylostella larvae (14,
38, 41). Cry1A-resistant P. xylostella is
cross-resistant to Cry1Fa toxin apparently because of the absence of
available toxin binding sites shared by the Cry1A and Cry1Fa in
susceptible larvae (39). However, the extent of toxin
binding does not always correlate with insecticidal activity (15,
42).
One explanation for binding that does not result in insect mortality is
the fact that some Cry1 toxins bind to the brush border membrane but do
not readily insert into the membrane of the insect midgut. This
phenomenon is reflected by a toxin's inability to bind to the brush
border membrane irreversibly (20, 24). The relevance of
"irreversible" binding was demonstrated clearly by Rajamohan et al.
(34). Cry1Ab toxin domain II mutants that were 400-fold less
toxic to Manduca sexta exhibited total binding to brush border membrane vesicles (BBMV) similar to the total
binding of wild-type Cry1Ab toxin. However, recombinant toxin binding was reversible and thus differed from the irreversible binding of the
wild-type Cry1Ab toxin.
After Cry1 toxin binds to the brush border membrane, a large portion of
the molecule inserts into the membrane, forming ion channels. Carroll
and Ellar (6) developed a straightforward assay for
measuring Cry1 toxin-induced permeability in BBMV. When BBMV are
diluted in a hyperosmotic solution, they rapidly shrink, which results
in an increase in scattered light. If the solute (such as KCl) is
permeable through toxin-induced pores, the vesicles rapidly reswell.
Using this technique, Carroll and Ellar (6) found that
Cry1Ac significantly increased the membrane permeability of
M. sexta BBMV. In contrast, Cry1Ba, which is not toxic to
M. sexta, had no effect on BBMV permeability. The
correlation between toxicity and pore formation is now becoming better
established. For example, Lorence et al. (25) observed that
two insecticidal Cry toxins (Cry1Ca and Cry1D), but not inactive Cry1Ac
toxin, formed cation channels in the brush border membrane of
Spodoptera frugiperda. Also, mutations in Cry1Aa toxin that
resulted in a loss of toxicity to Bombyx mori larvae
impaired the ability of the toxin to form pores in the BBMV
(43).
The purpose of this study was to compare the binding and pore formation
properties of B. thuringiensis Cry1Ac, Cry1Bb, Cry1Ca, and
Cry1Fa by using BBMV from S. exigua and S. frugiperda. We established that the toxicities of the four toxins
examined to S. exigua and S. frugiperda
larvae differ significantly. Binding experiments showed that the
Cry1Ac, Cry1Ca, and Cry1Bb toxins bound with similar affinities to
S. exigua and S. frugiperda BBMV, although some of these toxins recognized distinct binding sites. Our
results demonstrated that toxin activities inferred from
membrane-permeabilizing capacities determined by the light scattering
assay are not necessarily related to 50% lethal concentrations
(LC50) determined by feeding bioassays.
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MATERIALS AND METHODS |
B. thuringiensis strains and toxin purification.
B. thuringiensis strains that harbored the
cry1Ca, cry1Fa, or cry1Bb gene were
provided by Ecogen, Inc. (Langhorne, Pa.). The GenBank accession number
of the cry1Ca gene cloned from B. thuringiensis
subsp. entomocidus is X07518. The GenBank accession numbers
of the cry1Fa gene cloned from B. thuringiensis
subsp. aizawai (7) and the cry1Bb gene
cloned from B. thuringiensis subsp. aizawai
EG5847 are M63897 and L32020, respectively. B. thuringiensis
subsp. kurstaki HD-73, which produces only one Cry protein,
Cry1Ac, was obtained from the Bacillus Genetic Stock Culture
Collection (Columbus, Ohio).
All of the B. thuringiensis strains were grown for 3 days at
28°C in 1 liter of half-strength L broth until sporulation and cell
lysis. The crystals, spores, and debris were collected by centrifugation at 7,500 × g for 30 min, and the pellet
was washed with 1 M NaCl containing 0.1% Triton X-100 and then with
distilled water. The crystals were dissolved in 50 ml of 50 mM
Na2CO3 (pH 9.6) containing 0.1%
2-mercaptoethanol by incubating the preparation for 2 h at room
temperature and then centrifuged at 27,000 × g for 30 min to remove the insoluble debris. The protoxin was treated with 10 mg
of L-1-tosylamide-2-phenylethylchloromethyketone-treated trypsin (Sigma) at room temperature for 15 min (Cry1Ca) or 30 min
(Cry1Fa, Cry1Bb, and Cry1Ac). Toxin samples were filtered through a
0.2-µm-pore-size filter (Nalge Co.) and then were loaded onto an
Econo-Pac Q anion-exchange column (Bio-Rad) that previously had been
equilibrated with Na2CO3 buffer (pH 9.6) and
was connected to a fast-performance liquid chromatograph (Pharmacia).
The toxin was eluted from the column with a 0 to 0.6 M NaCl gradient in 20 mM Na2CO3 (pH 9.6). Fractions containing
purified toxin were pooled and stored at 4°C. For the Cry1Ca toxin,
an additional purification step was performed to remove contaminating
small peptides. The Cry1Ca toxin that eluted from the Econo-Pac Q
column was dialyzed against 20 mM Na2CO3 buffer
(pH 9.6) overnight and then loaded onto a Mono Q HR 5/5 column
(Pharmacia). The bound toxin was eluted by using the same NaCl
gradient. Fractions containing purified Cry1Ca toxin were pooled and
stored at 
20°C until they were used. We found that this additional
purification step was important for labeling the Cry1Ca toxin to a high
specific activity.
Insect bioassays.
Toxin preparations were diluted with
phosphate-buffered saline (10 mM Na2HPO4, 1.7 mM KH2PO4, 2.7 mM KCl, 136.9 mM NaCl [pH 7.4]) containing 0.1% bovine serum albumin (BSA). Seven toxin concentrations were tested by using 20 neonate S. exigua or S. frugiperda larvae per concentration.
S. exigua was obtained from the USDA/ARS Southern Field
Crop Insect Management Laboratory, Stoneville, Miss., and S. frugiperda was obtained from the USDA/ARS Turf Grass Laboratory
Tifton, Ga. Cry1 toxin samples (50 µl) were applied uniformly to the
surface (area, 2 cm2) of an artificial diet preparation
(Southland Products, Lake Village, Ark.) and then allowed to dry. Each
larva was placed onto the diet surface and reared at 26°C and 70%
relative humidity with a photoperiod consisting of 12 h of light
and 12 h of darkness. Insect mortality was scored after 7 days,
and the data were analyzed with the POLO-PC computer program
(9).
Iodination.
Cry1Ac was iodinated by the chloramine T method
described by Garczynski et al. (15). Cry1Ca, Cry1Fa, and
Cry1Bb were iodinated by the Iodobead (Pierce) method as described
previously (27), except that each reaction vial was gently
shaken during iodination. The specific radioactivities were 20, 6.1, 10.1, and 8.6 µCi/µg of input toxin for Cry1Ac, Cry1Ca, Cry1Fa, and
Cry1Bb, respectively. Cry1Bb, Cry1Ca, and Cry1Fa were labeled with Nal
by the Iodobead (Pierce) method (27) in order to examine the
effects of iodination on toxin activity. Briefly, 50 µl of 50 mM Nal
(pH 9.0) was added to a reaction mixture containing 300 to 400 µg of
toxin. After incubation for 10 min at room temperature, the free Nal
molecules were removed by three 1-ml washes with 20 mM
Na2CO3 (pH 9.6) by using a Centriprep-30
ultrafiltration device (Amicon) at 4°C, and then the toxins were
concentrated to concentrations of 300 to 400 µg/ml. The protein
concentrations were determined after the preparations were washed with
a Bio-Rad protein assay kit; BSA was used as the standard as described
by Bradford (5).
Gel electrophoresis.
Cry1 toxin preparations (approximately
10 µg for gels that were stained with Coomassie brilliant blue R-250
or 105 cpm for 125I-labeled proteins) were
analyzed by sodium dodecyl sulfate-10% polyacrylamide gel
electrophoresis (SDS-PAGE). The gels were stained with Coomassie
brilliant blue R-250 or were exposed to a Kodak XAR-5 film with an
intensifying screen at 80°C for 3 h.
Preparation of BBMV.
Midguts were excised from fifth-instar
S. exigua and S. frugiperda larvae and
frozen on dry ice. BBMV were prepared by the MgCl2
precipitation method (44), as modified by Ferre et al. (14). Each final BBMV pellet was suspended in 0.3 M
mannitol-5 mM EGTA-17 mM Tris-Cl (pH 7.5) and stored at 80°C
until it was used.
Membrane vesicle binding assays.
Binding assays were
performed as described by Garczynski et al. (15).
Qualitative binding experiments were done to determine if each labeled
Cry1 toxin bound to S. exigua and S. frugiperda BBMV and to identify a BBMV concentration suitable for
competition binding experiments. Duplicate samples of various
concentrations of BBMV from S. exigua and S. frugiperda were incubated with 0.1 nM 125I-labeled
toxin in 100 µl of phosphate-buffered saline containing 0.1% BSA at
room temperature for 30 min (Cry1Ac, Cry1Fa, and Cry1Bb) or 60 min
(Cry1Ca). The samples were centrifuged at 15,000 × g for 5 min, and then the pellets were washed twice with binding buffer.
The radioactivity was measured with a Beckman model Gamma 4000 detector.
To evaluate binding at a quantitative level, we performed homologous
competition assays (in which competition between a labeled
ligand and
an unlabeled ligand was examined) with different amounts
of unlabeled
Cry1Ac, Cry1Ca, and Cry1Bb as described above by
using BBMV from
S. exigua and
S. frugiperda. Using the
results
of these binding experiments, we calculated the dissociation
constants
(
Kd) and the binding site
concentrations (
Bmax) with the LIGAND
computer
program (Biosoft). We performed heterologous competition
binding assays
(in which competition between a labeled toxin and
a different unlabeled
ligand was examined) with different amounts
of unlabeled Cry1Ac,
Cry1Bb, Cry1Ca, and Cry1Fa toxins to investigate
whether these toxins
recognized the same binding
site.
To measure toxin dissociation, BBMV (10 µg of protein) were incubated
with
125I-labeled toxins (concentration, 0.1 nM) at room
temperature for
30 min (Cry1Ac) or 60 min (Cry1Ca and Cry1Bb) in order
to achieve
equilibrium binding, and then unlabeled toxins (final
concentration,
100 nM) were added into the reaction mixtures. The
amounts of
the bound
125I-labeled toxins were then
determined at different times after
the excess unlabeled toxins were
added as described by Van Rie
et al. (
40).
Permeability assays.
Light scattering assays were performed
with a stop flow spectrofluorimeter (model RSM 1000; On-line Instrument
Systems, Bogart, Ga.). BBMV from S. exigua and
S. frugiperda were diluted to a concentration of 0.5 mg/ml with 10 mM HEPES-Tris (pH 8.0) containing 0.1% BSA. Aliquots
(1.0 ml) of BBMV were incubated with Cry1Ac, Cry1Bb, Cry1Ca, or Cry1Fa
toxin (final concentration, 2.5 µg/ml) at room temperature for 30 min. The toxin-BBMV mixture and 0.5 M KCl in 10 mM HEPES-Tris (pH 8.0)
were manually transferred into separate reservoirs by using syringes.
Assays were initiated by simultaneously injecting 35 µl of a
toxin-BBMV mixture and 35 µl of KCl into the cuvette in the
spectrofluorimeter sample compartment. Incident 450-nm light was
scattered at 90° from incidence and was monitored for 120 s by
obtaining five measurements per s. The model RSM-1000
spectrofluorimeter has a time lag of about 3 ms between the injection
time and the onset of data collection by the photodetector. To
determine the amount of light scattered by control BBMV before
osmotically induced shrinkage (i.e., in buffer alone), measurements
were obtained after simultaneous injection of 35 µl of 10 mM
HEPES-Tris (pH 8.0) and 35 µl of BBMV in 10 mM HEPES-Tris (pH 8.0).
To determine the maximum amount of light scattered after BBMV
shrinkage, the scattered light was measured after BBMV and 0.5 M KCl
were injected simultaneously. The cuvette was washed three times with
50 mM Tris (pH 8.0) between measurements involving different insect
BBMV. Assays were conducted in triplicate for both species for each toxin.
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RESULTS |
Toxicity of four B. thuringiensis toxins to
S. exigua and S. frugiperda.
Figure 1 shows the purity of the Cry1
toxins used in this study. Each toxin appeared as a single band on an
SDS-PAGE gel after staining for unlabeled toxins and after
autoradiography for 125I-labeled toxins. In bioassays,
Cry1Ac did not kill S. exigua or S. frugiperda (Table 1). Cry1Ca was
highly toxic to S. exigua (LC50, 127 ng/cm2) and weakly toxic to S. frugiperda
(LC50, 1,144 ng/cm2). In contrast, Cry1Bb was
active against S. frugiperda (LC50, 308 ng/cm2) but only marginally active against
S. exigua (LC50, 1,468 ng/cm2).
Only Cry1Fa exhibited high levels of activity against both S. exigua and S. frugiperda
(LC50, 177 and 109 ng/cm2, respectively).
Bioassays performed with iodinated Cry1Bb, Cry1Fa, and Cry1Ca showed
that the effects of iodination on toxin activity differed between
toxins. I-labeled Cry1Bb was not toxic to S. frugiperda, I-labeled Cry1Fa exhibited greatly reduced toxicity against both Spodoptera species compared with unlabeled
Cry1Fa, and I-labeled Cry1Ca retained insecticidal activity (Table 1).
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FIG. 1.
SDS-PAGE and autoradiography analyses of purified Cry1
toxins. (A) Coomassie blue-stained SDS-PAGE gel. Lane 1, molecular size
markers; lane 2, Cry1Ac; lane 3, Cry1Ca; lane 4, Cry1Fa; lane 5, Cry1Ba. (B) Autoradiograph of 125I-labeled toxins. Lane 2, Cry1Ac; lane 3, Cry1Ca; lane 4, Cry1Fa; lane 5, Cry1Ba. The numbers on
the left are molecular masses (in kilodaltons).
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Maximal binding of labeled toxins to BBMV from S. exigua and S. frugiperda.
Qualitative
binding experiments were done to determine if each labeled Cry1 toxin
bound to S. exigua and S. frugiperda BBMV and to identify a BBMV concentration
suitable for competition binding experiments. 125I-labeled
Cry1Ac, Cry1Bb, Cry1Ca, and Cry1Fa were incubated with various
concentrations of BBMV from S. exigua and S. frugiperda. Maximal binding of Cry1Ac, Cry1Ca, and Cry1Bb was
observed at concentrations between 200 and 300 µg of vesicle protein
per ml (Fig. 2). Cry1Ca and Cry1Bb bound
similarly to S. exigua and S. frugiperda BBMV (Fig. 2), although their toxicities against the two species were different (Table 1). Cry1Ac, which is not toxic to
S. exigua and S. frugiperda, bound
strongly to BBMV from both species (27 and 28% of the input
125I-Cry1Ac bound to S. exigua and
S. frugiperda BBMV, respectively) (Fig. 2).
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FIG. 2.
Binding of 125I-labeled Cry1Ac ( ), Cry1Bb
( ), Cry1Ca ( ), and Cry1Fa ( ) to S. exigua BBMV
(A) and S. frugiperda BBMV (B). Vesicles at the
concentrations indicated were incubated with 125I-labeled
toxins at a concentration of 0.1 nM. Each data point is a mean based on
the results for duplicate samples.
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Much less
125I-Cry1Fa than other Cry1 toxins bound to BBMV
from
Spodoptera species. The maximal levels of binding to
both species
were only 4 to 5% of the input
125I-labeled
toxin (Fig.
2). Perhaps attachment of
125I to the Cry1Fa
toxin reduces its toxicity to larvae and its binding
to BBMV. Even when
the lower toxicity of labeled Cry1Fa was taken
into account, toxicity
and maximal binding were not well correlated
for the toxins. In
particular, labeled Cry1Fa was more toxic to
both species than Cry1Ac
was, yet Cry1Fa exhibited much lower
maximal binding than Cry1Ac
exhibited.
Homologous and heterologous competition binding.
As shown in
Table 2, three toxins bound to BBMV from
S. exigua and S. frugiperda with
similar Kd and Bmax
values. We could not calculate Kd and
Bmax values for Cry1Fa due to the low level of
binding of 125I-labeled Cry1Fa to BBMV from the two
insects.
In both
S. exigua and
S. frugiperda,
unlabeled Cry1Bb and Cry1Ca did not compete with bound
125I-Cry1Ac (Fig.
3A and B).
Similarly, unlabeled Cry1Ac did not
compete with
125I-Cry1Ca and
125I-Cry1Bb (Fig.
3C to F).
Cry1Fa exhibited high-affinity competition
for the binding sites of
125I-Cry1Ac (Fig.
3A and B),
125I-Cry1Bb
(Fig.
3C and D), and
125I-Cry1Ca (Fig.
3E and F). In both
S. exigua and
S. frugiperda,
unlabeled
Cry1Ca exhibited significant competition for bound
125I-Cry1Bb (Fig.
3E and F). Similar competition
was also observed
with labeled Cry1Ca and unlabeled Cry1Bb (Fig.
3C and D). These
binding data revealed that there are two high-affinity
Cry1 binding
sites in
S. exigua and
S. frugiperda. Cry1Ac and Cry1Fa recognize
one site, and Cry1Bb,
Cry1Ca, and Cry1Fa recognize a second site.
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FIG. 3.
Competition between 125I-labeled Cry1Ac (A
and B), Cry1Bb (C and D), and Cry1Ca (E and F) toxins and unlabeled
Cry1Ac (), Cry1Bb (), Cry1Ca (), and Cry1Fa () toxins.
S. exigua BBMV (A, C, and E) and S. frugiperda BBMV (B, D, and F) were incubated with
125I-labeled toxins at a concentration of 0.1 nM plus
different concentrations of unlabeled toxins. Binding was expressed as
a percentage of the maximum amount of toxin bound during incubation
with labeled toxin. Each data point is a mean based on the results for
duplicate samples.
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Dissociation binding assays.
Since Cry1Ac, Cry1Ca, and Cry1Bb
had different toxicities for S. exigua and
S. frugiperda larvae but bound with similar
Kd and Bmax values to
BBMV from the two insects, we investigated whether the extents of
dissociation of membrane-bound toxins are major factors in their
toxicities. As shown in Fig. 4, the
extents of Cry1Ac, Cry1Ca, and Cry1Bb toxin dissociation from
S. exigua and S. frugiperda BBMV were
not different (Fig. 4A and B). For example, while the LC50
of Cry1Ca for S. exigua was 127 ng/cm2 and
the LC50 of Cry1Ca for S. frugiperda was
1,144 ng/cm2, the amount of 125I-Cry1Ca
displaced by excess unlabeled Cry1Ca was the same for both species.
Also, Cry1Ac was the least toxic of the three toxins tested, yet its
dissociation was intermediate between the dissociation of Cry1Bb and
the dissociation of Cry1Ca. Furthermore, binding of the three toxins to
BBMV was for the most part irreversible after incubation for 70 min
with excess unlabeled toxins (Fig. 4A and B). The difference between
the insecticidal and noninsecticidal Cry1 toxins could not be accounted
for by dissociation of inactive toxins from the membrane surface.
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FIG. 4.
Dissociation of Cry1Ac (), Cry1Bb (), and Cry1Ca
() from S. exigua BBMV (A) and S. frugiperda BBMV (B). BBMV (10 µg of protein) were incubated with
125I-labeled toxin at a concentration of 0.1 nM for 30 min
(Cry1Ac) or 60 min (Cry1Bb and Cry1Ca) in order to obtain equilibrium
binding, and then unlabeled toxins (concentration, 100 nM) were added
to the reaction mixtures. The bound toxins were measured at different
times. Each data point is a mean based on the results of two
independent experiments.
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Permeability assays.
Since reversible and irreversible binding
of Cry1 toxins to BBMV from S. exigua and S. frugiperda did not account for the differences in toxicity, we
analyzed the next step in toxin action, pore formation. When external
KCl was added to BBMV, the amount of scattered light rapidly increased
due to vesicle shrinkage. The shrinkage response (measured by
determining the increase in the scattered light signal, expressed in
volts) was determined by comparing the light scattered from BBMV that
were coinjected with buffer having the same osmotic strength (HEPES
buffer) and the light scattered from BBMV that were coinjected with 0.5 M KCl (Fig. 5A). The decrease in the
signal after 30 s may have been due to endogenous ion channels in
the BBMV. When S. frugiperda BBMV were made more
permeable to KCl (for example, with insecticidal toxins), the size of
the vesicles changed rapidly and less light was scattered compared to
the untreated control membranes (Fig. 5A). The BBMV response to toxin
shown in Fig. 5A was deduced by comparing the results for BBMV mixed
with HEPES buffer, BBMV mixed with KCl, and toxin-treated BBMV mixed
with KCl. The light scattering value (in volts) for the no-salt
treatment (buffer alone) (NS) was subtracted from the value for the
salt treatment (S): S NS = X. This was done to correct for
the salt effect. The value for the no-salt treatment (buffer alone) was
subtracted from the values for toxin treatments (T) (in volts): T NS = Y. This was done to correct for the buffer effect.
(Y/X) × 100 was calculated in order to determine the percent
change in light scattering corrected for both salt and buffer effects.
Figure 5B shows the proportion of scattered light change for
toxin-treated BBMV compared to untreated BBMV from
S. frugiperda for three separate experiments. The
results of the light scattering assays indicated that membrane
permeability induced by Cry1 toxins in S. frugiperda
increased in the following increasing order: Cry1Ca, Cry1Bb, Cry1Fa.
Cry1Ca and Cry1Bb are not significantly different from each other.
Cry1Fa is significantly different from Cry1Ca and Cry1Bb. Cry1Ca,
Cry1Bb, and Cry1Fa are all significantly different from Cry1Ac. Cry1Fa,
the most insecticidal toxin, changed membrane permeability the most,
and the nontoxic compound Cry1Ac had no overall effect on membrane
permeability.
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FIG. 5.
Scattered light signal of toxin-treated S. frugiperda BBMV after an increase in medium osmolarity. (A) Direct
traces of scattered light from toxin-treated BBMV. BBMV were
resuspended at a concentration of 0.5 mg/ml in 10 mM HEPES-Tris (pH
8.0) containing 0.1% BSA. Aliquots (1 ml) of BBMV were incubated with
toxins (2.5 µg/ml) at room temperature for 30 min. Assays were
initiated by simultaneously injecting 35 µl of a toxin-BBMV mixture
and 35 µl of 0.5 M KCl into a cuvette in the spectrofluorimeter
sample compartment. Light scattered at 90° from incidence was
monitored for 120 s by using five measurements per s. In the HEPES
sample, untreated BBMV were coinjected with 10 mM HEPES-Tris (pH 8.0)
containing 0.1% BSA. The KCl sample contained untreated BBMV
coinjected with 0.5 M KCl. (B) Proportion of scattered light signal
change for toxin-treated S. frugiperda BBMV compared to
KCl-treated BBMV, calculated from three independent assays. Five
scattered light signal values (in volts) were extracted from raw data
obtained at 1, 10, 20, 30, and 40 s in each assay, and then an
average was calculated from these values. The standard deviations were
calculated from the means of the three independent assays and are
indicated as error bars.
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Since
125I-Cry1Bb was able to bind specifically to
S. frugiperda BBMV but was not insecticidal in feeding
bioassays, pore formation
assays were performed with iodinated Cry1Bb
to determine if iodination
of this toxin affected the
membrane-permeabilizing capacities
in vitro. We expected that iodinated
Cry1Bb toxin would not increase
membrane permeability in the light
scattering assay, as iodinated
Cry1Bb toxin activity is dramatically
reduced in vivo compared
with Cry1Bb toxin activity. However, there was
no difference between
the ability of native Cry1Bb and the ability of
iodinated Cry1Bb
(data not shown) to increase membrane permeability in
S. frugiperda BBMV. The membrane-permeabilizing effects
of iodinated Cry1Bb
were not examined with
S. exigua
BBMV since the LC
50 of this toxin
is relatively high for
this insect compared to
S. frugiperda (Table
1). In
addition, the membrane-permeabilizing effects of iodinated
Cry1Ca were
not examined with
S. exigua and
S. frugiperda BBMV
since the activity of iodinated Cry1Ca was not
affected by labeling,
as determined by feeding bioassays (Table
1).
The tracings obtained in the
S. exigua light scattering
experiments are shown in Fig.
6A, and the
relative changes are shown
in Fig.
6B. The light scattering assays
showed that Cry1Ac had
no effect on
S. exigua membrane
permeability. The three other
toxins increased the membrane
permeability of
S. exigua BBMV in
the following
increasing order: Cry1Bb, Cry1Ca, Cry1Fa. The membrane
permeabilities
induced by Cry1Bb, Cry1Ca, and Cry1Fa were significantly
different from
each other, and the effects of these toxins were
significantly
different from the effect of Cry1Ac.
View larger version (36K):
[in this window]
[in a new window]
|
FIG. 6.
Change in scattered light signal of toxin-treated
S. exigua BBMV. (A) Direct traces of scattered light
from toxin-treated BBMV. Data were collected as described in the legend
to Fig. 4. (B) Proportion of scattered light signal change for
toxin-treated BBMV compared to KCl-treated BBMV, calculated as
described in the legend to Fig. 4.
|
|
The relationship between toxin concentration and the magnitude of light
scattering was tested by using the most insecticidal
toxin (Cry1Fa) and
S. exigua BBMV. The data in Fig.
7 confirm
that an increase in the
concentration of Cry1Fa resulted in a
decrease in scattered light. This
dose-dependent response is an
indirect measure of pore formation in
S. exigua BBMV.
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 7.
Relationship between Cry1Fa concentration and light
scattering signal. S. frugiperda BBMV were preincubated
with different concentrations of Cry1Fa. Experiments were conducted as
described in the legend to Fig. 4.
|
|
| |
DISCUSSION |
The effects of iodination on Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba,
Cry1Ca, Cry1D, and Cry1E have been reported previously (11, 15,
17, 18, 40). Only Cry1Ba was found to lose its toxicity and its
ability to bind to Ostrinia nubilalis and Pieris
brassicae when it was labeled with 125I
(17). In this study, we found that the toxicities of Cry1Bb and Cry1Fa decreased significantly when they were labeled with 125I, while Cry1Ca retained its insecticidal activity when
it was labeled with 125I (Table 1). Binding assays showed
that labeling Cry1Fa with Na125I resulted in a significant
decrease in binding to BBMV from both Spodoptera species
(Fig. 2). However, the ability of Cry1Bb to bind to BBMV from either
Spodoptera species was not altered by 125I
labeling (Fig. 2). Cummings and Ellar (8) reported that
chemical modification of 7 of 21 tyrosine residues in the Cry1Ac toxin reduced its binding and toxicity to M. sexta. Therefore,
some tyrosine residues are critical to toxin conformation and receptor recognition. If Cry1Fa tyrosine residues that are involved in receptor
binding are modified by attachment of 125I, the toxin
binding and membrane-permeabilizing abilities of this toxin could be
inhibited. Since 125I-Cry1Bb exhibited high-affinity
binding and 126I-Cry1Bb was an effective pore former (data
not shown), iodination of Cry1Bb tyrosine residues seems to alter a
step in the intoxication process prior to interaction with the brush
border membrane. Perhaps iodinated Cry1Bb is more susceptible to
proteinases than noniodinated Cry1Bb is and is inactivated in the
midgut lumen. These data also demonstrate that the effects of
iodination on binding and toxicity of Cry1 toxins may be different.
Multiple binding sites for the B. thuringiensis Cry1 toxins
are present in many insects (2, 13-15, 22, 32, 40). For example, Aranda et al. (2) reported that Cry1Ab and Cry1Ca recognized different binding sites on the brush border membrane of
S. frugiperda. In P. xylostella, Cry1Ab
and Cry1Fa recognized the same binding site, while Cry1Ca bound to a
distinct site (14, 45). The results of the present study
show that there are at least two high-affinity Cry1 binding sites in
S. exigua and S. frugiperda; one site
is for Cry1Ac, and the other site is for Cry1Bb and Cry1Ca. Cry1Fa
competed not only with Cry1Ac but also with Cry1Bb and Cry1Ca in both
Spodoptera species (Fig. 3). This suggests that Cry1Fa binds
to both sites. One possible explanation for this duality is that the
Cry1Fa toxin molecule has two binding determinants; one determinant
might recognize a Cry1Ac receptor, while the other might bind to a
Cry1Bb-Cry1Ca receptor. Indeed, de Maagd et al. (10)
reported that domain II of Cry1Ab and Cry1Ac recognized the same
binding molecule, while domain III of these toxins bound to a distinct
molecule in S. exigua and M. sexta. Alternatively, Cry1Fa might have a binding determinant that recognizes only one binding site. Although our data show that Cry1Fa binding competes with binding of Cry1Ac, Cry1Bb, and Cry1Ca, they do not indicate whether these toxins bind to the same epitope or bind to
different sites on the same molecule. Masson et al. (28) reported that purified Cry1Ac-binding aminopeptidase N from M. sexta has multiple binding sites for three Cry1A toxins. If both Cry1Ac and Cry1Ca receptor molecules had a separate binding site for
Cry1Fa toxin, Cry1Fa might compete with Cry1Ac, Cry1Bb, and Cry1Ca
binding by steric hindrance.
B. thuringiensis toxin binding to BBMV is a two-step process
which involves (i) recognition of a receptor and (ii) irreversible association with the membrane (20, 24). The irreversible
binding of toxins to BBMV has been reported to correlate with
insecticidal potencies of B. thuringiensis toxins (20,
24). However, this positive relationship was not observed in our
experiments. We found that Cry1Ac, Cry1Ca, and Cry1Bb irreversibly
bound to BBMV from S. exigua and S. frugiperda (Fig. 4), although the toxicities of these toxins
against both Spodoptera insects were different (Table
1). This phenomenon has been described for other insects. Lee et
al. (23) reported that Cry1Ac irreversibly bound to
susceptible and resistant strains of Heliothis virescens.
Therefore, the relationship between irreversible binding and
insecticidal activity requires further investigation.
Several workers have identified pore formation as a key step in
B. thuringiensis Cry1 toxin action (12, 33). The
results of our light scattering assays indicate that Cry1Bb, Cry1Ca,
and Cry1Fa significantly alter the membrane permeability of
S. exigua and S. frugiperda BBMV,
whereas Cry1Ac does not (Fig. 5 and 6). These results are consistent
with previous reports that BBMV permeability is related to Cry1 toxin
insecticidal potencies (6, 25, 43). However, our results
also indicate that the ability of a Cry1 toxin to increase membrane
permeability in vitro does not always correspond to toxin activity
measured in vivo. For example, Fig. 5 shows that there was no
significant difference in the abilities of Cry1Ca and Cry1Bb to
increase membrane permeability in S. frugiperda, but
the LC50 of these two toxins differed fourfold for this
insect (Table 1). In addition, we observed that while the activity of iodinated (cold-labeled) Cry1Bb decreases in vivo, this toxin retains
the ability to bind to BBMV and increase membrane permeability in vitro
(Table 1 and Fig. 2; data not shown). The data obtained with iodinated
Cry1Bb suggest that iodination caused a structural modification of the
toxin molecule that was detected by the insect bioassay but not by
binding or light scattering assays. Wolfersberger et al.
(43), in their analyses of mutant Cry1Aa toxins by a light
scattering assay, noted a similar phenomenon. These authors commented
that while the results of light scattering assays might reflect the
true membrane-permeabilizing potencies of the toxins, feeding bioassay
results not only reflect membrane-permeabilizing potencies but also
include other factors, such as toxin resistance to midgut proteases.
In our binding experiments, Cry1Ac irreversibly bound to S. exigua and S. frugiperda BBMV (Fig. 2 to 4).
However, Cry1Ac did not permeabilize BBMV from either
Spodoptera species (Fig. 5 and 6). These results suggest
that some membrane components are required for Cry1Ac-induced pore
formation. We do not know what the Spodoptera membrane
components are and why they do not facilitate Cry1Ac channel formation
in Spodoptera BBMV. In M. sexta, components associated with the 120-kDa aminopeptidase modify Cry1Ac-induced pore
formation. The 120-kDa Cry1A-binding aminopeptidase is isolated as a
complex with other membrane proteins and lipids (26). When incorporated into planar lipid bilayers, this complex catalyzed channel
formation by Cry1Ac with physical properties that were similar to those
of whole BBMV proteins but distinct from those of the purified 115-kDa
aminopeptidase (35; unpublished data). Other
as-yet-unidentified membrane components seem to be critical factors
that determine the ability of toxins to permeabilize the brush border membrane.
In conclusion, our results confirm that Cry1 toxin binding is necessary
but not sufficient for toxicity (15, 42). The similarity of
the data obtained from the Cry1 toxin binding experiments (as
determined by saturation, homologous competition, and heterologous competition assays) performed with Spodoptera BBMV is
striking. Our light scattering results support the conclusion that this assay could be a useful tool for identifying active toxins, with the
caveat that in vivo toxicity is determined not just by
membrane-permeabilizing capacity but also by other factors in the
intoxication process.
| |
ACKNOWLEDGMENTS |
We thank Jim Baum, Bruce Tabashnik, and Michael Wolfersberger for
critically reading a draft of the manuscript.
The toxin binding experiments were partially funded by Ecogen Inc. This
research was also supported by the NRI Competitive Grants Program of
the U.S. Department of Agriculture.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Entomology, Biosciences Building, Room 413, University of Georgia, 125 Cedar Street, Athens, GA 30602-2603. Phone: (706) 542-2436. Fax: (706)
542-2640. E-mail: Adang{at}arches.uga.edu.
| |
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Abstract
Introduction
