©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Kinetics of Bacillus thuringiensis Toxin Binding with Brush Border Membrane Vesicles from Susceptible and Resistant Larvae of Plutella xylostella(*)

Luke Masson (1)(§), Alberto Mazza (1), Roland Brousseau (1), Bruce Tabashnik (2)

From the (1) Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada and the (2) Department of Entomology, University of Hawaii, Honolulu, Hawaii 96822

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

An optical biosensor technology based on surface plasmon resonance was used to determine the kinetic rate constants for interactions between the CryIA(c) toxin from Bacillus thuringiensis and brush border membrane vesicles purified from susceptible and resistant larvae of diamondback moth (Plutella xylostella). CryIA(c) association and dissociation rate constants for vesicles from susceptible larvae were determined to be 4.5 10M s and 3.2 10 s, respectively, resulting in a calculated affinity constant of 7 nM. CryIE toxin did not kill susceptible or resistant larvae and did not bind to brush border vesicles. Contrary to expectations based on previous studies of binding in resistant P. xylostella, the binding kinetics for CryIA(c) did not differ significantly between susceptible larvae and those that were resistant to CryIA(c). Determination of the number of CryIA(c) receptors revealed an approximately 3-fold decrease in total CryIA(c) receptor numbers for resistant vesicles. These results suggest that factors other than binding may be altered in our resistant diamondback moth strain. They also support the view that binding is not sufficient for toxicity.


INTRODUCTION

The Gram-positive, spore-forming bacterium Bacillus thuringiensis is the most widely used microbial pest control agent (1-3). Many strains of B. thuringiensis contain parasporal crystals comprised of one or more insecticidal crystal proteins. After ingestion by insects, the crystals are solubilized in the midgut and the proteins are activated by gut proteases. These activated toxins bind to specific high affinity sites on the midgut epithelial cell layer in susceptible insects. A breakdown in gut integrity and eventual larval death follows the initial binding event (4, 5, 6) . The toxin inserts into the membrane of cultured insect cells (7) or lipid bilayers (8, 9, 10) and subsequently forms ion channels. In larvae of the moth Manduca sexta, a membrane-bound aminopeptidase N is the receptor for the CryIA(c) toxin of B. thuringiensis(11, 12) .

Demand for B. thuringiensis is growing rapidly because of its environmental compatibility and amenability to genetic engineering (13). Whether B. thuringiensis toxins are delivered in conventional sprays or in transgenic plants, the greatest threat to their continued success is the evolution of resistance in pests (14, 15) . Many insects have evolved with a resistance to B. thuringiensis in response to laboratory selection; the diamondback moth, Plutella xylostella, is the first pest to develop resistance to B. thuringiensis in open field populations (14-16).

Results from P. xylostella and the indian meal moth, Plodia interpunctella, suggest that reduced binding of toxins to midgut receptors is a primary mechanism of resistance to B. thuringiensis(17, 18, 19, 20) . One resistant strain of P. interpunctella also showed reduced proteolytic activation of a B. thuringiensis toxin (21) . In laboratory-selected resistant strains of Heliothis virescens, reduced binding was not associated with resistance to B. thuringiensis(22, 23) , suggesting that changes in postbinding events like calcium influx and loss of pH regulation (7) could also contribute to resistance.

Previous studies comparing the binding of B. thuringiensis toxins to resistant and susceptible strains were performed at equilibrium using BBMVs() and radioactively labeled toxins (17, 18, 20, 22, 23) . The calculated affinity constants have thus been based on the net effects of association and dissociation of toxin-receptor complexes. Information on the rate of both complex formation and dissociation would be valuable because dramatic rate alterations may not always be evident at equilibrium.

Independent measurement of the rates of association and dissociation of toxin-receptor complexes can be made with an optical detection system that exploits a phenomenon called surface plasmon resonance (24, 25, 26) . In this system, one of the reactants (either toxin or receptor) is immobilized to a hydrophilic dextran layer on the surface of a gold sensor chip in a microflow cell (27) . As binding occurs, the increase in total mass attached to the sensor chip changes the angle of polarized light reflected from the surface of the chip. These changes are detected by a diode array. By linking this system to a personal computer, one can measure complex association and dissociation in real time. We previously used this approach to determine association and dissociation rates for the B. thuringiensis toxin CryIA(b) and immobilized BBMVs from larvae of Choristoneura fumiferana(26) .

To achieve a better understanding of the interactions between CryIA(c) and its specific binding protein on the brush border surface, the kinetic binding rates of activated CryIA(c) toxin to BBMVs isolated from P. xylostella larvae were determined. The aim of this work was to assess if the CryIA(c) resistance phenotype in a field-resistant population could be correlated to alterations in toxin binding rates.


MATERIALS AND METHODS

Insects

We studied larvae from two Hawaiian strains of diamondback moth. The susceptible strain (LAB-P) was derived from individuals collected from Pulehu, Maui (16) . The susceptible strain had been reared in the laboratory for >80 generations without exposure to any insecticide. The resistant strain (NO-QA) was derived from individuals collected from a watercress farm near Pearl City, Oahu. Repeated exposure to sprays of commercial formulations of B. thuringiensiskurstaki in the field produced a 20-fold increase in the LC (concentration needed to kill 50% of larvae) of the resistant strain relative to the susceptible strain (16).

We increased the LC of the resistant strain in the laboratory by selecting it repeatedly with Dipel (28, 29) , a commercial formulation of the HD-1 strain of B. thuringiensiskurstaki that contains spores and insecticidal crystal proteins CryIA(a), CryIA(b), CryIA(c), CryIIA, and CryIIB (1, 3, 30) . The concentration of Dipel ranged from 25.6 to 2560 mg of active ingredient / liter. In most selected generations, mortality was between 40 and 70%. Larvae were reared on cabbage, and colonies were maintained as described previously (16, 20) .

Insect Bioassays

CryIA(b), CryIA(c), CryIC, and CryIE protoxins were tested against third instar larvae of the susceptible and resistant strains using leaf residue bioassays (16, 31) . For each toxin, resistant and susceptible strains were tested simultaneously. Four replicates of an average of 10 larvae (total of 40) from each strain were tested at l0 and 100 µg of protoxin/ml. Mortality was recorded 2 days after larvae were placed on treated leaves. For CryIB, mortality at 10 and 100 µg of protoxin/ml was estimated from previously reported probit regression lines (32) .

BBMV Purification

Late instar larvae from each strain were frozen. After approximately 500-800 larvae were thawed, their guts were removed, placed in ice-cold buffer A (300 mM mannitol, 5 mM EGTA, and 17 mM Tris base, pH 7.5), and then frozen on dry ice. BBMVs were purified from frozen tissue by homogenization and selective magnesium precipitation as described by Wolfersberger et al.(33) . All protein concentrations were determined by the method of Bradford (34) . As a control, BBMVs were prepared from excised small intestines from 6-8-week-old FVB mice.

BBMV Biotinylation and Enzyme Characterization

Purified BBMVs were biotinylated by co-sonication with N-biotin-phosphatidylethanolamine in a ratio of 1:100 (w/w; phosphatidyl N-biotinethanolamine:BBMV protein) and 0.01% cholesterol (Sigma) as described elsewhere (26) . Following sonication, biotinylated BBMVs were centrifuged at 100,000 g for 1 h, and the pellet was resuspended in HBS. The resuspended pellet was then sonicated briefly and re-centrifuged at 100,000 g for 1 h, and the protein concentration of the final resuspension was determined and adjusted to a final concentration of 1.0 mg of vesicle protein/ml. The preparation was aliquoted and stored at -80 °C for up to 6 months. The amount of contaminating non-gut membrane material varied among the different BBMV preparations. To normalize susceptible and resistant vesicle preparations for the determination of maximal ligand binding (R), the specific activity of the gut membrane enzyme marker leucine aminopeptidase in washed, freshly prepared, biotinylated BBMVs was determined by the spectrophotometric assay of Tuppy et al. (35) using leucine p-nitroanilide as a substrate. We arbitrarily defined 1 unit of leucine aminopeptidase enzyme activity as A/min = 1.0.

Toxin Purification

The cryIA(b) and cryIA(c) genes, cloned from the NRD-12 strain of B. thuringiensis, were expressed in Escherichia coli HB101 (36). The cryIC and cryIE genes were subcloned into a B. thuringiensis/E. coli shuttle vector and expressed in a crystal minus mutant of B. thuringiensis. Intracellular inclusion bodies or crystals were further purified by Renografin (Squibb) gradients (36) . All purified protoxins were solubilized in 40 mM carbonate buffer, pH 10.5, and treated with trypsin (final concentration, 0.1% (w/v)). The mixture was incubated for 3 h at ambient temperature, after which the 65,000-dalton trypsin-resistant protein was purified by ion exchange liquid chromatography using either Mono Q or Q-Sepharose anion exchangers (Pharmacia Biotech Inc.) buffered with 40 mM carbonate buffer, pH 10.5. Bound toxins were eluted using a 50-500 mM NaCl gradient and dialyzed immediately into distilled water. The activated toxins were freshly diluted into HBS before use in our binding studies.

Instrumentation and Reagents

The BIAcore system and CM5 sensor chips were obtained from Pharmacia. Surfactant-free HBS was used as the running and diluting buffer for all vesicle experiments. Bovine serum albumin (BSA) (fraction 5, radioimmunoassay grade) was purchased from Sigma. Rabbit anti-biotin (IgG fraction) was purchased from Rockland (Gilbertsville, PA). Phosphatidyl N-biotinethanolamine (lyophilized) was purchased from Avanti Polar Lipids (Alabaster, AL). The buffers used with the BIAcore machine contained the following: HBS (10 mM HEPES, pH 7.4, 150 mM NaCl, and 3.4 mM EDTA), HBS-P20 (HBS containing 0.05% BIAcore Surfactant P20), regeneration buffer (50 mM CAPS, pH 11.0, and 150 mM NaCl), detergent wash (HBS + 0.1% (v/v) Triton X-100), carboxymethylated dextran activation solutions (A = 0.1 MN-hydroxysuccinimide, B = 0.1 MN-ethyl-N`-(3-diethylaminopropyl)carbodiimide), coupling buffer (20 mM ammonium acetate, pH 4.5), and deactivation solution (1 M ethanolamine, pH 8.5). All protein chemical immobilizations were done using the standard BIAcore amine coupling protocol provided with the Pharmacia coupling kit.

Sensor Chip Configuration and Binding Specificity

We used two sensor chip configurations: immobilized vesicle and immobilized toxin. In the immobilized vesicle configuration, 20,000 RU of the rabbit anti-biotin immunoglobulin dissolved in 20 mM ammonium acetate, pH 5 (equivalent to 20 ng of protein) was immobilized by standard amine coupling (25) . Sonicated vesicles containing the biotin anchor were adjusted to a protein concentration of 1 mg/ml as described elsewhere (26) and injected over the antibody surface. A vesicle level of approximately 1700-1800 RU was reproducibly obtained. The immobilized vesicles were then washed until a stable baseline was obtained, after which toxin (freshly prepared in HBS containing 0.01% (w/v) BSA) was injected. In the immobilized toxin configuration, approximately 5000 RU of toxin (dissolved as a 0.1 mg/ml stock solution in 20 mM ammonium acetate, pH 5) was amine-coupled to the dextran. BSA (0.01% (w/v)) was added to all vesicle preparations immediately prior to injection over the toxin surface to reduce nonspecific interactions. In all experiments, a reagent flow rate of 5 µl/min was used.

Nonspecific binding of B. thuringiensis toxins to exposed plasma membranes has been noted by Ryerse et al.(37) . To assess the extent of nonspecific binding of Cry toxins to BBMVs from P. xylostella, we used papain-activated CryIIIA, which does not bind to the midgut epithelial cells of P. xylostella (19). CryIIIA was either immobilized to the dextran layer on the CM5 sensor chip surface (immobilized toxin configuration) or injected over immobilized P. xylostella BBMVs (immobilized vesicle configuration). To further examine toxin binding specificity to BBMVs, homologous/heterologous toxin competition experiments were used. Preincubation with a toxin homologous to that found on the immobilized surface should specifically block BBMV binding to the surface, whereas using a heterologous toxin having its own specific receptor should not compete, resulting in BBMV binding to the sensor chip surface.

Kinetic Rate Analysis

The general rate equation to describe the interaction between two molecular species can be written as dR/dt = kC(R - R) - kR, where R is the response, dR/dt is the rate of toxin-receptor complex formation, C is the concentration of injected toxin, R - Ris the amount of free remaining receptor sites at time t (expressed in RU), kis the association rate constant (expressed as M s), and kis the dissociation rate constant (expressed as s) (25) . By rearranging the rate equation to dR/dt = kCR - (kC + k)R, one sees that the rate of toxin binding is linearly related to the response (25) . When the binding rate of each of the response curves is plotted against the response (dR/dt versus R), the association rate kcan be determined by plotting the slope of the dR/dt versus R curves against toxin concentration. To determine k, we used four concentrations of activated CryIA(c) injected over 1800 RU of BBMVs immobilized by rabbit anti-biotin IgG.

Dissociation rate constants were determined by a different method than was described previously for BBMVs (26) . Because the level of nonspecific binding is lowest in the immobilized toxin configuration, all dissociation experiments were performed by immobilizing CryIA(c) to the sensor chip surface and saturating the surface with BBMVs. The absence of vesicles when the flow is replaced with buffer allows the determination of kd by plotting the log of the drop in response against the time interval, ln(Rt/Rt) = k(t- t), where Rt is the response at an arbitrarily chosen starting time (t) after the toxin is replaced by buffer and Rtand tare subsequent data points chosen along the dissociation curve. Ideally, the chosen start point should be close to the end of the vesicle flow to prevent any influences by vesicle rebinding. Our arbitrary start point was generally chosen where the ln(Rt/Rt) versus (t- t) curve initially became linear but before 5-10% of the bound vesicles had dissociated.

RESULTS

Toxicity to Susceptible and Resistant Larvae

CryIA(b) and CryIA(c) were extremely toxic to larvae from the susceptible strain of diamondback moth but did not kill larvae from the resistant strain (). Two other lepidopteran-specific toxins, CryIB and CryIC, were highly toxic to both susceptible and resistant larvae. At the concentrations tested, CryIE caused little or no mortality to susceptible or resistant larvae. The relative toxicity of CryIA(b), CryIB, CryIC, and CryIE to the susceptible and resistant larvae studied here corresponds well with the pattern of toxicity of the independently derived susceptible and resistant strains of diamondback moth studied by Ferré et al.(18) .

Cry Binding Characteristics

In the immobilized toxin configuration, BBMVs from susceptible larvae showed no significant binding when injected over a surface of immobilized CryIIIA toxin (Fig. 1). In the immobilized vesicle configuration, CryIIIA demonstrated limited binding to the P. xylostella BBMVs. The observed binding was considered to be nonspecific as a result of the nearly linear (non-saturable) response. The CryIIIA toxin could remain stably bound to the vesicles for more than 15 min, thereby making it an ideal candidate for blocking nonspecific CryI toxin binding.


Figure 1: Nonspecific binding of CryIIIA to P. xylostella BBMVs. P. xylostella BBMVs isolated from susceptible larvae were passed over a surface containing 6000 RU of immobilized CryIIIA (B). On a second surface, approximately 1800 RU of biotin-anchored BBMVs from the same preparation were immobilized on a surface of immobilized anti-biotin antibodies. A solution of CryIIIA toxin (1000 nM) was then injected over the BBMV surface (A). All response curves in this and all subsequent figures were adjusted for the mass action of the injected buffer components, and the resonance units were normalized to zero at the start of injection for comparative purposes.



To examine the relationship between toxicity and ligand binding, immobilized BBMV surfaces prepared from mouse small intestine or from susceptible larvae were used (Fig. 2). We have found that CryIA(c) or CryIE could bind, to a limited extent (i.e. approximately 200 RU), to these vesicles.() A preinjection of CryIIIA over the immobilized mouse BBMV surface followed by an injection of CryIA(c) resulted in a very low level of bound CryIA(c) toxin (<40 RU). An injection of trypsin-activated CryIE, which is essentially non-toxic for P. xylostella (), over a surface of immobilized BBMVs from susceptible P. xylostella resulted in a small but linear response. The CryIE toxin complex dissociated completely immediately after toxin flow was replaced by buffer, suggesting that the observed increase in RU was nonspecific in nature. By contrast, an injection of an identical concentration of CryIA(c) over immobilized BBMVs from susceptible P. xylostella larvae resulted in a high level of binding that appeared to be nearing saturation. When the toxin flow was replaced by buffer, the toxin-BBMV complex (approximately 350 RU) started to slowly dissociate. Surprisingly, CryIA(c) toxin also bound to BBMVs from resistant P. xylostella larvae, suggesting that a functional CryIA(c) receptor is present on the surface of the resistant vesicles.


Figure 2: Specificity of CryI toxin binding to BBMVs. BBMVs from either P. xylostella larvae or mouse intestine were immobilized to an anti-biotin surface. A 1000 nM solution of CryIIIA was preinjected to reduce nonspecific binding over the BBMV surface for 400 s followed by a 1000 nM solution of a trypsin-activated lepidopteran-specific toxin. The curves are labeled with the injected toxin followed by the type of BBMV. R, BBMVs from resistant P. xylostella; S, BBMVs from susceptible P. xylostella; M, BBMVs from mouse small intestine.



In light of the CryIE results presented above, we determined whether CryI trypsin-activated toxins can bind to themselves to form dimers or other multimeric forms by passing CryIA(c) toxin over an immobilized homologous toxin surface. As shown in Fig. 3 , approximately 500 RU of toxin remained bound after the injection. When the toxin flow was replaced by buffer, the toxin complexes dissociated, rapidly dropping to 50% of the total bound after 120 s. The gentler slope of the observed biphasic dissociation curve is due to rebinding of the toxin to freed immobilized binding sites.


Figure 3: Binding of CryIA(c) to immobilized homologous toxin. Approximately 6000 RU of trypsin-activated CryIA(c) were immobilized and washed with HBS to remove unbound toxin until a stable baseline was reached. A 400-s injection of a 1000 nM CryIA(c) toxin stock solution was passed over the toxin surface. After the end of toxin injection, the flow was switched to HBS for an extended period.



To further characterize the specificity of toxin binding to BBMVs from resistant P. xylostella, the vesicles were preincubated with either a homologous or heterologous toxin (CryIA(c) or CryIC, respectively) before injection over an immobilized CryIA(c) surface (Fig. 4). Preincubation of BBMVs from resistant larvae with CryIC, a heterologous protein highly toxic to both susceptible and resistant P. xylostella larvae, resulted in net binding to the CryIA(c) surface. The binding curve is similar to that generated when the vesicles were preincubated with BSA. However, little or no net capture of BBMVs occurred at the end of the vesicle injection when preincubated with the homologous toxin. The specific blocking of BBMV binding to an immobilized CryIA(c) surface suggests that BBMVs from resistant P. xylostella larvae possess a receptor that specifically binds CryIA(c) toxin.


Figure 4: Homologous/heterologous competition of CryI toxins for BBMVs purified from resistant P. xylostella larvae. Vesicles from resistant insects were preincubated for 30 min on ice with 5 mM of BSA, CryIC, or CryIA(c). The BBMVs were then injected over a surface of immobilized CryIA(c) (approximately 6000 RU).



Determination of Kinetic Rate Constants

Typical CryIA(c) response curves using immobilized BBMVs from susceptible P. xylostella larvae are shown in Fig. 5A. The four different toxin concentration curves showed that the rate of increase in RU, which represents toxin binding to the vesicles, generally decreased with time. The association rate can be calculated by determining the slopes from the linear part of the curve produced by plotting the binding rates (dR/dt) of the four separate toxin concentrations used against the response (R).() By plotting these four slopes against the toxin concentration, the kcan be calculated from the slope of the fitted line (Fig. 5B) and was determined to be 4.4 10M s. A similar binding response was observed with CryIA(c) using immobilized BBMVs from resistant larvae (Fig. 6A). The plot of the four dR/dt versus R slopes against toxin concentration produced a k(3.6 10M s) for BBMVs from resistant larvae (Fig. 5B) that was not significantly different than that determined for BBMVs from susceptible larvae.


Figure 5: Association rate kinetics of CryIA(c) with immobilized BBMVs from susceptible P. xylostella larvae. A, typical response curve showing injections of four different CryIA(c) concentrations. The data obtained in A were transformed into binding rate versus response curves (not shown) as described under ``Materials and Methods.'' B, slopes of the four curves plotted against toxin concentration. The slope of the fitted linear curve represents the association rate constant.




Figure 6: Association rate kinetics of CryIA(c) with immobilized BBMVs from resistant P. xylostella larvae. A, typical response curve showing injections of four different CryIA(c) concentrations. The data obtained in A were transformed into binding rate versus response curves (not shown) as described under ``Materials and Methods.'' B, slopes of the four curves plotted against toxin concentration. The slope of the fitted linear curve represents the association rate constant.



Results from a typical experiment used to determine dissociation rate constants using BBMVs from both susceptible and resistant insects are shown in Fig. 7. A 600-s BBMV injection from either susceptible or resistant insects sufficient to saturate an immobilized CryIA(c) surface was replaced by a continuous flow of buffer (Fig. 7). The dissociation of the vesicle from the toxin surface is manifested by the slow drop in RU with time after the buffer switch. The slope of the transformed dissociation curve (Fig. 8A) calculated from the susceptible larval BBMV curve in Fig. 7represents the dissociation rate constant and was calculated to be 3.94 10 s. This kis not significantly different from that calculated for the resistant larval BBMVs (Fig. 8B) (2.57 10 s). Because the affinity constant (K) is a ratio of the dissociation and association rate constants, the Kof CryIA(c) for its receptor can be determined. As summarized in , CryIA(c) showed similar high affinity binding to BBMV preparations from susceptible and resistant larvae.


Figure 7: Dissociation curves of CryIA(c) with susceptible and resistant P. xylostella BBMVs. Approximately 6000 RU of activated CryIA(c) toxin were immobilized, and BBMVs from either susceptible or resistant P. xylostella larvae were injected until the surfaces were saturated (usually 600 s). Typical curves showing vesicle saturation and dissociation (after HBS buffer replaced vesicle flow) are shown. The area of the dissociation segment of the curve used to calculate k was selected by choosing an arbitrary starting point (t = 0) a few minutes after the start of buffer flow but before 5% of the total amount of bound vesicle was dissociated.




Figure 8: Calculation of dissociation rate constant. The dissociation rate constant was calculated from the slope of the fitted line (solid black line) in ln(Rt/Rt) versus (t - t) plots for BBMVs purified from either susceptible larvae (A) or from resistant larvae (B). The small dots represent the data points (log of the drop in response) calculated at 1-s intervals using data from the segment of the dissociation curve described in Fig. 7.



In the immobilized vesicle configuration experiments, even though the same number of BBMV resonance units was immobilized between the two preparations, BBMVs from resistant larvae tended to bind slightly higher levels of toxin. By assaying the levels of a gut-specific membrane marker and normalizing the maximal binding of the toxin to immobilized BBMVs, a comparative estimate of receptor levels (R) was determined. The results presented in revealed only a 3-fold decrease in relative number of receptors in resistant BBMVs.

DISCUSSION

We determined the kinetic binding characteristics of the CryIA(c) toxin from B. thuringiensis to BBMVs purified from both susceptible and CryIA(c)-resistant larvae of P. xylostella. The results presented here show that CryIA(c) can specifically recognize and bind to BBMVs prepared from resistant as well as susceptible diamondback moth larvae. Although the kinetic parameters were similar for the two larval types, a lower number of receptors in the resistant BBMVs were observed. However, the minor difference in receptor numbers does not seem sufficient to account for the major difference in CryIA(c) susceptibility.

Binding to BBMVs from resistant P. xylostella was unexpected because several studies using B. thuringiensis-resistant P. xylostella and CryIA(b) (18, 19) or CryIA(c) toxin (20) demonstrated little or no binding of these toxins to BBMVs. In particular, the resistant larvae tested here were derived from the same strain that showed very little binding of radiolabeled CryIA(c) to BBMVs (20) . The difference between the present work and that with radiolabeled toxin is difficult to explain, especially because we do not yet understand the relationship between results of surface plasmon resonance kinetic studies compared with results of equilibrium binding studies. One of the problems with both the equilibrium binding studies and the present work is that toxin-vesicle interactions are presumably biphasic. The first phase consists of toxin-receptor binding, whereas the second phase involves integration of the toxin into the membrane. Indeed, it has been shown that B. thuringiensis toxins can integrate into lipid bilayers in the absence of a specific receptor (8-10). Because one cannot discriminate between the two phases, most binding calculations represent an amalgamation of the two, with the possible reappearance of free receptor to further confound the results. To determine the extent of the membrane integration problem, solubilization of the CryIA(c) receptor could be achieved by cleaving the glycosylphosphatidylinositol anchor (38) , thus stripping it from the vesicle and eliminating interference from the lipid environment.

A possible explanation for the contrasting binding results may rest with the additional BBMV processing required by the surface plasmon resonance technique. BBMVs are sonicated to enable insertion of the biotin-fatty acid anchor and to reduce the vesicle size so that the BBMVs are small enough to pass through the 0.5-µm diameter flow tubing in the apparatus. Cation-precipitated vesicles used directly in receptor studies normally range in size from 0.1 to 1 µm and possess different morphologies (33, 39) . It is possible that the sonication step may disrupt the masking of the receptor by one or more tightly associated proteins in the vesicles prepared from resistant larvae. It has been shown that the 120-kDa receptor from M. sexta is tightly associated with a number of other membrane proteins.()

CryIC toxin also bound to BBMVs from either susceptible or resistant larvae but did not compete with CryIA(c) in heterologous competition experiments, indicating that this toxin recognizes a different receptor than CryIA(c). Activated CryIE toxin, which is nontoxic to P. xylostella, showed no net binding (i.e. the amount of RU remaining bound after toxin flow is switched to buffer) to immobilized BBMVs in agreement with previous results, indicating that binding is a prerequisite for toxicity (5, 19, 40) . The association and dissociation rate constants and consequently the calculated affinity constant for CryIA(c) toxin to BBMVs from both susceptible and resistant larvae were not significantly different. The high affinity binding of CryIA(c) to the receptor from resistant larvae supports the view that toxin binding does not necessarily result in toxicity (23, 41, 42) . The Kdetermined by our optical technique correlated well (3-4-fold higher) with that calculated at equilibrium for BBMVs from susceptible larvae using radiolabeled toxins (20) .

One interesting discovery from our immobilized toxin experiments was the capacity of toxin molecules to multimerize on an immobilized toxin surface. Although CryIA(c) toxin associates rapidly with the CryIA(c) toxin immobilized to the hydrogel on the sensor chip surface, the formed protein complexes dissociate rapidly. What role these toxin-toxin interactions may play in toxicity is uncertain at this point, although one could speculate that, like the role proposed for the receptor (43) , toxin-toxin binding could increase the effective concentration of the toxin at the microvillar membrane surface. It is unknown if the toxin acts in the form of a monomer or a multimer at the cell surface to create a pore, but the appearance of clearly defined ion channel substates in planar lipid bilayers suggests that the toxin may act in an multimeric form (9) similar to the hexameric -toxin pore (44) or the heptameric aerolysin channel (45) . Because the concept of surface multimerization may have important implications in the toxin mode of action, further experiments are planned to determine if the multimeric binding is specific and how CryIA(c) toxin interacts with heterologous toxins.

  
Table: 1635000358p4in Estimated from probit analysis of previously reported data (32).(119)

  
Table: Summary of CryIA(c) kinetic rate constants and calculated affinity constants



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: National Research Council of Canada, Biotechnology Research Institute, 6100 Royalmount Ave., Montreal, Quebec, H4P 2R2 Canada. Tel.: 514-496-6150; Fax: 514-496-6213; E-mail: Masson@biotech.lan.nrc.ca.

The abbreviations used are: BBMV, brush border membrane vesicle; HBS, HEPES-buffered saline; RU, resonance unit(s); BSA, bovine serum albumin; CAPS, 3-(cyclohexylamino)propanesulfonic acid.

L. Masson, unpublished data.

L. Masson, unpublished data.

M. Adang, personal communication.


ACKNOWLEDGEMENTS

We thank R. Ménard and M. O'Connor-McCourt for helpful discussions, N. Finson, T. Gonsalves, and B. Helvig for rearing insects, A. M. Mes-Masson for providing the dissected mouse intestines, R. Frutos for providing the recombinant cryIC and cryIE genes in B. thuringiensis, and W. J. Moar for providing CryIC toxin.


REFERENCES
  1. Feitelson, J. S., Payne, J., and Kim, L.(1992) Bio/Technology 10, 271-275
  2. Lambert, B., and Peferoen, M.(1992) Bioscience 42, 112-122
  3. Rodgers, P. B.(1993) Pestic. Sci. 39, 117-129
  4. Lüthy, P., and Ebersold, H. R.(1981) Pathogenesis of Invertebrate Microbial Diseases (Davidson, E. W., ed) pp. 235-267, Allanheld, Osmun Publishers, Totowa, NJ
  5. van Rie, J., Jansens, S., Höfte, H., Degheele, D., and van Mellaert, H.(1989) Eur. J. Biochem. 186, 239-247 [Abstract]
  6. Knowles, B.(1994) Adv. Insect Physiol. 24, 275-308
  7. Schwartz, J. L., Garneau, L., Masson, L., and Brousseau, R.(1991) Biochim. Biophys. Acta 1065, 250-260 [Medline] [Order article via Infotrieve]
  8. Slatin, S. L., Abrams, C. K., and English, L.(1990) Biochem. Biophys. Res. Commun. 169, 765-772 [CrossRef][Medline] [Order article via Infotrieve]
  9. Schwartz, J. L., Garneau, L., Masson, L., Brousseau, R., and Rousseau, E.(1993) J. Membr. Biol. 132, 53-62 [Medline] [Order article via Infotrieve]
  10. Walters, F. S., Slatin, S. L., Kulesza, C. A., and English, L. H. (1993) Biochem. Biophys. Res. Commun. 196, 921-926 [CrossRef][Medline] [Order article via Infotrieve]
  11. Sangadala, S., Walters, F. W., English, L. H., and Adang, M. J.(1994) J. Biol. Chem. 269, 10088-10092 [Abstract/Free Full Text]
  12. Knight, P. J. K., Crickmore, N., and Ellar, D. J.(1994) Mol. Microbiol. 11, 429-436 [Medline] [Order article via Infotrieve]
  13. Entwistle, P. F., Cory, J. S., Bailey, M. J., and Higgs, S. (eds) (1993) Bacillus thuringiensis, an Environmental Biopesticide: Theory and Practice, John Wiley & Sons, Inc., New York
  14. McGaughey, W. H., and Whalon, M.(1992) Science 258, 1451-1455
  15. Tabashnik, B. E.(1994) Annu. Rev. Entomol. 39, 47-79 [CrossRef]
  16. Tabashnik, B. E., Cushing, N. L., Finson, N., and Johnson, M. W. (1990) J. Econ. Entomol. 83, 1671-1676
  17. van Rie, J., McGaughey, W. H., Johnson, D. E., Barnett, B. D., and van Mellaert, H. V.(1990) Science 247, 72-74 [Medline] [Order article via Infotrieve]
  18. Ferré, J., Real, M. D., van Rie, J., Jansens, S., and Peferoen, M.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5119-5123 [Abstract]
  19. Bravo, A., Jansens, S., and Peferoen, M.(1992) J. Invertebr. Pathol. 60, 237-246 [CrossRef]
  20. Tabashnik, B. E., Finson, N., Groeters, F. R., Moar, W. J., Johnson, M. W., Luo, K., and Adang, M. J.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4120-4124 [Abstract]
  21. Oppert, B., Kramer, K. J., Johnson, D. E., MacIntosh, S. C., and McGaughey, W. H.(1994) Biochem. Biophys. Res. Commun. 198, 940-947 [CrossRef][Medline] [Order article via Infotrieve]
  22. MacIntosh, S. C., Stone, T. B., Jokerst, R. S., and Fuchs, R. L.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8930-8933 [Abstract]
  23. Gould, F., Martinez-Ramirez, A., Anderson, A., Ferré, J., Silva, F. J., and Moar, W. J.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7986-7990 [Abstract]
  24. Jönsson, U., Fägerstam, L., Ivarsson, B., Johnsson, B., Karlsson, R., Lundh, K., Löf, S., Persson, B., Roos, H., Rönnberg, I., Sjölander, S., Stenberg, E., St, R., Urbaniczky, C., stlin, H., and Malmqvist, M.(1991) BioTechniques 11, 620-627 [Medline] [Order article via Infotrieve]
  25. Karlsson, R., Michaelsson, A., and Mattsson, L.(1991) J. Immunol. Methods 145, 229-240 [CrossRef][Medline] [Order article via Infotrieve]
  26. Masson, L., Mazza, A., and Brousseau, R.(1994) Anal. Biochem. 218, 405-412 [CrossRef][Medline] [Order article via Infotrieve]
  27. Löf, S., and Johnsson, B.(1990) J. Chem. Soc. Chem. Commun. 21, 1526-1528
  28. Tabashnik, B. E., Finson, N., and Johnson, M. W.(1991) J. Econ. Entomol. 84, 49-55
  29. Tabashnik, B. E., Finson, N., Johnson, M. W., and Heckel, D. G.(1995) J. Econ. Entomol. 88, 219-224
  30. Abbott, W. S.(1925) J. Econ. Entomol. 18, 265-267
  31. Tabashnik, B. E., Finson, N., Johnson, M. W., and Moar, W. J.(1993) Appl. Environ. Microbiol. 59, 1334-1335
  32. Tabashnik, B. E., Finson, N., Johnson, M. W., and Heckel, D. G.(1994) Appl. Environ. Microbiol. 60, 4627-4629 [Abstract]
  33. Wolfersberger, M., Luethy, P., Maurer, A., Parenti, P., Sacchi, F. V., Giordana, B., and Hanozet, G. M.(1987) Comp. Biochem. Physiol. 86A, 301-308 [CrossRef]
  34. Bradford, M. M.(1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  35. Tuppy, H., Weisbauer, U., and Wintersberger, E.(1962) Hoppe-Seyler's Z. Physiol. Chem. 329, 278-288
  36. Masson, L., Préfontaine, G., Péloquin, L., Lau, P. C. K., and Brousseau, R.(1990) Biochem. J. 269, 507-512 [Medline] [Order article via Infotrieve]
  37. Ryerse, J. S., Beck, J. R., Jr., and Lavrik, P. B.(1990) J. Invert. Pathol. 56, 86-90 [Medline] [Order article via Infotrieve]
  38. Garczynski, S. F., and Adang, M. J.(1995) Insect Biochem. Mol. Biol., in press
  39. Biber, J., Stieger, B., Haase, W., and Murer, H.(1981) Biochim. Biophys. Acta 647, 169-176 [Medline] [Order article via Infotrieve]
  40. Hofmann, C., Vanderbruggen, H., Höfte, H., van Rie, J., Jansens, S., and van Mellaert, H.(1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7844-7848 [Abstract]
  41. Wolfersberger, M. G.(1990) Experientia 46, 475-477 [Medline] [Order article via Infotrieve]
  42. Garczynski, S. F., Crim, J. W., and Adang, M. J.(1991) Appl. Environ. Microbiol. 57, 2816-2820 [Medline] [Order article via Infotrieve]
  43. Knowles, B. H., and Ellar, D. J.(1987) Biochim. Biophys. Acta 924, 509-518
  44. Bhakdi, S., and Tranum-Jensen, J.(1991) Microbiol. Rev. 55, 733-751
  45. Wilmsen, H. U., Leonard, K. R., Tichelaar, W., Buckley, J. T., and Pattus, F.(1992) EMBO J. 11, 2457-2463 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.