WO2009076558A1 - Methods to enhance effectiveness of bacillus thuringiensis toxins - Google Patents

Abstract

Methods to enhance the effectiveness of Bacillus thuringiensis toxins by preventing endocytosis of Bt receptors are disclosed.

Description

METHODS TO ENHANCE EFFECTIVENESS OF BACILLUS THURINGIENSIS TOXINS

Cross-Reference to Related Applications

[0001] This application claims priority from U.S. provisional application 61/007,332 filed 11 December 2007. The contents of this document are incorporated herein by reference.

Technical Field

[0002] The invention relates to screening methods and enhancement methods that rely on the finding that toxicity is enhanced by exocytosis of receptors for Bacillus thuringiensis (Bt) type toxins.

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[0003] The soil bacterium Bacillus thuringiensis (Bt) produces a variety of protein toxins (Cry toxins) that are insecticidaJ. The unique insecticidal activity of Cry toxins makes Bt an effective insecticide for agriculturally important insect pests and vectors of human and animal disease. The toxins have been used in formulations for application to crops and other target areas and plants have been modified to produce these toxins in situ.

[0004] In general, Cry toxins bind to receptors specific for these proteins to initiate a cascade which leads to cell death. (Such receptors for Cry toxins in general are denoted Bt-R.) Fcr example. Cry IAb toxin produced by Bacillus thuringiensis exerts insecticidal action upon binding to BT-Rj. a cadherin receptor localized in the midgut epithelium of the tobacco hornworm Manduca sexta. The univalent binding of toxin to receptor transmits a. death signal m'O the cell and turns on a multi-step signal transduction pathway involving adenylyl cyclase (AC) and protein kinase A (PKA), which drives the biochemical events that culminate in oncotic cell death.

[0005] The Cry 1 Ab toxin binds to a highly conserved structural motif in the cadherin reeeptor(Vadlamudi et al., 1995; Griko et ah, 2007), which, in turn, triggers a signaling event that leads to oncotic-Hkε cell dεath(Zhang et al., 2005). The resulting signal stimulates heterotrimeric G protein and adenylyl cyclase (AC) with an accompanying dramatic increase in production of cAMP. cAMP activates protein kinase A CPKA)₅ bringing about an array of cellular changes including cytoskeletal rearrangement and ion fluxing. Acceleration of this second messenger pathway alters the chemistry of the cell to elicit cell death (Zhang et al., 2005; Zhang et al., 2006). Toxin action can be inhibited by blocking the binding of the toxin to BT-R₁ either in an insect (Dorsch et al., 2002) or in cultured insect cells (Tsuda et al., 2003; Zhang et al., 2005), demonstrating that specific toxin-receptor interaction is the primary determinant of toxicity.

Disclosure of the Invention

[0006] The invention is based on the discovery that Bt toxins promote the translocation of BT receptor_! from intracellular membrane vesicles to the plasma membrane. Movement of the receptor is mediated by toxin-induced signal transduction, and amplification of this signaling is directly correlated to the execution of cell death. Apparently, toxin-induced cell signaling engages exocytotic trafficking of receptor_! to the cell surface. Enrichment of receptor on the cell surface recruits additional toxin, which, in turn, amplifies the signal cascade initialized by the toxin-receptor interaction. This process can be enhanced by providing endocytosis inhibitors.

[0007] In one aspect, the invention is directed to a method to enhance the effectiveness of Bt related toxin insecticides which method comprises applying, along with said toxin, an effective amount of an inhibitor of endocytosis. Thus, the insecticide along with the inhibitor can be applied directly to areas of insect infestation or the toxin may be expressed recombinantly in plants designed to resist insect infestation and the inhibitor applied separately.

[0008] In another aspect, the invention is directed to a method to identify a potential insecticide with enhanced toxicity which method comprises treating cells that display receptors for at least one Bt toxin at their surface with a candidate compound and assessing the surface of the cells for the presence of an increased number of said receptors. Typically, these cells are insect cells that natively produce the receptor or they can be recombinant cells modified to produce it.

Brief Description of the Drawings

[0009] Figures IA and IB show the effect of endocytosis inhibitors and of Exol on the effectiveness of CrylAb toxin.

[0010] Figures 2A-2C show the dependence of the location of Bt receptors at the cell surface on the number of cell passages.

[0011] Figures 3A-3B show the stimulation of exocytosis of BT-Ri by CrylAb toxin and the effect of Exol thereon. [0012] Figure 4 is a graph showing the dependence of translocation of the receptor on the presence of toxin and Exol.

[0013] Figures 5A-5B show the correlation between enhanced exocytosis of BT-R and binding of Cry IAb toxin.

[0014] Figure 6 shows inhibition of toxin-induced exocytosis of the receptor by transduction inhibitors EDTA and PKAi.

[0015] Figure 7 shows the mechanism whereby enhanced exocytosis is effected after initial binding with Cry toxin.

Modes of Carrying Out the Invention

[0016] The examples below demonstrate that for a typical Bt toxin, the effectiveness of cell killing is enhanced by virtue of the ability of the toxin to effect translocation of additional receptors for the toxin to the cell surface. This permits additional toxin molecules to be bound, thus enhancing the effectiveness of the toxin itself. Inhibitors of endocytosis, which would reverse this process, therefore enhance the effect of the toxin.

[0017] A substantial number of Cry toxins produced by B. thuringiensis is known in the art. Agaisse, H, et al, J. Bacteriol (1995) 177:6027-6032. While there is some overlap, a multiplicity of receptors is also known which bind one or more of these Cry toxin types. The examples hereinbelow which employ CrylAb toxin and its corresponding receptor Bt-Ri are thus applicable to any cell which comprises receptors of this type.

[0018] The present work provides the first description of a bacterial toxin promoting exocytosis through receptor-mediated activation of a protein kinase signaling pathway. The results reported here reveal that activation of the AC/PKA signal transduction pathway by the CrylAb toxin of Bt drives the exocytosis of BT-Ri as well as other downstream biochemical events. Importantly, the AC/PKA-mediated exocytosis of BT-Ri is required for effective cell killing because inhibition of receptor transport by Exol blocks toxin-induced cell death. Accumulation of receptor on the cell surface provides a stage for additional toxin binding.

[0019] Based on these observations, an updated model for Cry toxin action that includes enhanced exocytosis of BT-R is shown in Figure 7. The model depicts progression of cell death triggered by the univalent binding of Cry toxin monomer to BT-R. This binding, which is highly specific, transmits a death signal into the cell and establishes a multi-step signal transduction pathway, manifested by activation of AC and PKA. A major consequence is stimulation of the exocytosis of BT-R from intracellular vesicles to the plasma membrane. The enhanced display of BT-R receptor on the cell surface facilitates recruitment of additional toxin molecules which, in turn, amplifies the original signal in a cascade-like manner. The signaling kinase PKA modifies downstream effectors/executioners that drive the biochemical events which dismantle the cell, culminating in oncotic cell death. Cry toxin-induced oncosis is a multi-step process typified by signaling protein kinases, in contrast to apoptosis that requires signaling proteases known as caspases.

[0020] In Figure 7, the univalent binding of Cry toxin monomer to BT-R initiates the progression of cell death by transmitting a death signal into the cell (Step 1). A signal transduction pathway, involving AC and PKA, is activated (Step 2). Activation of the signaling pathway mediates exocytosis of the BT-R receptor from intracellular vesicles to the cytoplasmic membrane (Step 3) within 15 min of toxin exposure. The resulting enhanced display of the BT-R receptor on the cell surface facilitates recruitment of additional toxin molecules which, in turn, amplifies the original signal in a cascade-like manner. The signaling kinase PICA modifies downstream effectors/executioners that drive the biochemical events which dismantle the cell, culminating in oncotic cell death.

[0021] In general, in the invention method, the toxin may be supplied in a composition that is externally applied to areas where an insecticidal effect is to be provided, including crop growing areas, areas in which desired decorative plants are being grown, and surfaces in the environment that will be occupied by animals or humans where it is desired that insects be eliminated, such as livestock growing areas, poultry houses, outdoor patios, picnic grounds, or even interior surfaces of commercial buildings and homes.

[0022] In one embodiment, the toxin may be supplied through recombinant modification of plants to be protected from insect infestation, as Bt toxin is conventionally supplied as known in the art. Many plants have been modified to produce various Bt proteins, the specific nature of which is dependent on the insect to be deterred.

[0023] Thus, whether the toxin is supplied as an insecticidal composition or is endogenously produced by a genetically modified plant, the effectiveness of the toxin can be enhanced by the addition of an inhibitor of endocytosis. If the toxin is supplied as an insecticide, the endocytosis inhibitor can be supplied as a separate preparation or in the same composition as the toxin. If the toxin is endogenously produced by plants, the plants can be sprayed or otherwise treated with the inhibitor. The inhibitor may even be absorbed through the root system when applied to the soil.

[0024] The criterion of exocytosis may also be used to identify inhibitors of Exol. In general, this method will compare the levels of exocytosis observed when cells are treated with toxin in the presence of Exol and in the presence or absence of a candidate inhibitor. If desired, exocytosis in the presence of toxin alone may be used as an additional standard. Thus, if a candidate compound enhances the level of exocytosis observed when cells are treated in the presence of both toxin and Exol in the presence of the compound as compared to its absence, it is identified as an inhibitor. If, for some reason, an environment to be treated is contaminated with Exol, such inhibitors may be used to enhance the effect of the toxin.

[0025] Exocytosis can also be used as a criterion for identifying effective insecticides. In one simple type assay, cells that display the receptor at their surface are plated on a surface and treated with a candidate compound. The migration of the receptor protein from an intracellular vesicle to the cell surface is then monitored using any one of a variety of techniques. The ability of a compound to effect migration of the receptor to the surface or to increase the number of receptors on the surface identifies it as a suitable insecticide.

[0026] Any method that detects the level of Bt receptors in the plasma membrane or on the cell surface may be used. For example, the cells may be treated with an antibody or an immunoreactive portion thereof coupled directly or indirectly to a detectable label and the level of exocytosis measured by the quantity of detectable label associated with the cells. Typically, the cells are treated with a detection reagent, washed to remove any unbound reagent, and examined as appropriate. The detectable label may be a radioisotope, a fluorophore, an enzyme, a reactive compound, or any other detectable label available in the art. A plethora of such labels is known.

[0027] Alternatively, extracts may be prepared of the plasma membrane and, if desired, of intracellular organelles or vesicles and the levels of receptor in each cellular location determined by any acceptable method, including Western Blot, or other forms of labeling such as those described above.

[0028] Another illustrative method for determining the level of exocytosis employs microscopic observations of the cells optionally supplied with labels specific for receptors. [0029] The following examples are intended to illustrate but not to limit the invention. [0030] The abbreviations used in the examples are:

Exol, 2-(4-fluorobenzoylamino)-benzoic acid methyl ester;

Noc, Nocodazole;

Cyd, Cytochalasin D;

Baf, Bafilomycin A;

H5, High Five cells;

S5, BT-Ri-transfected High Five cells;

PVDF, polyvinyl difluoride; TBS, Tris HCl-buffered saline; HRP, horseradish peroxidase; PFA, paraformaldehyde.

[0031] In the examples below the following procedures were followed.

[0032] Preparation of Cry IAb toxin: Cry IAb toxin, obtained from parasporal crystals of Bt subsp. berliner, was activated as described (Griko et al., 2004) by trypsin digestion. The activated CrylAb protein was purified by anion-exchange chromatography using a MonoQ HR 10/10 column with an AP-Biotech FPLC system. Quantification of the purified CrylAb toxin protein was measured by the bicinchoninic acid method (Pierce).

[0033] Cell cultures: High Five™ insect cells (Invitrogen, cabbage looper cells) were cultured in insect-Xpress medium (Cambrex, East Rutherford, NJ) supplemented with gentamycin (10 μg/ml, Sigma). High Five (H5) cells transfected with full-length BT-Ri cDNA (GenBank accession No. AF319973), designated as S5 cells (Zhang et al., 2005), were maintained in the same medium plus G418 (800 μg/ml, ISC BioExpress, Kaysville, UT).

[0034] Assay for cytotoxicity: H5 and S5 cells were seeded in 96-well plates (IxIO⁴ cells per well) and grown overnight to form cell monolayers at the bottom of the wells. The cell monolayers were then preincubated for 30 min with Exol (72 μM), nocodazole (20 μM), cytochalasin D (10 μM), bafilomycin A (200 nM), EDTA (5 mM), EGTA (5 mM), PKAi 14- 22-amide (PKAi, 8 μM), depending on the protocol, before the addition of CrylAb toxin (120 nM). After 20 minutes of toxin addition, phase-contrast microscopy was performed with a Nikon TE600 microscope and an RTE/CCD-1300 camera (Roper Scientific) at 20Ox to record cellular morphological changes. Ten microliters of trypan blue (0.4%, wt/vol) was added to each well and incubated for 5 min to determine cell death. Photomicrographs for stained cells were taken with an RTE/CCD-1300 camera (Roper Scientific, Trenton, NJ) at 20Ox by using a microscope (Nikon TE600). Photomicrographs were then analyzed by imaging software (METAMORPH 4, Universal Imaging, Downington, PA) to count the number of dead cells (NB, blue stained cells) and viable cells (NT, transparent cells), respectively. Cytotoxicity of CrylAb toxin was calculated by the ratio of dead cell (NB) over the number of total cell (NB + NT).

[0035] Immunofluore scent staining: H5 and S5 cells were seeded in 8-well glass chambers and grew overnight to form a monolayer that attached to the bottom of the glass chamber. After washed in PBS, the attached cells were fixed in 4% paraformaldehyde solution (PFA) and permeabilized with 0.2% Triton^® X-100 at room temperature. Then cells were rinsed three times in PBS and blocked with 1% BSA in PBS for 30 min. Antibodies against BT-R₁, produced by injecting purified BT-R₁ into New Zealand white rabbits, were added to treated cells, followed by Alexa Fluor -488 chicken anti-rabbit IgG antibody (Molecular Probes), for immunofluorescent staining. A fluorescence microscope (Nikon TE600) was used to view the stained samples. Microphotographs for stained cells were taken with an RTE/CCD-1300 camera (Roper Scientific) at 40Ox.

[0036] Preparation of cell extracts: Cells were seeded in 6-well plates (1x106 cells per well) and allowed to grow attached to the bottom surface of the plates overnight. Cells then were washed in PBS (4°C) and lysed in CytoBuster™ protein extraction reagent (Novagen). Cell lysates were centrifuged at 13,000 xg for 10 min at 4°C and the supernatants, which contained the intracellular membrane vesicles and soluble proteins, were collected. The pellets, mostly plasma membrane, were dissolved in membrane protein buffer (5 M urea, 2 M thiourea, 2% (w/v) 3[(3-cholamidopropyl)dimethylammonio]-propanesulfonic acid (CHAPS), 40 mM Tris-HCl).

[0037] The S5 and H5 cells were incubated with Exol (72 μM), EDTA (5 mM), EGTA (5 mM), PKAi 14-22-amide (PKAi, 8 μM), respectively, followed by CrylAb toxin (120 nM) treatment before preparation of cell extracts for measurement of BT-R₁ translocation. The freshly prepared cell extracts were used for Western blotting analysis.

[0038] Western blotting analysis: Equal amounts of protein (10 μg) for each sample were separated on 7% SDS-PAGE and were transferred to polyvinyl difluoride (PVDF) membrane (Millipore) for Western blotting analysis. The volume of membrane pellet sample that was loaded on the gels was eight times that of the supernatant sample. Before loading on the gels, same volume of 2x loading buffer (2% (w/v) SDS, 5M urea, 2M thiourea, 4% (w/v) CHAPS, 100 mM Tris HCl, 20% glycerol, 10 mM fi-ME and 0.03% (w/v) bromophenol blue) was added to protein samples and vortexing for 1 min at room temperature with or without boiling. Detection of BT-R₁ was accomplished by using BT-R₁ antibody and horseradish peroxidase- coupled secondary antibodies (Sigma). Proteins were finally detected by ECL plus reagent (Amersham).

[0039] Calculation OfBT-R₁ distribution: A relatively distribution of BT-R₁ between plasma membrane and intracellular vesicles was calculated based on the results obtained from the western blotting profiles. Briefly, the intensity of each band on the West blots which represented BT-R₁ in those lanes corresponding to either plasma membrane (P) or vesicular membrane (V) were scanned and quantified by ImageQuant software (Molecular Dynamics, Version 5.3). Because the sample volume of P that was loaded on the gels was eight times that of V, the quantified data from ImageQuant software was normalized as P to 8* V for a sample pair. Therefore, the distribution Of BT-R₁ was calculated as P/(P+8*V) for P samples and 8*V/(P+8*V) for V samples.

Example 1 Effect of Exocytosis and Endocytosis Inhibitors on CrylAb Toxin-Induced Cell Death

[0040] S5 cells transfected with full-length cDNA encoding BT-R₁ (Genbank accession No. AF319973) are sensitive to CrylAb toxin and exhibit distinctive cytological changes, i.e., membrane blebbing, cell swelling and lysis, with the progression of toxin-induced cell. As can be seen in Figure IA, toxin-treated (120 nM) S5 cells became swollen and misshaped (center upper) compared to untreated healthy S5 cells (left upper). When S5 cells were incubated with Exol, a specific cell permeable exocytosis inhibitor (Feng et al., 2003), followed by the addition of toxin, they did not undergo obvious morphological changes (right upper) and appeared as untreated healthy cells (left upper). On the other hand, cells pre-treated with the endocytosis inhibitors nocodazole, bafilomycin A and cytochalasin D did exhibit cytological changes (Figure IA, left lower, center lower and right lower, respectively) much like those cells exposed to CrylAb toxin (center upper). Furthermore, the endocytosis inhibitors did not inhibit toxin-induced cell death, as determined by Trypan blue staining, whereas Exol dramatically prevented cell death (Figure IB). The results of the endocytosis inhibitor experiments are consistent with our previous observation that CrylAb is not internalized during the morphological changes associated with cytotoxicity, i.e., blebbing and swelling (Zhang et al, 2006).

[0041] Direct delivery of the CrylAb toxin into untransfected (receptor-free) H5 cells by the protein delivery reagent Chariot(Morris et al., 2001) also did not induce cell death, so there is no receptor-mediated endocytosis of CrylAb. However, exocytotic activity appears to be associated with toxin action.

[0042] Figure 1 thus shows the involvement of exocytosis in the cytotoxic action of CrylAb. (a) S5 cells exposed to toxin (120 nM) in the presence of Exol (right upper), nocodazole (left lower), bafilomycin A (center lower) and cytochalasin D (right lower). S5 cells treated with CrylAb toxin (center upper) and untreated S5 cells (left upper). Only those cells pre-treated with Exol (right upper) showed similar morphological characteristics as untreated cells (left upper). Bar = 40μm. (b) Quantitative assessment of exocytosis and endocytosis inhibitors on cell death. The bars represent percentage of dead cells (determined by Trypan blue staining of nuclei) in cell cultures treated with toxin (120 nM) in the presence of inhibitors and CrylAb toxin treatment as described in (a). Only Exol inhibited CrylAb-induced cell death (compare to untreated control). Data represent the mean ± SD of six experiments. Exol= 2-(4-fluorobenzoylamino)-benzoic acid methyl ester; Noc = Nocodazole; Cyd = Cytochalasin D; Baf = Bafilomycin A.

Example 2 Constitutive Expression and Localization of BT-R^ in S5 Cells

[0043] The BT-R₁ cadherin receptor is located in the plasma membrane as well as in intracellular vesicles of transfected cells (Zhang et al., 2005). The dynamics of BT-Ri distribution during exocytosis which is involved in the progression of toxin-induced cell death were therefore investigated. The pattern of BT-Ri localization in S5 cells without exposure to the Cry IAb toxin was determined as follows:

[0044] BT-Ri was detected by immuno-fluorescent staining using anti-BT-Ri antibody (green) in both intracellular membrane vesicles (Figure 2A, left panel, white arrow) and plasma membrane (Figure 2A, left panel, black arrow). There was no BT-Ri expressed in untransfected H5 cells (Figure 2A, right panel). Western blotting with anti-BT-Ri antibody also revealed the presence of BT-Ri in the cytoplasm containing intracellular membrane vesicles (Figure 2B, V, white arrows) and the plasma membrane (Figure 2B, P, black arrows).

[0045] The amount of BT-Ri localized in the plasma membrane of freshly transfected cells consistently increased as they underwent cell division. The increase occurred during ten generations post transfection (Figure 2B, G₂, G₅ and Gi ₀), after which it stabilized (Figure 2B, Gio, Gi5 and G30). The amount of BT-Ri in the plasma membrane relative to the total amount distributed in intracellular membranes and the plasma membrane increased significantly from Go to Gio, reaching a maximum of -13% at Gio and remaining constant throughout the next 20 generations (Figure 2C, Gi₀-G₃₀). The consistency in the pattern of distribution and turnover rate of BT-Ri in dividing S5 cells demonstrates the constitutive nature of receptor translocation when the cells are not under toxin pressure.

[0046] Figure 2 thus shows stabilized expression and localization of BT-Ri in S5 cells, (a) S5 and H5 cells were immunostained by using BT-Ri antibody (green). The black arrow points to BT-Ri localized on the cell surface (plasma membrane) and the white arrow points to BT-Ri localized inside the cell (left panel). No BT-Ri was detected in H5 cells (right panel). Bars = 5μm. (b) BT-Ri (210 kDa) in plasma membrane (P, black arrows) and intracellular vesicle preparations (V, white arrows) from S5 cells were analyzed by Western blotting using BT-Ri antibody. G₂-G₃₀ represents protein patterns from the 2nd through the 30th generation of S5 cells after transfection. G₀ represents the protein pattern of H5 cells before transfection. The amount of BT-R₁ in the plasma membrane (P) of S5 cells increased with a corresponding decrease in the amount in intracellular vesicles (V). The lower molecular weight protein in the V lanes represents a protein that binds nonspecific ally to anti-BT-Ri antibody, (c) The relative percentage of BT-Ri in the plasma membrane was calculated based on the expression profiles of S5 cells in (b). The expression of BT-R₁ increased along with its localization in the plasma membrane through the 10th generation (Gio) which remained constant through at least G₃₀.

Example 3 Stimulation of BT-R₁ Exocytosis by Cryl Ab Toxin

[0047] Rapid (15 min) translocation of the receptor from intracellular vesicles to the cell surface when BT-R₁ antibody was used in Western blot analysis to examine distribution of BT-R₁ in Cryl Ab toxin-treated S5 cells. The change in distribution of BT-R₁ between the plasma membrane (Figure 3A, P, black arrow) and the intracellular membrane vesicles of toxin- treated cells (Figure 3A, V, white arrow) was dramatic, compared to the distribution in untreated cells (Figure 3A, V and P). It is noteworthy that there was no obvious change in the total amount of BT-R₁ detected within this short time frame (15 min), despite the remarkable shift in receptor distribution.

[0048] When S5 cells were incubated with the exocytosis inhibitor Exol 30 min before addition of Cryl Ab, the distribution Of BT-R₁ (Figure 3B, Exol+CrylAb) was the same as that in untreated cells (Figure 3A, no CrylAb). This result is in contrast to the pattern observed in toxin-treated cells without Exol (Figure 3A and B, CrylAb).

[0049] Thus Figure 3 shows stimulation of the exocytosis of BT-R₁ by CrylAb toxin, (a) The distribution of BT-R₁ in toxin-treated S5 cells was analyzed by Western blotting using BT-R₁ antibody. Upon toxin treatment (120 nM), most of the BT-R₁ was detected in the plasma membrane preparation (P, black arrow). There was a dramatic decrease of BT-R₁ in the intracellular vesicle preparations (V, white arrow), compared to samples from untreated cells. The lower molecular weight protein in the V lanes represents a protein that binds nonspecifically to anti-BT-Ri antibody, (b) S5 cells were incubated with the exocytosis inhibitor Exol (20 μM) 30 min before addition of CrylAb toxin, and protein extracts were analyzed by Western blotting as mentioned above.

[0050] Mathematical analysis of the protein profiles in Figure 3A and B revealed that the ratio of BT-R₁ in intracellular vesicle membranes (V) to plasma membrane (P) is approximately 8:1 in untreated cells (Figure 4, no CrylAb), 1:4 in CrylAb-treated cells (Figure 4, CrylAb) and 8:1 in CrylAb-treated cells pre-incubated with Exol (Figure 4, Exol+CrylAb). Apparently, CrylAb toxin stimulates rapid accumulation of BT-R₁ on the cell surface and the translocation of receptor correlates with exocytotic activity.

[0051] Thus, Figure 4 shows comparative distribution of BT-R₁ in untreated (no CrylAb), CrylAb-treated (CrylAb) and CrylAb-treated S5 cells pre-incubated with Exol (Exol+CrylAb). V represents intracellular vesicles and P, cytoplasmic membrane. Exol effectively inhibited toxin- stimulated translocation of BT-R₁ (compare Exol +CrylAb to no CrylAb). The distribution of BT-R₁ was calculated based on the results obtained from the western blot profiles in Figure 3 and calculated as P/(P+8*V) for P samples and 8*V/(P+8*V) for V samples as described above.

Example 4 Correlation of Enhanced Exocytosis to the Binding of Cryl Ab to BT-R₁

[0052] The initial stage of cytotoxicity involves the univalent binding of CrylAb toxin to a conserved structural motif in BT-R₁ and the subsequent transmission of the death signal inside the cell (Griko et al., 2007). Binding of CrylAb to BT-R₁ reaches saturation immediately before membrane blebbing (Zhang et al., 2005; Zhang et al., 2006). Likewise, Exol blocks cytotoxicity before the cells begin to bleb (data not shown). To determine whether the binding of toxin to receptor affects the exocytotic transport of BT-R₁ or vice versa, we analyzed the dynamics of toxin-induced exocytosis of BT-R₁ (Figure 5A). Translocation of the receptor began within 5 min after addition of CrylAb toxin (Figure 5 A, 5 min, V and P), reaching saturation in 15 min. By then, 80-90% of the BT-R₁ was detected in plasma membrane (Figure 5A, 15 min). Interestingly, CrylAb monomer (60 kDa) increased to a maximum level on the cell surface within the same time frame. The relative percent saturation of BT-R₁ and CrylAb in the plasma membrane was the same throughout toxin exposure (Figure 5B, 40 to 100% in 15 min), indicating that CrylAb toxin binds instantaneously to receptor presented on the cell surface. Furthermore, the number of toxin molecules bound is limited by the number of receptor molecules present at any given time. The correlation between CrylAb monomer binding and the enhanced display of BT-R₁ on the cell surface (Figure 5B) most likely explains why effective cell-killing depends on receptor translocation (Figure 1), i.e., additional receptor on the cell surface is necessary for maximum toxin binding and subsequent action.

[0053] Figure 5 shows correlation between enhanced exocytosis of BT-R₁ and monomeric binding of CrylAb toxin, (a) The distribution of BT-R₁ in the plasma membrane (P) and intracellular vesicles (V) of S5 cells (G15) was assessed at different times (5-15 min) of toxin treatment (120 nM) by Western blotting using BT-R₁ antibody. Saturation of the receptor in P occurred within 15 min. (b) Quantitative assessment of the amount of BT-R₁ and Cryl Ab monomer on the plasma membrane is based on the time-dependent profiles of BT-Ri as seen in (a) and in Figure 3c (Zhang et al. 2005). The black bars represent the relative percentage of BT-Ri on the plasma membrane at the indicated time of toxin exposure. The gray bars represent the relative percentage of membrane-associated Cryl Ab monomer at different times of toxin exposure (Fig. 3c, Zhang et al. 2005). The exocytosis of BT-Ri correlates with the increased association of Cryl Ab monomer on the cell surface in a time-dependent manner. Both the accumulation of BT-Ri on cell surface and the membrane bound toxin monomer reached saturation within about 15 min of toxin exposure.

Example 5 Exocvtosis Of BT-R₁ Mediated by CrylAb-Induced Signal Transduction

[0054] Because BT-Ri is a cadherin that serves as the receptor for Cry IA toxins, we determined whether Cryl Ab-induced exocytosis of BT-Ri is driven by the activation of AC/PKA signal transduction in S5 cells that ultimately brings about cell death. The translocation of BT-Ri after toxin addition in the presence of a specific protein kinase A inhibitor, PKAi 14-22- Amine (PKAi), and the divalent cation chelators, EDTA and EGTA was determined (Figure 6). EDTA chelates Mg²⁺ and Ca²⁺ whereas EGTA preferentially chelates Ca²⁺. Both EDTA and PKAi effectively interrupt AC/PKA signal transduction stimulated by Cryl Ab in S5 cells and block the progression of cell death before S5 cells enter the blebbing stage (Zhang et al, 2005; Zhang et al, 2006). EGTA has no such effect (Zhang et al, 2005). S5 cells were incubated with EDTA (5 mM), EGTA (5 mM) and PKAi (8 μM) for 30 min separately before addition of toxin. In the EDTA- and PKAi-pretreated cells (Figure 6, EDTA+Cryl Ab and PKAi+Cryl Ab, respectively), the ratio of BT-Ri in plasma membrane (P) to intracellular vesicles (V) was the same as that for untreated cells (Figure 6, no CrylAb). In other words, the distribution of BT-Ri did not change upon toxin treatment in the presence of EDTA and PKAi. However, BT-Ri was redistributed in EGTA-pretreated cells upon toxin addition in a manner similar to toxin-treated cells alone (Figure 6, EGTA+CrylAb and CrylAb, respectively). These results indicate that blocking activation of intracellular AC/PKA signal transduction by EDTA and PKAi, not EGTA, prevents the rapid exocytosis of BT-Ri occasioned by CrylAb toxin.

[0055] Figure 6 shows inhibition of toxin-induced exocytosis of BT-Ri by EDTA and PKAi. Cells were treated with EDTA and EGTA (5 mM) and PKAi (8 μM) separately before addition of CrylAb toxin (120 nM). The distribution of BT-Ri in the plasma membrane (P) and the intracellular vesicles (V) was assessed by Western blotting using BT-R₁ antibody. EDTA and PKAi effectively blocked toxin-induced exocytosis of BT-Ri whereas EGTA had no such effect.

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Claims

Claims

1. A method to enhance the effectiveness of Bacillus thuringiensis toxin (Bt toxin) which method comprises supplementing said toxin with an endocytosis inhibitor.

2. The method of claim 1 wherein the endocytosis inhibitor is nocodazole, bafilomycin A or cytochalasin.

3. The method of claim 1 wherein said Bt toxin is produced endogenous Iy in a plant.

4. The method of any of claims 1-3 wherein the inhibitor is applied as a spray.

5. An insecticidal composition which comprises an effective amount of a Bt toxin in combination with an effective amount of an endocytosis inhibitor.

6. The composition of claim 5 wherein the endocytosis inhibitor is nocodazole, bafilomycin A or cytochalasin.

7. A method to provide an insecticidal effect to an environment which method comprises applying to said environment an effective amount of a Bt toxin in combination with an effective amount of an endocytosis inhibitor.

8. A method to identify an insecticide with enhanced toxicity which method comprises treating cells that display receptors for at least one Bt toxin at their surface with a candidate compound, and assessing the surface of the cells for the presence of an increased number of said receptors, whereby an increase in said number identifies said candidate as an insecticide with enhanced toxicity.

9. The method of claim 8 wherein the cells are insect cells that natively produce the receptor.

10. The method of claim 8 wherein said cells are recombinant cells modified to produce said receptor.