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Applied and Environmental Microbiology, June 2007, p. 3623-3629, Vol. 73, No. 11
0099-2240/07/$08.00+0     doi:10.1128/AEM.01056-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Binding of Cyt1Aa and Cry11Aa Toxins of Bacillus thuringiensis Serovar israelensis to Brush Border Membrane Vesicles of Tipula paludosa (Diptera: Nematocera) and Subsequent Pore Formation

Jesko Oestergaard,¹ Ralf-Udo Ehlers,^1* Amparo C. Martínez-Ramírez,² and Maria Dolores Real²

Institute for Phytopathology, Department of Biotechnology and Biological Control, Christian Albrechts University, Kiel, Germany,¹ Departamento de Genetica, Facultat de Ciències Biològiques, Universitat de Valencia, Burjassot, Valencia, Spain²

Received 8 May 2006/ Accepted 27 March 2007

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Bacillus thuringiensis serovar israelensis (B. thuringiensis subsp. israelensis) produces four insecticidal crystal proteins (ICPs) (Cry4A, Cry4B, Cry11A, and Cyt1A). Toxicity of recombinant B. thuringiensis subsp. israelensis strains expressing only one of the toxins was determined with first instars of Tipula paludosa (Diptera: Nematocera). Cyt1A was the most toxic protein, whereas Cry4A, Cry4B, and Cry11A were virtually nontoxic. Synergistic effects were recorded when Cry4A and/or Cry4B was combined with Cyt1A but not with Cry11A. The binding and pore formation are key steps in the mode of action of B. thuringiensis subsp. israelensis ICPs. Binding and pore-forming activity of Cry11Aa, which is the most toxic protein against mosquitoes, and Cyt1Aa to brush border membrane vesicles (BBMVs) of T. paludosa were analyzed. Solubilization of Cry11Aa resulted in two fragments, with apparent molecular masses of 32 and 36 kDa. No binding of the 36-kDa fragment to T. paludosa BBMVs was detected, whereas the 32-kDa fragment bound to T. paludosa BBMVs. Only a partial reduction of binding of this fragment was observed in competition experiments, indicating a low specificity of the binding. In contrast to results for mosquitoes, the Cyt1Aa protein bound specifically to the BBMVs of T. paludosa, suggesting an insecticidal mechanism based on a receptor-mediated action, as described for Cry proteins. Cry11Aa and Cyt1Aa toxins were both able to produce pores in T. paludosa BBMVs. Protease treatment with trypsin and proteinase K, previously reported to activate Cry11Aa and Cyt1Aa toxins, respectively, had the opposite effect. A higher efficiency in pore formation was observed when Cyt1A was proteinase K treated, while the activity of trypsin-treated Cry11Aa was reduced. Results on binding and pore formation are consistent with results on ICP toxicity and synergistic effect with Cyt1Aa in T. paludosa.

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The European crane fly Tipula paludosa (Diptera: Nematocera) is the major pest of pasture, meadows, and turf grass in temperate climates in northwest Europe (5) and is an invasive pest in North America (20, 32). T. paludosa is univoltine, laying eggs in August/September. Larvae hatch in September/October and develop through four instars until May, diapause during June and July, and pupate in August (5). Larvae of T. paludosa (leatherjackets) cause damage mainly by feeding above ground on leaves of pasture (39). Secondary damage is caused by birds, like crows, which destroy the grass searching for insect larvae. Since parathion (E 605 forte) was banned by the European Commission in January 2002, no chemical control measures are available in Europe. An alternative approach could be the use of biological control agents based on Bacillus thuringiensis serovar israelensis (B. thuringiensis subsp. israelensis), which is toxic to T. paludosa larvae (29, 37, 40).

Bacillus thuringiensis serovar israelensis (B. thuringiensis subsp. israelensis), a gram-positive, spore-forming bacterium, is an effective control agent against mosquitoes and black flies (4, 21). The toxicity of B. thuringiensis subsp. israelensis is based on insecticidal crystal proteins (ICPs), which are produced during sporulation (8, 10, 16). The ICPs are composed of four major proteins, Cyt1Aa, Cry11Aa, Cry4Ba, and Cry4Aa (1, 15), which differ in toxicity (14). The B. thuringiensis subsp. israelensis crystal proteins are solubilized under alkaline conditions of the insect midgut (24) and proteolytically activated from a protoxin into a toxin with specific binding properties to different receptor molecules on the midgut epithelium of mosquitoes (17, 44). The 28-kDa protein Cyt1Aa can be activated by proteinase K, resulting in a 25-kDa toxic fragment (11). Trypsin is the major proteolytic enzyme in mosquito larval midgut (6, 45) and is also present in the gut fluid of Tipula spp. (36). The Cry11Aa protoxin proteolytically activated with trypsin splits into two fragments of 32 and 36 kDa (13, 44).

Cry11Aa is the most toxic protein for Aedes aegypti, Anopheles stephensi, and Culex pipiens, whereas the Cyt1Aa protein is nontoxic or less toxic but synergizes the toxic effect of Cry proteins (14, 23, 42, 43). Cry4A and Cry4B have also been shown to react synergistically in mosquito larvae (2).

In vivo binding of Cry11Aa to the apical brush border membrane of the gastric ceca and posterior stomach has been shown to occur in histological sections of intoxicated Anopheles gambiae (35). Feldmann et al. (17) observed a very low toxicity of Cry11Aa against Tipula oleracea and identified different receptor proteins for Cry11Aa in Anopheles stephensi and T. oleracea. Strong binding to a single 148-kDa receptor protein of A. stephensi brush border membrane vesicles (BBMVs) was observed, whereas in T. oleracea a 78-kDa receptor protein was identified. However, no binding of the trypsinized Cry11Aa toxin in T. oleracea was observed (17).

The aim of this study was to define the toxicities of the different B. thuringiensis subsp. israelensis toxins and to analyze the binding capacities and pore-forming activities of Cyt1Aa and Cry11Aa proteins in T. paludosa midgut BBMVs.

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Culture of Tipula paludosa.
To obtain larvae for laboratory bioassays and the preparation of BBMVs, T. paludosa adults were collected during late August and September from turf and meadows in Aukrug, Germany. Males and females were combined for copulation. After insemination, females were decapitated, legs and wings were removed, and single torsos were placed onto 1.5% agar petri dishes (diameter, 9 cm) with a 2-mm layer of tap water for egg laying. Up to 300 eggs were harvested per petri dish. The first instars (L1) hatched after 10 days and were first fed with chickweed (Stellaria media) in plastic boxes (32 by 17 cm) half filled with sandy soil and kept at 15°C. The second instars were fed with S. media and lettuce (Lactuca sativa var. capitata), and the third and fourth instars received lettuce only. The soil was kept moist by spraying tap water over the surface of the soil every 2 days. The different larval stages were distinguished according to the method of Pritchard (33). L1 larvae lack anal papillae, and older stages have fields of bristles instead. The second to fourth instars were distinguished by the size of their head capsule. To assess toxicity of the Cry proteins, 3- to 4-day-old first instars were used, whereas for preparation of the BBMVs fourth instars were used.

Culture of B. thuringiensis subsp. israelensis and recombinant B. thuringiensis strains.
The IPS82 B. thuringiensis subsp. israelensis standard strain (Institute Pasteur, Paris, France) was propagated in a 5-liter fermentor (Meredos, Göttingen, Germany) in tryptone-yeast medium (10 g/liter tryptone, 5 g/liter corn starch, 2 g/liter yeast extract, 28 mM glucose, 6 mM K₂HPO₄, 7 mM KH₂PO₄) at 30°C for 24 h. Oxygen saturation was controlled at 40%. Spray drying to obtain technical powder was done according to the method of Lisansky et al. (27). Recombinant B. thuringiensis subsp. israelensis strains producing either the Cyt1A or the Cry11A protein were obtained from Brian Federici (University of California) and those producing Cry4A or Cry4B from Ray Akhurst (CSIRO, Canberra, Australia). Recombinant strains were prepared from electrocompetent B. thuringiensis 4Q8 cells (Bacillus Genetic Stock Center, Ohio), produced in liquid media according to the method of Huges et al. (25), and spray dried.

Quantification of ICPs.
Toxin quantities were determined by a double sandwich enzyme-linked immunosorbent assay (ELISA) by use of specific monoclonal antibodies as described by Oestergaard et al. (30). Technical powder (50 mg) was suspended in water and centrifuged at 20,000 x g and the supernatant decanted. The pellet was suspended in 13 ml solubilization buffer containing 0.05 M Na₂CO₃ and 0.01 M dithiothreitol (Merck, Darmstadt, Germany) and homogenized using a dispersing machine (ART Miccra D-8) at 19,000 rpm for 30 s. The buffer was adjusted to pH 11 for solubilization of Cyt1Aa and to pH 10.5 for solubilization of Cry11Aa und Cry4 proteins. The samples were then heat treated at 60°C for 60 min and centrifuged at 14,000 x g for 10 min. Then, 1:50, 1:250, 1:500, 1:1,000, 1:2,000, and 1:3,000 dilutions of the ICP solutions were added to multiwell plates in aliquots of 100 µl and incubated for 30 min. One hundred microliters of the detection antibody (30) specific to the ICP proteins was added and incubated for 30 min. In the next step, 100 µl goat anti-mouse immunoglobulin G1 antibody (Roche-Boehringer, Mannheim, Germany) conjugated with horseradish peroxidase was supplemented. Thirty minutes later, 100 µl ABTS (2,2'-azino-di-3-ethylbenzthiazoline sulfonate) (Roche-Boehringer, Mannheim, Germany) was added and the enzyme reaction quantified at an optical density at 405 nm after 30 min of incubation and gentle agitation (400 rpm).

Toxicity of the single toxins and their combinations.
T. paludosa L1 larvae were fed with leaves of S. media sprayed with a suspension of the technical B. thuringiensis subsp. israelensis powder of either one of the different recombinant strains or the B. thuringiensis subsp. israelensis strain IPS82 and combinations of the different recombinant strains. For each variant, 120 larvae were each fed with one treated leaf. Mortality was determined after 2 days. IPS82 was applied at 16 µg cm^–2, equivalent to 0.58 µg cm^–2 Cyt1 toxin, 0.7 µg cm^–2 Cry11 toxin, and 0.07 µg cm^–2 Cry4 toxin (according to the ELISA quantification). The different recombinant strains were applied at equal amounts, as assessed for IPS82. For the combined use of the different recombinant strains, the leaves were sprayed two, three, or four times according to the number of strains combined in one treatment. Data on mortality were Abbott corrected and compared using chi-square analysis. To analyze whether synergistic effects occurred with the combined treatments, the expected additive mortality was calculated according to the equation E = P1 + P2 x (1 – P1), where E is the expected additive mortality in the combined treatment, P1 is the observed mortality in the treatment with Cyt1, and P2 is the observed mortality caused by the respective recombinant strain (3). The observed mortality was compared with the calculated additive mortality by use of chi-square analysis.

Purification, solubilization, and processing of ICPs.
Crystalline inclusions of recombinant B. thuringiensis subsp. israelensis strains, which produce only Cyt1Aa or Cry11Aa, and of B. thuringiensis subsp. israelensis IPS82 were purified from cell debris and spores by centrifugation in discontinuous sucrose gradients as described by Thomas and Ellar (38). One-milliliter fractions of the density gradient were carefully removed from the top to the bottom and collected. Fractions containing the crystals were diluted 1:10 with distilled water, mixed, and afterwards centrifuged for 20 min at 20,000 x g to collect the pellet. The crystalline proteins in the pellet were solubilized in alkaline Na₂CO₃ buffer (50 mM, pH 11.2) for 90 to 120 min at 37°C in a water bath. Samples were then centrifuged, and the pH of the supernatant was adjusted to 8 with 1 M Tris-HCl before use. The concentrations of the different solubilized toxins were determined according to the method of Bradford (7). The purity of the Cyt1Aa and Cry11Aa toxins obtained from the recombinant strains was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Crystals and solubilized proteins were heated at 95°C in Laemmli buffer before being loaded onto a 12% gel following the instruction manual of the Mini-Protean III electrophoresis cell (Bio-Rad). For the proteolytic activation of toxins, solubilized Cyt1Aa and the crystalline proteins of IPS82 were treated with proteinase K for 1 h at 30°C (30:1) according to the method of Chow et al. (11). The Cry11Aa toxin was incubated with trypsin for 2 h at 25°C (50:1) according to the methods of Dai and Gill (13) and Yamagiwa et al. (44). The Cry3A toxin used as a negative control was purified from strain BTS1 and alkali solubilized for 90 min by following the method described above for the recombinant strains.

Preparation of BBMVs.
Several hundred T. paludosa L4 larvae were frozen at –20°C before dissection. The midgut was separated from the hindgut, the fat body, and the rest of the larva. The first segment (with the head capsule) and the last segment (with the stigmata) were cut. The dermal muscular tunica was slit, and the gut was removed from the rest of the larvae. The midguts were washed with MET buffer (300 mM mannitol, 5 mM EGTA, and 200 mM Tris-HCl, pH 7.4) and frozen in an Eppendorf tube by transfer into liquid nitrogen for 10 s before being stored at –80°C. The BBMVs were prepared according to the methods of Wolfersberger et al. (41). Midguts (2.5 g) were mixed with 10 volumes MET buffer and homogenized using a dispersing instrument (ART Miccra) at 6,000 rpm for 1 min. After addition of the same volume of 24 mM MgCl₂, the samples were incubated on ice for 15 min. After centrifugation at 20,000 x g for 30 min, the supernatant was removed and the pellet was resuspended carefully in half of the volume of MET buffer; the same volume of 24 mM MgCl₂ was added. After 15 min of incubation on ice, the sample was centrifuged for 10 min at 3,000 x g. The supernatant was centrifuged again at 20,000 x g for 30 min. The supernatant was removed, and the pellet containing the BBMVs was resuspended in 500 µl MET buffer and stored at –80°C until use.

Binding assays with isolated BBMVs.
Solubilized Cyt1Aa and Cry11Aa proteins were biotinylated using biotinyl-N-hydroxysuccinimide (RPN28; Amersham) as described in the manufacturer's instructions. After biotinylation, the protein concentration was determined according to the method of Bradford (7). For the binding experiments, 10 ng of the biotinylated toxins was mixed with a BBMV fraction containing 10 µg of membrane proteins in a final volume of 100 µl. After 1 h of incubation at room temperature, unbound toxin was removed by centrifugation and washed as described by Rausell et al. (34). BBMVs were suspended in Laemmli buffer (20 µl) and heated at 95°C before being loaded onto a 12% gel by following the instruction manual of the Mini-Protean III electrophoresis cell (Bio-Rad). After SDS-PAGE, the proteins were blotted to a nitrocellulose membrane for 1 h at 250 mA. The biotinylated toxins bound to the BBMVs were visualized by incubating the nitrocellulose membrane with streptavidin-peroxidase conjugate for 1 h before the addition of Supersignal West Pico chemiluminescent substrate (Pierce) by following the instructions of the manufacturer. For competition experiments, a 500-fold excess of the corresponding nonlabeled toxin was added to the original binding mix.

In vitro pore formation assays.
The pore formation activities of Cyt1Aa, Cry11Aa, and B. thuringiensis subsp. israelensis strain IPS82 were determined by calcein leakage experiments performed according to the method of Rausell et al. (34). BBMVs containing 300 µg membrane proteins were mixed with 1 ml calcein solution {80 mM calcein, 150 mM KCl, 10 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), pH 9} and sonicated three times for 30 s each to load the BBMVs with the fluorescent calcein. The nonentrapped calcein was separated from the loaded BBMVs by gel filtration on Sephadex G-50 using a 3-cm column with an elution buffer containing 150 mM KCl, 10 mM CHAPS, pH 9. Fifteen micrograms of BBMV was added to 2 ml of the elution buffer. Toxin (50 nM) was added and mixed with BBMV, and the increase in fluorescence due to the release of calcein from BBMVs was monitored with a fluorescence spectrophotometer at an excitation wavelength of 490 nm, an emission wavelength of 520 nm, and a slit of 10 nm (Cary Eclipse; Varian). Assays were performed with solubilized Cry11Aa toxin, solubilized Cry11Aa toxin (trypsin treated), solubilized Cyt1Aa toxin, solubilized Cyt1Aa toxin (proteinase K treated), solubilized B. thuringiensis subsp. israelensis IPS82 toxin, and B. thuringiensis subsp. israelensis IPS82 toxin (proteinase K treated). Protease treatments were completed as described above.

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Toxicity of ICPs for T. paludosa first instars.
Results on the mortality of first-instar T. paludosa caused by ICPs of recombinant strains and wild-type IPS82 of B. thuringiensis serovar israelensis are presented in Fig. 1A to C. Mortality recorded for IPS82 was 76% and did not significantly differ from mortality obtained with the combination of all four toxins (73%). Of the single recombinant strains, the Cyt1A-producing strain caused the highest mortality (45%), whereas almost no mortality was recorded when larvae were exposed to Cry11A- and Cry4-producing strains. Mortality of T. paludosa exposed to any of the combinations of the Cry proteins was lower than 12%, which was not significantly different (P < 0.05, chi-square test) from the mortality (0.025%) obtained with the untreated controls (Fig. 1B). A combination of Cyt1A toxin with Cry4A toxin proved to be more toxic to T. paludosa (65.6%) than either of the single toxins (Fig. 1C). The calculated additive effect for this combination was 47%. No synergistic effect was recorded when Cry4B toxin was added to the mixture, as 55% mortality was not significantly different from the 45.2% mortality recorded for Cyt1A alone. No additive effects were calculated for combinations of Cyt1A and Cry4 toxins (Fig. 1C). None of the combinations surpassed the activity of the combination of all B. thuringiensis subsp. israelensis toxins or the wild-type strain, indicating that no antagonistic effects occurred. These results were confirmed when L1 larvae were exposed to half of the amount of toxins (data not shown).

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FIG. 1. Abbott-corrected mortality of first-instar T. paludosa, caused by ICPs of B. thuringiensis serovar israelensis. Shown are results from strain IPS82 producing all four ICPs and combinations of different recombinant strains producing one of the toxins Cry4A, Cry4B, Cry11A, or Cyt1A. Comparison of IPS82 and combinations of all recombinant strains with (A) single recombinant strains, (B) combinations of Cry4- and Cry11-producing recombinants, and (C) combinations of Cyt1A-, Cry4-, and Cry11-producing recombinants. For each variant, 120 insects were used. The technical powder was applied on leaves of S. media at 16 µg cm–2, equivalent to 0.58 µg cm–2 Cyt1, 0.7 µg cm–2 Cry11, and 0.07 µg cm–2 Cry4. The amount of technical powder of the recombinant strains was adjusted to the same amount of toxins, as quantified by ELISA, in IPS82 technical powder. Bars with different letters indicate significant differences (chi-square test with Yates' correction, P 0.05).

Binding experiments.
The Cry11Aa (72-kDa) protein was purified by density gradient centrifugation (Fig. 2A) and solubilized for 120 min under alkaline conditions. The solubilization procedure of Cry11Aa resulted in two fragments, with molecular masses of 32 and 36 kDa (Fig. 2B). Both fragments were biotin labeled and detected with streptavidin-peroxidase after blotting (Fig. 3A). No binding of the 36-kDa fragment to T. paludosa BBMVs was detected. The 32-kDa fragment was able to bind to T. paludosa BBMVs, but only a partial reduction of binding of this fragment was observed in competition experiments in the presence of a 500-fold excess of unlabeled Cry11Aa toxin (Fig. 3A). The Cyt1Aa (28-kDa) protein was purified by sucrose gradient centrifugation (Fig. 2C) and solubilized. A 90-min solubilization in alkaline buffer resulted in a single 28-kDa polypeptide, whereas a prolonged solubilization (120 min) gave rise to two fragments, of 28 and 25 kDa (Fig. 2D). Two different Cyt1Aa preparations were used for the binding and competition experiments of the Cyt1Aa toxin. With the first preparation (90-min solubilization time), specific binding of the 28-kDa Cyt1Aa toxin to T. paludosa BBMVs was observed (Fig. 3B). The 25- and 28-kDa proteins of the second preparation (120-min solubilization time) also showed specific binding to the BBMVs of T. paludosa (Fig. 3C).

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FIG. 2. SDS-PAGE analysis of Cry11Aa and Cy1Aa toxins. Lanes 1, molecular mass markers (250, 150, 100, 75, 50, 37, 25, and 20 kDa); lanes 2, (A) purified Cry11Aa toxin (apparent molecular mass of 72 kDa), (B) Cry11Aa after 120 min of alkaline solubilization (apparent molecular masses of 32 and 36 kDa), (C) purified Cyt1Aa toxin (apparent molecular mass of 28 kDa), and (D) Cyt1Aa after 120 min of alkaline solubilization (apparent molecular masses of 25 and 28 kDa).

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FIG. 3. Western blot analysis of biotin-labeled toxins after binding assays and homologous competition experiments on BBMVs of T. paludosa larvae. (A) Binding of biotin-labeled Cry11Aa toxin (120 min of alkali solubilization). (B) Binding of biotin-labeled Cyt1Aa toxin (90 min of alkali solubilization). (C) Binding of biotin-labeled Cyt1Aa toxin (120 min of alkali solubilization). Lanes 1, biotin-labeled toxin loaded directly on the SDS-PAGE gel; lanes 2, biotin-labeled toxin bound to T. paludosa BBMVs; lanes 3, biotin-labeled toxin bound to T. paludosa BBMVs in the presence of a 500-fold excess of nonlabeled toxin.

Pore-forming activity.
To investigate whether the toxins that bound to T. paludosa BBMVs were also able to form pores, calcein leakage experiments with the solubilized toxins were performed as described above. As a negative control, Cry3A toxin, which is known to be nontoxic for dipteran insects (26), was also included. It has been proven by adding proteinase K and trypsin to the BBMVs that the two toxin-activating enzymes have no effect on the BBMVs (data not shown). The pore-forming activity of solubilized Cry11Aa is shown in Fig. 4A. The addition of Cry11Aa toxin to T. paludosa BBMVs caused a considerable increase in fluorescence, due to the release of calcein, compared to the level for the Cry3A toxin control. It has been reported that, in mosquitoes, the 34- and 32-kDa Cry11Aa fragments obtained by gut extract proteolytic activation are both essential for toxicity, as are the 36- and 32-kDa tryptic fragments (44). In order to analyze the effect of Cry11Aa proteolytic treatment on pore-forming activity in T. paludosa BBMVs, the trypsinized Cry11Aa-inducing release of calcein was studied. The level of calcein released by trypsinized Cry11Aa protein (34 fluorescence arbitrary units [FAU]) was lower than that released by non-trypsin-treated Cry11Aa (42 FAU) (Fig. 4A).

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FIG. 4. Analysis of calcein leakage from T. paludosa calcein-loaded BBMVs induced by (A) Cry11Aa, (B) Cyt1Aa, and (C) B. thuringiensis subsp. israelensis IPS82 toxins. Toxins were added after a 50-s measurement. Cry11, solubilized Cry11Aa; T Cry11, solubilized and trypsin-treated Cry11Aa; Cry3, solubilized Cry3A; PK Cyt1, solubilized and proteinase K-treated Cyt1Aa; Cyt1, solubilized Cyt1Aa; PK IPS, solubilized and proteinase K-treated B. thuringiensis subsp. israelensis IPS82; IPS, solubilized B. thuringiensis subsp. israelensis IPS82. Conditions of toxin solubilization and trypsin activation are described in Materials and Methods.

A similar study was carried out to analyze the pore-forming activity of Cyt1Aa toxin. In this case, the protein was treated with proteinase K, previously reported to activate Cyt1Aa (31). Solubilized and proteinase K-activated Cyt1Aa toxins were able to induce pore formation in T. paludosa BBMVs (12 FAU), although much more efficiently when proteinase K-treated toxin was used (41 FAU) (Fig. 4B). These results indicate that activation of Cyt1Aa is essential for pore formation. Cry3A toxin, nontoxic to dipteran insects and also used as a negative control, did not produce any calcein leakage with T. paludosa BBMVs. It has been reported that, in mosquitoes, the Cyt1Aa protein synergizes the toxicity of the other Cry toxins in B. thuringiensis subsp. israelensis (42). In this study, we demonstrated that Cyt1Aa is the most toxic protein in T. paludosa and that the insect is not susceptible to Cry11Aa. To analyze whether there is enhanced pore-forming activity in T. paludosa, calcein release experiments with solubilized and proteinase K-activated B. thuringiensis subsp. israelensis IPS82 were also carried out. With the same amounts of total protein used in the assay, solubilized and proteinase K-activated IPS82 caused a higher calcein leakage with T. paludosa BBMVs (53 FAU) than Cyt1A (41 FAU) (Fig. 4C).

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Toxicity tests with single toxins indicated that, of all B. thuringiensis subsp. israelensis toxins, Cyt1A was the most toxic component against leatherjackets (T. paludosa), whereas Cry11 and Cry4 proteins were virtually nontoxic. Low toxicity levels of Cry11A against T. oleracea have been also reported by Feldmann et al. (17), who found that T. oleracea susceptibility to Cry11 was 10,000-fold lower than that for the mosquito A. stephensi, and by Waalwijk et al. (40), who reported that Cry4B toxin is 10 times less toxic against T. oleracea than against A. stephensi. Statistical analysis revealed that Cyt1A has a synergistic effect when combined only with Cry4A and not with Cry4B or Cry11A. The mechanism of this Cyt1A synergism in tipulids is unknown, although it is probably different from that of mosquitoes, for which Cyt1A is nontoxic or only slightly toxic (14, 23) and Cry4 proteins are highly toxic (14). The Cry11Aa protoxin was processed into two fragments of 36 and 32 kDa by a 120-min alkali solubilization (Fig. 2B). The same pattern was also obtained when the protein was treated with trypsin (data not shown). When 50 mM dithiothreitol was added to the same solubilization buffer, a major 65-kDa protein was obtained (17), preventing the proteolysis of the protein. The 36-kDa fragment did not show any binding to the brush border membrane of T. paludosa, whereas binding of the 32-kDa fragment was detected (Fig. 3A). In contrast, for A. aegypti, binding of both fragments has been demonstrated previously (19). In midgut cells of this insect, a 65-kDa glycosylphosphatidylinositol-anchored alkaline phosphatase protein was characterized as a functional receptor of Cry11Aa toxin (18). This and other glycosylphosphatidylinositol-anchored proteins are proposed to be selectively included in lipid rafts (28). For other species susceptible to Cry11Aa, such as A. stephensi and T. oleracea, specific binding of Cry11Aa has also been demonstrated, whereas no binding of the trypsinized protein to BBMVs of these insects was observed (17).

The Cry11A competition experiments with unlabeled toxin resulted in a partial reduction of the 32-kDa fragment binding to T. paludosa BBMVs, an indication of a low specificity of the binding interaction that could be due to the proteolysis of the protein in the solubilization buffer. The lack of toxicity of Cry11Aa against T. paludosa (Fig. 1A) is in accordance with the absence of highly specific binding observed. However, solubilized Cry11Aa and trypsinized Cry11Aa were both able to form pores on T. paludosa BBMVs, although trypsinized Cry11Aa was less efficient than activated Cyt1A (Fig. 4A and B). If in T. paludosa, as in other insects, Cry11Aa interacts with lipid rafts, not only protein receptors but glycolipids that can also bind B. thuringiensis toxins (22) could mediate the interaction that leads to the pore formation.

Many possible explanations may account for the lack of Cry11Aa toxicity against T. paludosa even though it is able to induce pore formation. For instance, T. paludosa gut proteases could not be able to activate the protein properly and gut proteases could be very active and then degrade the toxin very quickly. This theory is supported by the fact that trypsinized Cry11Aa has a lower pore-forming activity than non-trypsin-treated toxin (Fig. 4A).

The principal insecticidal proteins of B. thuringiensis var. israelensis are four major ICPs: Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa. The pore-forming Cry proteins are toxic to dipteran larvae and show no amino acid sequence homology to the Cyt family proteins. The 28-kDa Cyt1Aa protein has a restricted in vivo activity spectrum, although in vitro it exhibits cytolytic activity against a variety of insect and mammalian cells. In mosquitoes, Cyt1A has been proposed to interact directly with membrane lipids, inserting into the membrane and forming pores or destroying the membrane in a detergent-like manner (9).

In this study, it was demonstrated for the first time that Cyt1Aa specifically binds to T. paludosa BBMVs (Fig. 3B and C), which indicates the presence of a Cyt1Aa receptor and an alternative insecticidal mechanism based on a receptor-mediated action, as described for Cry proteins. The toxicity of Cyt1Aa toxin to mosquito larvae is, on average, 1 order of magnitude lower than that of Cry4 or Cry11Aa toxins (12). However, for T. paludosa, Cyt1Aa is the most toxic protein (Fig. 1A). Differences in Cyt1Aa binding between mosquitoes and T. paludosa may account for the toxicity variation. As expected, calcein release experiments demonstrated the pore-forming capacity of Cyt1Aa protein. Proteinase K activation of Cyt1Aa drastically increased the calcein release (Fig. 4B), indicating that activation by gut proteases may be a determinant of the high Cyt1Aa toxicity observed for T. paludosa (Fig. 1A).

It has been reported previously that Cyt1Aa synergizes the mosquitocidal toxicity of Cry11Aa protein (31). All B. thuringiensis subsp. israelensis ICPs together, produced by the IPS82 strain, induced a higher calcein leakage than Cyt1Aa alone, even though the amount of Cyt1Aa in the B. thuringiensis subsp. israelensis IPS82 strain was much smaller than that from the pure Cyt1Aa experiment (Fig. 4B and C). Considering that the amount of total protein was the same for pure Cyt1Aa as for B. thuringiensis subsp. israelensis IPS82, the results are consistent with the toxicity-synergistic effect between the Cyt1Aa and Cry4Aa proteins recorded for T. paludosa (Fig. 1C). Even though less likely, another possible explanation which requires further investigation may be the proteinase K activation of Cry11Aa, inducing the increased pore formation activity observed as illustrated in Fig. 4C.

In contrast to results with mosquitoes, results with T. paludosa show that Cyt1Aa toxin, the major toxicity factor against T. paludosa, binds specifically to BBMVs and induces pore formation in the membrane. The toxin content and composition of commercial B. thuringiensis subsp. israelensis products are optimized so far for use against mosquitoes. New insights into the mode of action of the B. thuringiensis subsp. israelensis toxins in tipulids will promote the development of products better adapted for use against this pest.

 
We gratefully acknowledge financial support from the Deutsche Bundesstiftung Umwelt (DBU project 18965) and financial support of the first author from EU COST Action 862 "Bacterial Toxins for Insect Control" for a scientific mission to the University of Valencia.

We also thank Carolina Rausell for her assistance in calcein release experiments and Brian Federici and Ray Akhurst for providing recombinant B. thuringiensis subsp. israelensis strains.

 
* Corresponding author. Mailing address: Institute for Phytopathology, Department of Biotechnology and Biological Control, Christian Albrechts University, Hermann-Rodewald Str. 9, 24118 Kiel, Germany. Phone: 49 (0)431 880 4864. Fax: 49 (0)431 880 1583. E-mail: ehlers{at}biotec.uni-kiel.de

Published ahead of print on 6 April 2007.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Applied and Environmental Microbiology, June 2007, p. 3623-3629, Vol. 73, No. 11
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