Synergistic effect of the Bacillus thuringiensis toxins CryIAa and CryIAc on the gypsy moth, Lymantria dispar.

The insecticidal activity of toxins CryIAa, CryIAb, and CryIAc against Lymantria dispar (gypsy moth) and Bombyx mori (silkworm) was examined by force-feeding bioassays. Toxin CryIAa exhibited higher toxicity than toxins CryIAb and CryIAc for L. dispar and B. mori. To evaluate possible synergism among these toxins, bioassays were performed with mixtures of CryIAa and CryIAb, CryIAb and CryIAc, and CryIAa and CryIAc. Expected toxicity was calculated from the activity of each individual toxin and its proportion in the mixture by using the equation described by Tabashnik (B. E. Tabashnik, Appl. Environ. Microbiol. 58:3343-3346, 1992). Observed 50% growth-inhibitory doses were calculated from mixing experiments by probit analysis. In L. dispar bioassays, synergism was observed with a mixture of CryIAa and CryIAc while a mixture of CryIAa and CryIAb exhibited an antagonistic effect. No synergistic effect on B. mori was observed with any toxin combination. Voltage clamping assays of isolated L. dispar midguts also demonstrated that the mixture of CryIAa and CryIAc induced a greater slope of inhibition of short circuit current than did other toxin combinations.     

Toxicity of Bacillus thuringiensis Spore and Crystal Protein to Resistant Diamondback Moth (Plutella xylostella)

A colony of Plutella xylostella from crucifer fields in Florida was used in mortality bioassays with HD-1 spore, CryIA(a), CryIA(b), CryIA(c), CryIB, CryIC, CryID, CryIE, or CryIIA. The data revealed high levels of field-evolved resistance to HD-1 spore and all CryIA protoxins and no resistance to CryIB, CryIC, or CryID. CryIE and CryIIA were essentially not toxic. When HD-1 spore was combined 1:1 with protoxin and fed to susceptible larvae, spore synergized the activity of CryIA and CryIC 5- to 8-fold and 1.7-fold, respectively, and did not synergize the mortality of CryIIA. When fed to Florida larvae, spore failed to synergize the activity of all three CryIA protoxins, synergized the activity of CryIC 5.3-fold, and did not synergize the mortality for CryIIA. Binding studies with CryIA(b), CryIB, and CryIC were performed to determine possible mechanisms of resistance. The two techniques used were (i) binding of biotinylated toxin to tissue sections of larval midguts and (ii) binding of biotinylated toxin to brush border membrane vesicles prepared from whole larvae. Both showed dramatically reduced binding of CryIA(b) in resistant larvae compared with that in susceptible larvae but no differences in binding of CryIB or CryIC.     

Cry9Ca1 Toxin, a Bacillus thuringiensis Insecticidal Crystal Protein with High Activity against the Spruce Budworm (Choristoneura fumiferana)

The Cry9Ca1 toxin from Bacillus thuringiensis was significantly more toxic to spruce budworm (Choristoneura fumiferana) than the Cry1Ab6, Cry1Ba1, Cry1Ca2, Cry1Da1, Cry1Ea1, and Cry1Fa2 toxins. It displayed high activity against silkworm (Bombyx mori) but was not toxic to black army cutworm (Actebia fennica) or gypsy moth (Lymantria dispar). The Cry9Ca1 is the most effective spruce budworm toxin known to date and may offer promise for control and resistance management of that species.     

Domain III substitution in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition.

To test our hypothesis that substitution of domain III of Bacillus thuringiensis delta-endotoxin (Cry) proteins might improve toxicity to pest insects, e.g., Spodoptera exigua, in vivo recombination was used to produce a number of cryIA(b)-cryIC hybrid genes. A rapid screening assay was subsequently exploited to select hybrid genes encoding soluble protoxins. Screening of 120 recombinants yielded two different hybrid genes encoding soluble proteins with domains I and II of CryIA(b) and domain III of CryIC. These proteins differed by only one amino acid residue. Both hybrid protoxins gave a protease-resistant toxin upon in vitro activation by trypsin. Bioassays showed that one of these CryIA(b)-CryIC hybrid proteins (H04) was highly toxic to S. exigua compared with the parental CryIA(b) protein and significantly more toxic than CryIC. In semiquantitative binding studies with biotin-labelled toxins and intact brush border membrane vesicles of S. exigua, this domain III substitution appeared not to affect binding-site specificity. However, binding to a 200-kDa protein by CryIA(b) in preparations of solubilized and blotted brush border membrane vesicle proteins was completely abolished by the domain III substitution. A reciprocal hybrid containing domains I and II of CryIC and domain III of CryIA(b) did bind to the 200-kDa protein, confirming that domain III of CryIA(b) was essential for this reaction. These results show that domain III of CryIC protein plays an important role in the level of toxicity to S. exigua, that substitution of domain III may be a powerful tool to increase the repertoire of available active toxins for pest insects, and that domain III is involved in binding to gut epithelium membrane proteins of S. exigua.     

Location of a Bombyx mori receptor binding region on a Bacillus thuringiensis delta-endotoxin.

Receptor binding studies were performed with 125I-labeled trypsin-activated insecticidal toxins, CryIA(a) and CryIA(c), from Bacillus thuringiensis on brush-border membrane vesicles (BBMV) prepared from Bombyx mori larval midgut. Bioassays were performed by gently force feeding B. mori with diluted toxins. CryIA(a) toxin (LD50; 0.002 micrograms) was 200 times more active against B. mori larvae than CryIA(c) toxin (LD50; 0.421 micrograms) and showed high-affinity saturable binding. The Kd and the binding site concentration for CryIA(a) toxin were 3.5 nM and 7.95 pmol/mg, respectively. CryIA(c) toxin (Kd, 50.35 nM; Bmax, 2.85 pmol/mg) did not demonstrate high-affinity binding to B. mori BBMV. Control experiments with CryIA(a) and CryIA(c) toxins revealed no binding to mouse small intestine BBMV and nonspecific binding to pig kidney BBMV. These data provide evidence that binding to a specific receptor on the membrane of midgut epithelial cells is an important determinant with respect to differences in insecticidal spectrum of insecticidal crystal proteins. To locate a B. mori receptor binding region on the CryIA(a) toxin, homologous and heterologous competition binding studies were performed with a set of mutant proteins which had previously been used to define the B. mori "specificity domain" on this toxin (Ge, A. Z., Shivarova, N. I., and Dean, D. H. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4037-4041). These mutant proteins have had regions of their genes reciprocally exchanged with the cryIA(c) gene. A B. mori receptor binding region on CryIA(a) toxin includes the amino-terminal portion of the hypervariable region, amino acids 332-450, which is identical to the previously described B. mori specificity determining region. These data provide direct evidence that delta-endotoxins contain a tract of amino acids that comprise a binding region and as a results determines the specificity of a toxin.     

Aminopeptidase N purified from gypsy moth brush border membrane vesicles is a specific receptor for Bacillus thuringiensis CryIAc toxin.

We have evaluated the binding of Bacillus thuringiensis Cry toxins to aminopeptidase N (APN) purified from Lymantria dispar (gypsy moth) brush border membrane vesicle (BBMV). CryIAc toxin bound strongly to APN, while either the structurally related CryIAa and CryIAb toxins or CryIC, CryIIA, and CryIIIA toxins showed weak binding to APN. An in vitro competition binding study demonstrated that the binding of CryIAc to L. dispar BBMV was inhibited by APN. Inhibition of short circuit current for CryIAc, measured by voltage clamping of whole L. dispar midgut, was substantially reduced by addition of phosphatidylinositol-specific phospholipase C, which is known to release APN from the midgut membrane. In contrast, addition of phosphatidylinositol-specific phospholipase C had only a marginal effect on the inhibition of short circuit current for CryIAa. These data suggest that APN is the major functional receptor for CryIAc in L. dispar BBMV. A ligand blotting experiment demonstrated that CryIAc recognized a 120-kDa peptide (APN), while CryIAa and CryIAb recognized a 210-kDa molecule in L. dispar BBMV. In contrast, CryIAa and CryIAb bound to both the 120- and 210-kDa molecules in Manduca sexta BBMV, while CryIAc recognized only the 120-kDa peptide. The 120-kDa peptide (APN) in L. dispar BBMV reacted with soybean agglutinin, indicating that N-acetylgalactosamine is a component of this glycoprotein.     

Identification of putative insect brush border membrane-binding molecules specific to Bacillus thuringiensis delta-endotoxin by protein blot analysis.

Binding sites for insecticidal toxins of Bacillus thuringiensis are located in the brush border membranes of insect midguts. Two approaches were used to investigate the interactions of B. thuringiensis subsp. kurstaki HD-73 CryIA(c) toxin with brush border membrane vesicles from sensitive and naturally resistant insects: 125I-toxin-vesicle binding assays and protein blots probed with 125I-CryIA(c) toxin. In bioassays, Manduca sexta and Heliothis virescens larvae were highly sensitive, Helicoverpa zea larvae were moderately sensitive, and Spodoptera frugiperda larvae were resistant to CryIA(c) toxin. Studies of binding of 125I-CryIA(c) toxin to brush border membrane vesicles from the larval midguts revealed that all insects tested had high-affinity, saturable binding sites. Significantly, S. frugiperda larvae bind but are not killed by CryIA(c) toxin. Labeled CryIA(c) toxin incubated with protein blots identifies a major binding molecule of 120 kDa for M. sexta and 148 kDa for S. frugiperda. H. virescens and H. zea are more complex, containing 155-, 120-, 103-, 90-, and 63-kDa proteins as putative toxin-binding molecules. H. virescens also contains a minor toxin-binding protein of 81 kDa. These experiments provide information that can be applied toward a more detailed characterization of B. thuringiensis toxin-binding proteins.     

Identification of putative insect brush border membrane-binding molecules specific to Bacillus thuringiensis delta-endotoxin by protein blot analysis.

Binding sites for insecticidal toxins of Bacillus thuringiensis are located in the brush border membranes of insect midguts. Two approaches were used to investigate the interactions of B. thuringiensis subsp. kurstaki HD-73 CryIA(c) toxin with brush border membrane vesicles from sensitive and naturally resistant insects: 125I-toxin-vesicle binding assays and protein blots probed with 125I-CryIA(c) toxin. In bioassays, Manduca sexta and Heliothis virescens larvae were highly sensitive, Helicoverpa zea larvae were moderately sensitive, and Spodoptera frugiperda larvae were resistant to CryIA(c) toxin. Studies of binding of 125I-CryIA(c) toxin to brush border membrane vesicles from the larval midguts revealed that all insects tested had high-affinity, saturable binding sites. Significantly, S. frugiperda larvae bind but are not killed by CryIA(c) toxin. Labeled CryIA(c) toxin incubated with protein blots identifies a major binding molecule of 120 kDa for M. sexta and 148 kDa for S. frugiperda. H. virescens and H. zea are more complex, containing 155-, 120-, 103-, 90-, and 63-kDa proteins as putative toxin-binding molecules. H. virescens also contains a minor toxin-binding protein of 81 kDa. These experiments provide information that can be applied toward a more detailed characterization of B. thuringiensis toxin-binding proteins.     

Resistance to Toxins from Bacillus thuringiensis subsp. kurstaki Causes Minimal Cross-Resistance to B. thuringiensis subsp. aizawai in the Diamondback Moth (Lepidoptera: Plutellidae)

Repeated exposure in the field followed by laboratory selection produced 1,800- to >6,800-fold resistance to formulations of Bacillus thuringiensis subsp. kurstaki in larvae of the diamondback moth, Plutella xylostella. Four toxins from B. thuringiensis subsp. kurstaki [CryIA(a), CryIA(b), CryIA(c), and CryIIA] caused significantly less mortality in resistant larvae than in susceptible larvae. Resistance to B. thuringiensis subsp. kurstaki formulations and toxins did not affect the response to CryIC toxin from B. thuringiensis subsp. aizawai. Larvae resistant to B. thuringiensis subsp. kurstaki showed threefold cross-resistance to formulations of B. thuringiensis subsp. aizawai containing CryIC and CryIA toxins. This minimal cross-resistance may be caused by resistance to CryIA toxins shared by B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai.     

Specificity of Activated CryIA Proteins from Bacillus thuringiensis subsp. kurstaki HD-1 for Defoliating Forest Lepidoptera

The insecticidal activity of the CryIA(a), CryIA(b), and CryIA(c) toxins from Bacillus thuringiensis subsp. kurstaki HD-1 was determined in force-feeding experiments with larvae of Choristoneura fumiferana, C. occidentalis, C. pinus, Lymantria dispar, Orgyia leucostigma, Malacosoma disstria, and Actebia fennica. The toxins were obtained from cloned protoxin genes expressed in Escherichia coli. The protoxins were activated with gut juice from Bombyx mori larvae. Biological activity of the individual gene products as well as the native HD-1 toxin was assessed as the dose which prevented 50% of the insects from producing frass within 3 days (frass failure dose [FFD₅₀]). The three toxins were about equally active against M. disstria. In the Choristoneura species, CryIA(a) and CryIA(b) were up to fivefold more toxic than CryIA(c). In the lymantriid species, CryIA(a) and CryIA(b) were up to 100-fold more toxic than CryIA(c). The toxicity of HD-1 was similar to that of the individual CryIA(a) or CryIA(b) toxins in all of these species. None of the CryIA toxins or HD-1 exhibited and toxicity towards A. fennica. Comparison of the observed FFD₅₀ of HD-1 with the FFD₅₀ expected on the basis of its crystal composition suggested a possible synergistic effect of the toxins in the two lymantriid species. Our results further illustrate the diversity of activity spectra of these highly related proteins and provide a data base for studies with forest insects to elucidate the molecular basis of toxin specificity.     

Comparison of the response of midgut epithelial cells and cell lines from lepidopteran larvae to CryIA toxins from Bacillus thuringiensis

The cytotoxic responses of midgut epithelial cells (MEC) from spruce budworm (SBW), gypsy moth (GM) and silkworm (SW) larvae were compared with the cytotoxic response of lepidopteran cell lines (SF-9, SE-1a, and CF-1) to CryIA toxins from Bacillus thuringiensis. The MEC from SBW, SW and GM had binding proteins for CryIA(a,b,c) toxins, whereas the lepidopteran cell lines had binding proteins for CryIA(c). Single MEC exposed to CryIA(a,b,c) toxins in a qualitative lawn assay were equally susceptible to the toxins with a threshold response at about 1ng. The cell lines were not susceptible to CryIA(a,b) toxins in the dose range tested, but had threshold responses for CryIA(c) of 3.4ng for SF-9, 50.2ng for SE-1a and 5.9ng for CF-1. In the quantitative Live/Dead assay, MEC were equally susceptible to CryIA(a,b,c) toxins with a threshold effect at about 1ng and a maximum effect at about 10ng. CF-1 was most sensitive to CryIA(c) with a threshold effect at 0.39ng and a maximal effect at about 1ng. In contrast, a 25-50 times greater dose of CryIA(a) or CryIA(b) was required to elicit a similar response as CryIA(c) for CF-1. SF-9 and SE-1a were most susceptible to CryIA(c) with a threshold effect observed at about 0.5ng and maximal effects at about 2ng. SF-9 cells have a threshold and maximum response to CryIA(a,b) of about 10ng and 20ng, respectively. SE-1a cells have a threshold and maximal response to CryIA(a,b) of 5ng and 10ng, respectively. Intact midgut epithelium exposed to CryIA(a,b,c) toxins had a threshold dose of 2ng for CryIA(b), 10-30ng for CryIA(a) and 2-30ng for CryIA(c). This study has shown that MEC are affected by a broader spectrum of toxins compared to the lepidopteran larvae and insect cell lines.     

Brush border membrane aminopeptidase-n in the midgut of the gypsy moth serves as the receptor for the CryIA(c) δ-endotoxin of Bacillus thuringiensis

Aminopeptidase-N (AP-N) was purified from gypsy moth (Lymantria dispar, L.) brush border membrane vesicles (BBMV) proteins by mono-Q chromatography and Superdex-75 gel filtration in the presence of the zwitterionic detergent, CHAPS, using FPLC. The purified AP-N, identified by its enzymatic activity, had an apparent size of 100 kDa, and was identified as the unique Bacillus thuringiensis insecticidal toxin, CryIA(c), binding protein. AP-N clearly displayed strong binding to CryIA(c), exhibiting little or no binding to CryIA(a) or CryIA(b), and showing no binding for the coleopteran-specific toxin, CryIIIA. Protein blots of the BBMV proteins probed with biotin-labeled and ¹²⁵I-labeled insecticidal proteins revealed that CryIAc binds only to 120 kDa protein which is a slightly larger size in comparison to purified AP-N. Antibodies raised against the gypsy moth AP-N demonstrated that the purified AP-N and the 120 kDa CryIA(c) binding protein of total BBMV proteins are antigenically identical.     

Two Different Bacillus thuringiensis Delta-Endotoxin Receptors in the Midgut Brush Border Membrane of the European Corn Borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae)

Binding of three Bacillus thuringiensis insecticidal crystal proteins (ICPs) to the midgut epithelium of Ostrinia nubilalis larvae was characterized by performing binding experiments with both isolated brush border membrane vesicles and gut tissue sections. Our results demonstrate that two independent ICP receptors are present in the brush border of O. nubilalis gut epithelium. From competition binding experiments performed with I-labeled and native ICPs it was concluded that CryIA(b) and CryIA(c) are recognized by the same receptor. An 11-fold-higher binding affinity of CryIA(b) for this receptor correlated with a 10-fold-higher toxicity of this ICP compared with CryIA(c). The CryIB toxin did not compete for the binding site of CryIA(b) and CryIA(c). Immunological detection of ingested B. thuringiensis ICPs on gut sections of O. nubilalis larvae revealed binding only along the epithelial brush border membrane. CryID and CryIE, two ICPs that are not toxic to O. nubilalis, were not bound to the apical microvilli of gut epithelial cells. In vitro binding experiments performed with native and biotinylated ICPs on tissue sections confirmed the correlation between ICP binding and toxicity. Moreover, by performing heterologous competition experiments with biotinylated and native ICPs, it was confirmed that the CryIB receptor is different from the receptor for CryIA(b) and CryIA(c). Retention of activated crystal proteins by the peritrophic membrane was not correlated with toxicity. Furthermore, it was demonstrated that CryIA(b), CryIA(c), and CryIB toxins interact in vitro with the epithelial microvilli of Malpighian tubules. In addition, CryIA(c) toxin also adheres to the basement membrane of the midgut epithelium.     

Processing of delta-endotoxin from Bacillus thuringiensis subsp. kurstaki HD-1 and HD-73 by gut juices of various insect larvae.

Midgut juices were prepared from Adoxophyes sp., smaller tea tortrix (STT); Bombyx mori, silkworm (SW); Spodoptera litura, common cutworm (CCW); Plutella xylostella, diamondback moth (DBM); and Musca domestica, housefly (HF) and immobilized onto Sepharose 4B. delta-Endotoxins (ICPs) from Bacillus thuringiensis subsp. kurstaki HD-1 and HD-73 were digested by these immobilized gut juice proteases. All gut juices tested derived relatively proteolytic resistant cores from ICP. The molecular sizes of these cores, about 55 kDa in SDS-PAGE, were resulted. In the case of CCW, however, digestion was very strong and only 1/20 concentration of core protein remained relative to other digests. The N-terminal amino acid sequencing of the core proteins showed that they were truncated at the very end of the N-terminus of protoxin, CryIA, at different sites. Although housefly larvae were completely insensitive to active toxin, the gut juice produced the core, suggesting that the housefly may lack the binding sites for the core-active toxin.     

Removal of Adsorbed Toxin Fragments That Modify Bacillus thuringiensis CryIC delta-Endotoxin Iodination and Binding by Sodium Dodecyl Sulfate Treatment and Renaturation

We report that 10- and 25-kDa toxin fragments adhere to CryIC prepared from Bacillus thuringiensis insecticidal crystals, block iodination, and alter membrane binding. There is no apparent affect on CryIC toxicity against Spodoptera exigua. Associated peptides remained bound to CryIC in the presence of 50 mM dithiothreitol or 6 M urea. A novel detergent-renaturation procedure was developed for the purification of B. thuringiensis CryIC toxin. Sodium dodecyl sulfate (SDS) treatment followed by gel filtration chromatography yielded a homogeneous 62-kDa CryIC toxin. After removal of SDS and renaturation, the purified CryIC toxin was fully insecticidal to S. exigua larvae. I-labeled CryIC bound with high affinity to brush border membrane vesicles from S. exigua larvae.     

Mosquitocidal activity of the CryIC delta-endotoxin from Bacillus thuringiensis subsp. aizawai.

The cloned 135-kDa CryIC delta-endotoxin from Bacillus thuringiensis is a lepidopteran-active toxin, displaying high activity in vivo against Spodoptera litoralis and Spodoptera frugiperda larvae and in vitro against the S. frugiperda Sf9 cell line. Here, we report that the CryIC delta-endotoxin cloned from B. thuringienesis subsp. aizawai HD-229 and expressed in an acrystalliferous B. thuringiensis strain is also toxic to Aedes aegypti, Anophles gambiae, and Culex quinquefasciatus mosquito larvae. Furthermore, when solubilized and proteolytically activated by insect gut extracts, CryIC is cytotoxic to cell lines derived from the first two of these dipteran insects. This activity was not observed for two other lepidopteran-active delta-endotoxins, CryIA(a) and CryIA(c). However, in contrast to the case with a lepidopteran and dipteran delta-endotoxin cloned from B. thuringiensis subsp. aizawai IC1 (M.Z. Haider, B. H. Knowles, and D. J. Ellar, Eur. J. Biochem. 156:531-540, 1986), no differences in the in vitro specificity or processing of CryIC were found when it was activated by lepidopteran or dipteran gut extract. The recombinant CryIC delta-endotoxin expressed in Escherichia coli was also toxic to A. aegypti larvae. By contrast, a second cryIC gene cloned from B. thuringiensis subsp. aizawai 7.29 (V. Sanchis, D. Lereclus, G. Menou, J. Chaufaux, S. Guo, and M. M. Lecadet, Mol. Microbiol. 3:229-238, 1989) was nontoxic. DNA sequencing showed that the two genes were identical. However, CryIC from B. thuringiensis subsp. aizawai 7.29 had been cloned with a truncated C terminus, and when it was compared with the full-length CryIC delta-endotoxin, it was found to be insoluble under alkaline reducing conditions. These results show that CryIC from B. thuringiensis subsp. aizawai is a dually active delta-endotoxin.     

Removal of Adsorbed Toxin Fragments That Modify Bacillus thuringiensis CryIC δ-Endotoxin Iodination and Binding by Sodium Dodecyl Sulfate Treatment and Renaturation

We report that 10- and 25-kDa toxin fragments adhere to CryIC prepared from Bacillus thuringiensis insecticidal crystals, block iodination, and alter membrane binding. There is no apparent affect on CryIC toxicity against Spodoptera exigua. Associated peptides remained bound to CryIC in the presence of 50 mM dithiothreitol or 6 M urea. A novel detergent-renaturation procedure was developed for the purification of B. thuringiensis CryIC toxin. Sodium dodecyl sulfate (SDS) treatment followed by gel filtration chromatography yielded a homogeneous 62-kDa CryIC toxin. After removal of SDS and renaturation, the purified CryIC toxin was fully insecticidal to S. exigua larvae. ¹²⁵I-labeled CryIC bound with high affinity to brush border membrane vesicles from S. exigua larvae.     

Ionic permeabilities induced by Bacillus thuringiensis in Sf9 cells

The effect of Bacillus thuringiensis insecticidal toxins on the monovalent cation content and intracellular pH (pH _i ) of individual Sf9 cells of the lepidopteran species Spodoptera frugiperda (fall armyworm) was monitored with the fluorescent indicators potassium-binding benzofuran isophthalate (PBFI) and 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The sequential removal of K⁺ and Na⁺ from the medium, in the presence of CryIC, a toxin which is highly active against Sf9 cells, caused sharp shifts in the fluorescence ratio of PBFI, demonstrating a rapid efflux of these ions. In Sf9 cells, pH _i depends strongly on the activity of a K⁺/H⁺ exchanger. In the absence of toxin, removal of K⁺ from the external medium resulted in a reversible acidification of the cells. In the presence of CryIC, pH _i equilibrated rapidly with that of the bathing solution. This effect was both time- and concentration-dependent. In contrast with CryIC, CryIIIA, a coleopteran-specific toxin, and CryIA(a), CryIA(b) and CryIA(c), toxins which are either inactive or poorly active against Sf9 cells, had no detectable effect on pH _i . B. thuringiensis endotoxins thus appear to act specifically by increasing the permeability of the cytoplasmic membrane of susceptible cells to at least H⁺, K⁺ and Na⁺.     

Bivalent sequential binding model of a Bacillus thuringiensis toxin to gypsy moth aminopeptidase N receptor

Specificity for target insects of Bacillus thuringiensis insecticidal Cry toxins is largely determined by toxin affinity for insect midgut receptors. The mode of binding for one such toxin-receptor complex was investigated by extensive toxin mutagenesis, followed by real-time receptor binding analysis using an optical biosensor (BIAcore). Wild-type Cry1Ac, a three-domain, lepidopteran-specific toxin, bound purified gypsy moth (Lymantria dispar) aminopeptidase N (APN) biphasically. Site 1 displayed fast association and dissociation kinetics, while site 2 possessed slower kinetics, yet tighter affinity. We empirically determined that two Cry1Ac surface regions are involved in in vivo toxicity and APN binding. Mutations within domain III affected binding rates to APN site 1, whereas mutations in domain II affected binding rates to APN site 2. Furthermore, domain III contact is completely inhibited in the presence of N-acetylgalactosamine, indicating loss of domain III binding eliminates all APN binding. Based upon these observations, the following model is proposed. A cavity in lectin-like domain III initiates docking through recognition of an N-acetylgalactosamine moiety on L. dispar APN. Following primary docking, a higher affinity domain II binding mechanism occurs, which is critical for insecticidal activity.     

N-acetyl galactosamine is part of the receptor in insect gut epithelia that recognizes an insecticidal protein from Bacillus thuringiensis.

Proteins synthesized by the bacterium Bacillus thuringiensis are potent insecticides. When ingested by susceptible larvae they rapidly lyse epithelial cells lining the midgut. In vitro the toxins lyse certain insect cell lines and show saturable, high-affinity binding to brush-border membrane vesicles (BBMVs) prepared from insect midguts. We observed that the sugar N-acetyl galactosamine (GalNAc) specifically decreased the cytolytic activity of a CryIA(c) toxin towards Choristoneura fumiferana CF1 cells, completely abolished toxin binding to Manduca sexia BBMVs, partially inhibited binding to Heliothis virescens BBMVs and had no apparent effect on binding to Pieris brassicae BBMVs. In ligand blotting experiments the toxin bound proteins of 120 kDa in M. sexta, 125 kDa in P. brassicae and numerous proteins in H. zea. Toxin binding to these proteins was specifically inhibited by GalNAc. The toxin binding proteins of M. sexta and H. zea also bound the lectin soybean agglutinin. Taken together these findings suggest that N-acetyl galactosamine might be a component of a CryIA(c) toxin receptor of CF1 cells and of at least two of the insects tested.