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.     

Binding of Bacillus thuringiensis Cry1Ac toxin to Manduca sexta aminopeptidase-N receptor is not directly related to toxicity

Bacillus thuringiensis Cry1Ac delta-endotoxin specifically binds a 115-kDa aminopeptidase-N purified from Manduca sexta midgut. Cry1Ac domain III mutations were constructed around a putative sugar-binding pocket and binding to purified aminopeptidase-N and brush border membrane vesicles (BBMV) was compared to toxicity. Q509A, R511A, Y513A, and 509-511 (QNR-AAA) eliminated aminopeptidase-N binding and reduced binding to BBMV. However, toxicity decreased no more than two-fold, indicating activity is not directly correlated with aminopeptidase-N binding. Analysis of toxin binding to aminopeptidase-N in M. sexta is therefore insufficient for predicting toxicity. Mutants retained binding, however, to another BBMV site, suggesting alternative receptors may compensate in vivo.     

N-acetylgalactosamine on the putative insect receptor aminopeptidase N is recognised by a site on the domain III lectin-like fold of a Bacillus thuringiensis insecticidal toxin

Binding of the insecticidal Bacillus thuringiensis Cry1Ac toxin to the putative receptor aminopeptidase N is specifically inhibited by N-acetylgalactosamine (GalNAc), suggesting that this toxin recognises GalNAc on the receptor. A possible structural basis for involvement of domain III of the toxin in carbohydrate-mediated receptor recognition was noted in the similarity between the domain III fold of the related toxin Cry3A and a carbohydrate-binding domain in the 1,4-beta-glucanase from Cellulomonas fimi. This possibility was investigated by making selected mutations in domain III of the Cry1Ac delta-endotoxin. Mutagenesis of residues Asn506, Gln509 or Tyr513 resulted in toxins with reduced binding and a slower rate of pore formation in Manduca sexta midgut membrane vesicles compared to the wild-type Cry1Ac. These mutants also showed reduced binding to the 120 kDa Cry1Ac putative receptor aminopeptidase N. Unlike the wild-type toxin, binding of the triple mutant N506D,Q509E,Y513A (Tmut) to M. sexta midgut membrane vesicles could not be inhibited by GalNAc. These data indicate that GalNAc binding is located on domain III of Cry1Ac and therefore support a lectin-like role for this domain. A preliminary analysis of the Cry1Ac crystal structure locates Asn506, Gln509 and Tyr513 in a region on and adjacent to beta-16 in domain III, which has a unique conformation compared to the other known Cry structures. These residues are in a favourable position to interact with either soluble or protein-bound carbohydrate.     

Photorhabdus luminescens toxin-induced permeability change in Manduca sexta and Tenebrio molitor midgut brush border membrane and in unilamellar phospholipid vesicle

Photorhabdus luminescens, a Gram-negative bacterium, secretes a protein toxin (PL toxin) that is toxic to insects. In this study, the effects of the PL toxin on large receptor-free unilamellar phospholipid vesicles (LUVs) of Manduca sexta and on brush border membrane vesicles (BBMVs) of M. sexta and Tenebrio molitor were examined. Cry1Ac served as a positive control in our experiments due to its known channel-forming activity on M. sexta. Voltage clamping assays with dissected midguts of M. sexta and T. molitor clearly showed that both Cry1Ac and PL toxin caused channel formation in the midguts, although channel formation was not detected for T. molitor midguts under Cry1Ac and it was less sensitive to PL toxin than to Cry1Ac for M. sexta midguts. Calcein release experiments showed that both toxins made LUVs (unilamellar lipid vesicles) permeable, and at some concentrations of the toxins such permeabilizing effects were pH-dependent. The lowest concentrations of PL toxin were more than 600-fold and 24-fold lower to induce BBMV permeability of T. molitor and M. sexta than those to induce calcein release from LUVs of M. sexta. These further support that PL toxin is responsible for channel formation in the larvae midguts. The lower concentration to induce permeability in BBMV than in LUV is, probably, attributable to that BBMV has PL toxin receptors that facilitate the toxin to induce permeabilization. Furthermore, our results indicate that the effects of PL toxin on BBMV permeability of M. sexta were not significantly influenced by Gal Nac, but those of Cry1Ac were. This implies that PL toxin and Cry1Ac might use different molecular binding sites in BBMV to cause channel formation.     

Membrane insertion of the Bacillus thuringiensis Cry1Ab toxin: single mutation in domain II block partitioning of the toxin into the brush border membrane

The umbrella and penknife models hypothesize that insecticidal Bacillus thuringiensis Cry toxins partition into the apical membrane of the insect midgut by insertion of only two alpha-helices from domain I of the protein, alpha-helices 4 and 5 in the case of the umbrella model and alpha-helices 5 and 6 in the case of the penknife model. Neither model envisages membrane partitioning by domains II and III. In this study, we present data suggesting that mutations in the domain II residue, F371, affect insertion of the whole toxin into Manduca sexta brush border membrane vesicles (BBMVs). Using steady state fluorescence measurements combined with a proteinase K protection assay, we show that mutants of F371 have lost their ability to insert into the BBMV, even though binding to cadherin is almost unaffected. The study also identifies a difference in partitioning of toxins into artificial lipid vesicles (SUVs) as opposed to native BBMVs. While the F371 mutations block insertion of domains I and II into BBMVs, they only block domain II insertion into SUVs. Bioassay and voltage clamping of midguts also confirm the fluorescence data that the noninserting mutants are nontoxic. Our study leads us to propose that, in contrast to previous models of individual free helices inserting into the membrane, the toxin enters into the membrane as a whole molecule or oligomers of the molecule, wherein the domain II residue F371 has a vital role to play in membrane insertion.     

Selective antagonism to the cadherin BT-R1 interferes with calcium-induced adhesion of epithelial membrane vesicles

BT-R(1) is a member of the cadherin superfamily of proteins and is expressed in the midgut epithelium of Manduca sexta during larval development. Previously, we showed that calcium ions influence the structure and stability of BT-R(1) on brush border membrane vesicles (BBMVs) prepared from M. sexta midgut epithelium. In the present study, the effects of calcium and Cry1Ab toxin, produced by Bacillus thuringiensis, on the adhesive properties of BBMVs were investigated. Addition of calcium to a suspension of BBMVs promoted adhesion and aggregation of the vesicles. Treatment of BBMVs with trypsin or lowering the pH (pH 4.0) of the BBMV suspension abolished calcium-induced vesicle aggregation, whereas treatment with deglycosylating enzymes did not affect the aggregation of vesicles, indicating that adhesion and clustering of BBMVs involves protein-protein interactions. Preincubation of BBMVs with Cry1Ab toxin, which specifically binds to BT-R(1) with high affinity and disrupts the midgut epithelium of M. sexta, caused a 50% decrease in calcium-induced vesicle aggregation. The inhibitory effects of the Cry1Ab toxin on BBMV aggregation was blocked completely when the toxin was preincubated with a peptide containing the toxin-binding site of BT-R(1). Cry3A toxin, which is similar in molecular structure to Cry1Ab but does not bind to BT-R(1) and is not toxic to M. sexta larvae, did not affect BBMV aggregation. The results of this study demonstrate that the adhesive function of BT-R(1) is compromised by the Cry1Ab toxin, which acts as a selective antagonist, and supports the notion that BT-R(1) is critical in preserving the integrity of larval midgut epithelium in M. sexta.     

Specificity of Bacillus thuringiensis delta-endotoxins. Importance of specific receptors on the brush border membrane of the mid-gut of target insects

To study the molecular basis of differences in the insecticidal spectrum of Bacillus thuringienesis delta-endotoxins, we have performed binding studies with three delta-endotoxins on membrane preparations from larval insect mid-gut. Conditions for a standard binding assay were established through a detailed study of the binding of 125I-labeled Bt2 toxin, a recombinant B. thuringiensis delta-endotoxin, to brush border membrane vesicles of Manduca sexta. The toxins tested (Bt2, Bt3 and Bt73 toxins) are about equally toxic to M. sexta but differ in their toxicity against Heliothis virescens. Equilibrium binding studies revealed saturable, high-affinity binding sites on brush border membrane vesicles of M. sexta and H. virescens. While the affinity of the three toxins was not significantly different on H. virescens vesicles, marked differences in binding site concentration were measured which reflected the differences in in vivo toxicity. Competition experiments revealed heterogeneity in binding sites. For H. virescens, a three-site model was proposed. In M. sexta, one population of binding sites is shared by all three toxins, while another is only recognized by Bt3 toxin. Several other toxins, non-toxic or much less toxic to M. sexta than Bt2 toxin, did not or only marginally displace binding of 125I-labeled Bt2 toxin in this insect. No saturable binding of this toxin was observed to membrane preparations from tissues of several non-susceptible organisms. Together, these data provide new evidence that binding to a specific receptor on the membrane of gut epithelial cells is an important determinant with respect to differences in insecticidal spectrum of B. thuringiensis insecticidal crystal proteins.     

Pore formation activity of Cry1Ab toxin from Bacillus thuringiensis in an improved membrane vesicle preparation from Manduca sexta midgut cell microvilli

The pore formation activity of Cry1Ab toxin is analyzed in an improved membrane preparation from apical microvilli structures of Manduca sexta midgut epithelium cells (MEC). A novel methodology is described to isolate MEC and brush border membrane vesicles (BBMV) from purified microvilli structures. The specific enrichment of apical membrane enzyme markers aminopeptidase (APN) and alkaline phosphatase (APh) were 35- and 22-fold, respectively, as compared to the whole midgut cell homogenate. Ligand-blot and Western-blot experiments showed that Cry1A specific receptors were also enriched. The pore formation activity of Cry1Ab toxin was fourfold higher in the microvilli membrane fraction that showed low intrinsic K+ channels and higher APN and APh activities than in the basal-lateral membrane fraction harboring high intrinsic K+ channels. These data suggest that basal-lateral membrane was separated from apical membrane.This procedure should allow more precise studies of the interaction of Cry toxins with their target membranes, avoiding unspecific interaction with other cellular membranes, as well as the study of the pore formation activity induced by Cry toxins in the absence of endogenous channels from M. sexta midgut cells.     

Role of Bacillus thuringiensis Cry1 delta endotoxin binding in determining potency during lepidopteran larval development

Five economically important crop pests, Manduca sexta, Pieris brassicae, Mamestra brassicae, Spodoptera exigua, and Agrotis ipsilon, were tested at two stages of larval development for susceptibility to Bacillus thuringiensis toxins Cry1Ac, Cry1Ca, Cry1J, and Cry1Ba. Bioassay results for M. sexta showed that resistance to all four Cry toxins increased from the neonate stage to the third-instar stage; the increase in resistance was most dramatic for Cry1Ac, the potency of which decreased 37-fold. More subtle increases in resistance during larval development were seen in M. brassicae for Cry1Ca and in P. brassicae for Cry1Ac and Cry1J. By contrast, the sensitivity of S. exigua did not change during development. At both larval stages, A. ipsilon was resistant to all four toxins. Because aminopeptidase N (APN) is a putative Cry1 toxin binding protein, APN activity was measured in neonate and third-instar brush border membrane vesicles (BBMV). With the exception of S. exigua, APN activity was found to be significantly lower in neonates than in third-instar larvae and thus inversely correlated with increased resistance during larval development. The binding characteristics of iodinated Cry1 toxins were determined for neonate and third-instar BBMV. In M. sexta, the increased resistance to Cry1Ac and Cry1Ba during larval development was positively correlated with fewer binding sites in third-instar BBMV than in neonate BBMV. The other species-instar-toxin combinations did not reveal positive correlations between potency and binding characteristics. The correlation between binding and potency was inconsistent for the species-instar-toxin combinations used in this study, reaffirming the complex mode of action of Cry1 toxins.     

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.     

Resistance to Bacillus thuringiensis CryIA delta-endotoxins in a laboratory-selected Heliothis virescens strain is related to receptor alteration.

The Bacillus thuringiensis toxin-binding properties of midgut epithelial cells from two strains of Heliothis virescens were compared. One H. virescens strains (YHD2) which was selected against CryIAc toxin had over 10,000-fold resistance to CryIAc toxin relative to the susceptible strain and was cross-resistant to CryIAa and CryIAb. The second H. virescens strain (YDK) was susceptible to these toxins in the order CryIAc > CryIAb > CryIAa. Receptor-binding properties of CryIAa, CryIAb, and CryIAc toxins were compared between the susceptible and resistant strains. Saturation and competition-binding experiments were performed with brush border membrane vesicles prepared from midguts of the susceptible and resistant insects and 125I-labeled toxins. In the susceptible strain, saturable, specific, and high-affinity binding of all three toxins was observed. The relative binding-site concentration was directly correlated with toxicity (CryIAc > CryIAb > CryIAa). In the resistant strains, the binding affinities of CryIAb and CryIAc were similar to that observed with the susceptible strain and ony minor differences in binding-site concentration (Bmax) were observed. The major difference between the two strains was the total lack of binding of CryIAa toxin to the brush border membrane vesicles of the resistant strain. Heterologous competition-binding experiments and ligand blot analysis supported the hypothesis that there were multiple binding sites for the toxins. On the basis of results of the present study, we propose that alterations in binding proteins shared by all three toxins are a major factor in resistance. This suggests that not all receptors of CryIAc might be involved in toxic function.     

Role of interdomain salt bridges in the pore-forming ability of the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac

The four salt bridges (Asp(222)-Arg(281), Arg(233)-Glu(288), Arg(234)-Glu(274), and Asp(242)-Arg(265)) linking domains I and II in Cry1Aa were abolished individually in alpha-helix 7 mutants D222A, R233A, R234A, and D242A. Two additional mutants targeting the fourth salt bridge (R265A) and the double mutant (D242A/R265A) were rapidly degraded during trypsin activation. Mutations were also introduced in the corresponding Cry1Ac salt bridge (D242E, D242K, D242N, and D242P), but only D242N and D242P could be produced. All toxins tested, except D242A, were shown by light-scattering experiments to permeabilize Manduca sexta larval midgut brush border membrane vesicles. The three active Cry1Aa mutants at pH 10.5, as well as D222A at pH 7.5, demonstrated a faster rate of pore formation than Cry1Aa, suggesting that increases in molecular flexibility due to the removal of a salt bridge facilitated toxin insertion into the membrane. However, all mutants were considerably less toxic to M. sexta larvae than to the respective parental toxins, suggesting that increased flexibility made the toxins more susceptible to proteolysis in the insect midgut. Interdomain salt bridges, especially the Asp(242)-Arg(265) bridge, therefore contribute greatly to the stability of the protein in the larval midgut, whereas their role in intrinsic pore-forming ability is relatively less important.     

Ligand specificity and affinity of BT-R1, the Bacillus thuringiensis Cry1A toxin receptor from Manduca sexta, expressed in mammalian and insect cell cultures.

The Manduca sexta receptor for the Bacillus thuringiensis Cry1Aa, Cry1Ab, and Cry1Ac toxins, BT-R1, has been expressed in heterologous cell culture, and its ligand binding characteristics have been determined. When transfected with the BT-R1 cDNA, insect and mammalian cell cultures produce a binding protein of approximately 195 kDa, in contrast to natural BT-R1 from M. sexia, which has an apparent molecular weight of 210 kDa. Transfection of cultured Spodoptera frugiperda cells with the BT-R1 cDNA imparts Cry1A-specific high-affinity binding activity typical of membranes prepared from larval M. sexta midguts. Competition assays with BT-R1 prepared from larval M. sexta midguts and transiently expressed in cell culture reveal virtually identical affinities for the Cry1Aa, Cry1Ab, and Cry1Ac toxins, clearly demonstrating the absolute specificity of the receptor for toxins of the lepidopteran-specific Cry1A family. BT-R1 therefore remains the only M. sexta Cry1A binding protein to be purified, cloned, and functionally expressed in heterologous cell culture, and for the first time, we are able to correlate the Cry1Aa, Cry1Ab, and Cry1Ac toxin sensitivities of M. sexta to the identity and ligand binding characteristics of a single midgut receptor molecule.     

Binding of the CryIVD Toxin of Bacillus thuringiensis subsp. israelensis to Larval Dipteran Midgut Proteins

Ligand-blotting experiments on dipteran brush border membrane vesicles (BBMVs) showed binding of CryIVD toxin of Bacillus thuringiensis subsp. israelensis to proteins of 148 kDa in Anopheles stephensi and of 78 kDa in Tipula oleracea, both species being susceptible to CryIVD. Binding of CryIVD with BBMVs of A. stephensi resulted in a stronger signal than with BBMVs of T. oleracea. Likewise, larvae of A. stephensi are 10,000-fold more susceptible to the CryIVD toxin than are larvae of T. oleracea. Binding was also found with six proteins ranging in size from 48 to 110 kDa in BBMVs from the lepidopteran species Manduca sexta, but CryIVD was not toxic for M. sexta larvae. No binding of trypsinated CryIVD to BBMV proteins was observed. With the lepidopteran-specific toxin CryIA(b), no binding to dipteran BBMVs was found. Binding of CryIA(b) to nine different BBMV proteins ranging in size from 71 to 240 kDa was observed in M. sexta. The major binding signal was observed with a protein of 240 kDa for CryIA(b).     

Evidence for intermolecular interaction as a necessary step for pore-formation activity and toxicity of Bacillus thuringiensis Cry1Ab toxin

Based on the observation of large conductance states formed by Bacillus thuringiensis Cry toxins in synthetic planar lipid bilayers and the estimation of a pore size of 10-20 A, it has been proposed that the pore could be formed by an oligomer containing four to six Cry toxin monomers. However, there is a lack of information regarding the insertion of Cry toxins into the membrane and oligomer formation. Here we provide direct evidence showing that the intermolecular interaction between Cry1Ab toxin monomers is a necessary step for pore formation and toxicity. Two Cry1Ab mutant proteins affected in different steps of their mode of action (F371A in receptor binding and H168F in pore formation) were affected in toxicity against Manduca sexta larvae. Binding analysis showed that F371A protein bound more efficiently to M. sexta brush border membrane vesicles when mixed with H168F in a one to one ratio. These mutant proteins also recovered pore-formation activity, measured with a fluorescent dye with isolated brush border membrane vesicles, and toxicity against M. sexta larvae when mixed, showing that monomers affected in different steps of their mode of action can form functional hetero-oligomers.     

Specific binding of activated Vip3Aa10 to Helicoverpa armigera brush border membrane vesicles results in pore formation

Helicoverpa armigera is one of the most harmful pests in China. Although it had been successfully controlled by Cry1A toxins, some H. armigera populations are building up resistance to Cry1A toxins in the laboratory. Vip3A, secreted by Bacillus thuringiensis, is another potential toxin against H. armigera. Previous reports showed that activated Vip3A performs its function by inserting into the midgut brush border membrane vesicles (BBMV) of susceptible insects. To further investigate the binding of Vip3A to BBMV of H. armigera, the full-length Vip3Aa10 toxin expressed in Escherichia coli was digested by trypsin or midgut juice extract, respectively. Among the fragments of digested Vip3Aa10, only a 62 kDa fragment (Vip3Aa10-T) exhibited binding to BBMV of H. armigera and has insecticidal activity. Moreover, this interaction was specific and was not affected by the presence of Cry1Ab toxin. Binding of Vip3Aa10-T to BBMV resulted in the formation of an ion channel. Unlike Cry1A toxins, Vip3Aa10-T was just slightly associated with lipid rafts of BBMV. These data suggest that although activated Vip3Aa10 specifically interacts with BBMV of H. armigera and forms an ion channel, the mode of action of it may be different from that of Cry1A toxins.     

Association of Cry1Ac toxin resistance in Helicoverpa zea (Boddie) with increased alkaline phosphatase levels in the midgut lumen

Resistance to Bacillus thuringiensis Cry1Ac toxin was characterized in a population of Helicoverpa zea larvae previously shown not to have an alteration in toxin binding as the primary resistance mechanism to this toxin. Cry1Ac-selected larvae (AR1) were resistant to protoxins and toxins of Cry1Ab, Cry1Ac, and the corresponding modified proteins lacking helix α-1 (Cry1AbMod and Cry1AcMod). When comparing brush border membrane vesicles (BBMVs) prepared from susceptible (LC) and AR1 larval midguts, there were only negligible differences in overall Cry1Ac toxin binding, though AR1 had 18% reversible binding, in contrast to LC, in which all binding was irreversible. However, no differences were detected in Cry1Ac-induced pore formation activity in BBMVs from both strains. Enzymatic activities of two putative Cry1Ac receptors (aminopeptidase N [APN] and alkaline phosphatase [ALP]) were significantly reduced (2-fold and 3-fold, respectively) in BBMVs from AR1 compared to LC larvae. These reductions corresponded to reduced protein levels in midgut luminal contents only in the case of ALP, with an almost 10-fold increase in specific ALP activity in midgut fluids from AR1 compared to LC larvae. Partially purified H. zea ALP bound Cry1Ac toxin in ligand blots and competed with Cry1Ac toxin for BBMV binding. Based on these results, we suggest the existence of at least one mechanism of resistance to Cry1A toxins in H. zea involving binding of Cry1Ac toxin to an ALP receptor in the larval midgut lumen of resistant larvae.     

Role of tryptophan residues in toxicity of Cry1Ab toxin from Bacillus thuringiensis

Bacillus thuringiensis produces insecticidal proteins (Cry protoxins) during the sporulation phase as parasporal crystals. During intoxication, the Cry protoxins must change from insoluble crystals into membrane-inserted toxins which form ionic pores. The structural changes of Cry toxins during oligomerization and insertion into the membrane are still unknown. The Cry1Ab toxin has nine tryptophan residues; seven are located in domain I, the pore-forming domain, and two are located in domain II, which is involved in receptor recognition. Eight Trp residues are highly conserved within the whole family of three-domain Cry proteins, suggesting an essential role for these residues in the structural folding and function of the toxin. In this work, we analyzed the role of Trp residues in the structure and function of Cry1Ab toxin. We replaced the Trp residues with phenylalanine or cysteine using site-directed mutagenesis. Our results show that W65 and W316 are important for insecticidal activity of the toxin since their replacement by Phe reduced the toxicity against Manduca sexta. The presence of hydrophobic residue is important at positions 117, 219, 226, and 455 since replacement by Cys affected either the crystal formation or the insecticidal activity of the toxin in contrast to replacement by Phe in these positions. Additionally, some mutants in positions 219, 316, and 455 were also affected in binding to brush border membrane vesicles (BBMV). This is the first report that studies the role of Trp residues in the activity of Cry toxins.     

Different domains of Bacillus thuringiensis delta-endotoxins can bind to insect midgut membrane proteins on ligand blots.

We investigated the role of the constituent domains of the CryIA(b) and CryIA(c) delta-endotoxins in binding to midgut epithelial cell membrane proteins of Spodoptera exigua and Manduca sexta on ligand blots. A collection of wild-type and CryIC-CryIA hybrid toxins was used for this purpose. As demonstrated elsewhere (R. A. de Maagd, M. S. G. Kwa, H. van der Klei, T. Yamamoto, B. Schipper, J. M. Vlak, W. J. Stiekema, and D. Bosch, Appl. Environ. Microbiol. 62:1537-1543, 1996), CryIA(b) domain III recognized a 205-kDa protein on S. exigua blots, while no specific binding by domain I or II could be detected. In contrast, on ligand blots of M. sexta proteins CryIA(b) domain II recognized a 210-kDa protein and CryIA(b) domain III recognized a 250-kDa protein. Domain III is responsible for the interaction of CryIA(c) with 120-kDa major binding proteins of both S. exigua and M. sexta. In addition, in M. sexta CryIA(c) also reacts with a 210-kDa binding protein through its domain I and/or domain II. These results show that besides domain II, domain III of delta-endotoxins plays a major role in binding to putative receptors on ligand blots. However, for S. exigua there was no clear correlation between binding of toxins on ligand blots and the in vivo toxicity of the toxins. These and previous results suggest that interactions of insect membrane proteins with both domain II and domain III can occur and that detection of these interactions depends on the type of binding assay used.     

Susceptibility of Manduca sexta to Cry1Ab toxin of Bacillus thuringiensis correlates directly to developmental expression of the cadherin receptor BT-R(1)

The cadherin receptor BT-R(1), localized in the midgut epithelium of the tobacco hornworm, Manduca sexta, is coupled to programmed oncotic-like cell death, which is triggered by the univalent binding of the Cry1Ab toxin of Bacillus thuringiensis (Bt) to the receptor. Kinetic analysis of BT-R(1) expression during larval development reveals that the density of BT-R(1) on the midgut surface increases dramatically along with an equivalent rise in the concentration of Cry1Ab toxin molecules needed to kill each of the five larval stages of the insect. The increase in the number of BT-R(1) molecules per midgut surface area requires additional toxin molecules to kill older versus younger larvae, as evidenced by the corresponding LC(50) values. Based on these observations, we developed a mathematical model to quantify both the expression of BT-R(1) and the susceptibility of M. sexta larvae to the Cry1Ab toxin. Interestingly, the toxin-receptor ratio remains constant during larval development regardless of larval size and mass. This ratio apparently is critical for insecticidal activity and the decrease in toxin effectiveness during larval development is due primarily to the number of effective toxins and available receptors in the larval midgut. Evidently, susceptibility of M. sexta to the Cry1Ab toxin of Bt correlates directly to the developmental expression of BT-R(1) in this insect.