Reduction of Bacillus thuringiensis Cry1Ac toxicity against Helicoverpa armigera by a soluble toxin-binding cadherin fragment

A cadherin-like protein has been identified as a putative receptor for Bacillus thuringiensis (Bt) Cry1Ac toxin in Helicoverpa armigera and plays a key role in Bt insecticidal action. In this study, we produced a fragment from this H. armigera Cry1Ac toxin-binding cadherin that included the predicted toxin-binding region. Binding of Cry1Ac toxin to this cadherin fragment facilitated the formation of a 250-kDa toxin oligomer. The cadherin fragment was evaluated for its effect on Cry1Ac toxin-binding and toxicity by ligand blotting, binding assays, and bioassays. The results of ligand blotting and binding assays revealed that the binding of Cry1Ac to H. armigera midgut epithelial cells was reduced under denaturing or native conditions in vitro. Bioassay results indicated that toxicities from Cry1Ac protoxin or activated toxin were reduced in vivo by the H. armigera cadherin fragment. The addition of the cadherin fragment had no effect on Cry2Ab toxicity.     

Cry1B and Cry3A are active against Hypothenemus hampei Ferrari (Coleoptera: Scolytidae)

Cry1B and Cry3 proteins from Bacillus thuringiensis are toxic to beetles such as the colorado potato beetle and the cottonwood leaf beetle. We report the development of a suitable rearing, bioassay method and the toxicity of these Cry proteins to coffee berry borer first instar larvae.     

Loop residues of the receptor binding domain of Bacillus thuringiensis Cry11Ba toxin are important for mosquitocidal activity

Using a Cry11Ba toxin model, predicted loops in domain II were analyzed for their role in receptor binding and toxicity. Peptides corresponding to loops α8, 1 and 3, but not loop 2, competed with toxin binding to Aedes midgut membranes. Mutagenesis data reveal loops α8, 1 and 3 are involved in toxicity. Loops 1 and 3 are of greater significance in toxicity to Aedes and Culex larvae than to Anopheles. Cry11Ba binds the apical membrane of larval caecae and posterior midgut, and binding can be competed by loop 1 but not by loop 2 peptides. Cry11Ba binds the same regions to which anti-cadherin antibody binds, and this antibody competes with Cry11Ba binding suggesting a possible role of cadherin in toxication.     

The antimicrobial activity of the appetite peptide hormone ghrelin

The present study examined the antimicrobial activity of the peptide ghrelin. Both major forms of ghrelin, acylated ghrelin (AG) and desacylated ghrelin (DAG), demonstrated the same degree of bactericidal activity against Gram-negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), while bactericidal effects against Gram-positive Staphylococcus aureus (S. aureus) and Enterococcus faecalis (E. faecalis) were minimal or absent, respectively. To elucidate the bactericidal mechanism of AG and DAG against bacteria, we monitored the effect of the cationic peptides on the zeta potential of E. coli. Our results show that AG and DAG similarly quenched the negative surface charge of E. coli, suggesting that ghrelin-mediated bactericidal effects are influenced by charge-dependent binding and not by acyl modification. Like most cationic antimicrobial peptides (CAMPs), we also found that the antibacterial activity of AG was attenuated in physiological NaCl concentration (150mM). Nonetheless, these findings indicate that both AG and DAG can act as CAMPs against Gram-negative bacteria.     

Proteolytic processing of Bacillus thuringiensis toxin Cry1Ab in rice brown planthopper, Nilaparvata lugens (Stål)

To understand the low toxicity of Cry toxins in planthoppers, proteolytic activation of Cry1Ab in Nilaparvata lugens was studied. The proteolytic processing of Cry1Ab protoxin by N. lugens midgut proteases was similar to that by trypsin activated Cry1Ab. The Cry1Ab processed with N. lugens midgut proteases was highly insecticidal against Plutella xylostella. However, Cry1Ab activated either by trypsin or the gut proteases of the brown planthopper showed low toxicity in N. lugens. Binding analysis showed that activated Cry1Ab bound to brush border membrane vesicles (BBMV) from N. lugens at a significantly lower level than to BBMV from P. xylostella.     

Characterization and expression of cry4Cb1 and cry30Ga1 from Bacillus thuringiensis strain HS18-1

We characterized a novel Bacillus thuringiensis isolate native to China (HS18-1) that shows a spherical crystal harboring two major proteins of about 70 and 130 kDa, and contains three novel cry genes (cry4Cb1, cry30Ga1, cry54-type). Furthermore, the cry4Cb1 and cry30Ga1 genes were expressed in Escherichia coli BL21 (DE3): pLysS. Insecticidal activity tests showed that the cry4Cb1 protein exhibited larvicidal activity against Aedes aegypti (Diptera) and the cry30Ga1 protein was toxic to both A. aegypti and P. xylostella (Lepidoptera).     

Antisera-mediated in vivo reduction of Cry1Ac toxicity in Helicoverpa armigera

A functional assessment of Bacillus thuringiensis (Bt) toxin receptors in the midgut of lepidopteran insects will facilitate understanding of the toxin mode of action and provide effective strategies to counter the development of resistance. In this study, we produced anti-aminopeptidase (APN) and anti-cadherin sera with purified Cry1Ac toxin-binding APN or cadherin fragments from Heliocoverpa armigera. Antisera were evaluated for their effects on Cry1Ac toxicity through bioassays. Our results indicated that both the anti-APN and anti-cadherin sera reduced Cry1Ac toxicity in vivo, although cadherin antiserum reduced toxicity more than APN antiserum. These results suggest that both APN and cadherin are involved in Cry1Ac intoxication of H. armigera, evidence that the pore formation model may be representative of Cry1Ac toxin mode of action in this insect.     

Binding of Bacillus thuringiensis subsp. israelensis Cry4Ba to Cyt1Aa has an important role in synergism

Bacillus thuringiensis subsp. israelensis (Bti) produces at least four different crystal proteins that are specifically toxic to different mosquito species and that belong to two non-related family of toxins, Cry and Cyt named Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa. Cyt1Aa enhances the activity of Cry4Aa, Cry4Ba or Cry11Aa and overcomes resistance of Culex quinquefasciatus populations resistant to Cry11Aa, Cry4Aa or Cry4Ba. Cyt1Aa synergized Cry11Aa by their specific interaction since single point mutants on both Cyt1Aa and Cry11Aa that affected their binding interaction affected their synergistic insecticidal activity. In this work we show that Cyt1Aa loop β6-αE K198A, E204A and β7 K225A mutants affected binding and synergism with Cry4Ba. In addition, site directed mutagenesis showed that Cry4Ba domain II loop α-8 is involved in binding and in synergism with Cyt1Aa since Cry4Ba SI303-304AA double mutant showed decreased binding and synergism with Cyt1Aa. These data suggest that similarly to the synergism between Cry11Aa and Cyt1Aa toxins, the Cyt1Aa also functions as a receptor for Cry4Ba explaining the mechanism of synergism between these two Bti toxins.     

Comparative study of Bacillus thuringiensis Cry1Ia and Cry1Aa delta-endotoxins: Activation process and toxicity against Prays oleae

Cry1Ia and Cry1Aa proteins exhibited toxicities against Prays oleae with LC₅₀ of 189 and 116 ng/cm², respectively. The ability to process Cry1Ia11 protoxin by trypsin, chymotrypsin and P. oleae larvae proteases was studied and compared to that of Cry1Aa11. After solubilization under high alkaline condition (50 mM NaOH), Cry1Aa11 was converted into a major fragment of 65 kDa, whereas Cry1Ia11 protoxin was completely degraded by P. oleae larvae proteases and trypsin and converted into a major fragment of 70 kDa by chymotrypsin. Using less proteases of P. oleae juice, the degradation of Cry1Ia11 was attenuated. When the solubilization (in 50 mM Na₂CO₃ pH 10.5 buffer) and activation were combined, Cry1Ia11 was converted into a proteolytic product of 70 kDa after 3 h of incubation with trypsin, chymotrypsin and P. oleae juice. These results suggest that the in vivo solubilization of Cry1Ia11 was assured by larval proteases after a swelling of the corresponding inclusion due to the alkalinity of the larval midgut.     

Bacillus thuringiensis delta-endotoxin Cry1Ac domain III enhances activity against Heliothis virescens in some, but not all Cry1-Cry1Ac hybrids

We investigated the role of domain III of Bacillus thuringiensis delta-endotoxin Cry1Ac in determining toxicity against Heliothis virescens. Hybrid toxins, containing domain III of Cry1Ac with domains I and II of Cry1Ba, Cry1Ca, Cry1Da, Cry1Ea, and Cry1Fb, respectively, were created. In this way Cry1Ca, Cry1Fb, and to a lesser extent Cry1Ba were made considerably more toxic.     

Toxicity of Bacillus thuringiensis delta-endotoxins against bean shoot borer (Epinotia aporema Wals.) larvae, a major soybean pest in Argentina

The toxicity of seven Bacillus thuringiensis Cry protoxins was tested against neonate larvae of Epinotia aporema, a major soybean pest in Argentina and South America. The most active protoxins were Cry1Ab and Cry1Ac, with LC50 values of 0.55 and 1.39 microg/ml, respectively. Cry1Aa, Cry1Ba, Cry1Ca, and Cry9Ca protoxins were equally toxic with LC50 values about 4 microg/ml, whereas Cry1Da was not toxic. The synergistic activity of different protoxin-mixtures was also analyzed, no synergistic effect between the Cry proteins was observed, with the exception of the poorly toxic Cry1Ba/Cry1Da mixture that was slightly synergistic. The binding capacity of individual Cry1 and Cry9Ca toxins to brush border membranes of E. aporema was also determined. The non-toxic Cry1Da toxin was the only toxin unable to bind to E. aporema membranes. In addition the heterologous competition experiments showed that Cry1Ab and Cry1Ac toxins share a common binding site. Based on these data, we propose that Cry1Ab and Cry1Ac toxins could be used in the biological control of E. aporema.     

A trimeric building block model for Cry toxins in vitro ion channel formation

The crystal (Cry) insecticidal toxins, or δ-endotoxins, are lethal to a wide variety of insect larvae, and are therefore very important in insect control. Toxicity has been explained by formation of transmembrane oligomeric pores or ion channels and, more recently, by the ability of the monomeric toxin to subvert cellular signaling pathways. The structure, topology, and precise role of the putative pore in toxicity are not known. However, in vitro biophysical studies suggest that helices α4 and α5 in domain I insert into the lipid bilayer as an α-helical hairpin. Mutagenesis studies have assigned an important role to α5 in maintaining oligomerization, and to α4 in channel formation. To detect the possible homo-oligomerizing tendencies of these two helices, we have used the evolutionary conservation data contained in sixteen Cry homologs in order to filter non-native interactions found during a global conformational search. No conserved homo-oligomer was found for α4, but a right handed trimeric α5 model was present in the simulations of all Cry sequences. We propose a model for Cry toxin oligomerization based on sequence analysis and available mutagenesis data.     

Structural and functional analysis of the pre-pore and membrane-inserted pore of Cry1Ab toxin

Bacillus thuringiensis produces insecticidal Cry proteins that are active against different insect species. The primary action of Cry toxins is to lyse midgut epithelial cells in the target insect by forming lytic pores on the apical membrane. After interaction with cadherin receptor, Cry proteins undergo conformational changes from a monomeric structure to a pre-pore-oligomeric form that is able to interact with a second GPI-anchored aminopeptidase-N receptor and then insert into lipid membranes. Here, we review the recent advances in the understanding of the structural changes presented by Cry1Ab toxin upon membrane insertion. Based on analysis of the Trp fluorescence of pure monomeric and oligomeric Cry1Ab structures in solution and in membrane-bound state we reported that oligomerization caused 27% reduction of Trp exposed to the solvent. After membrane insertion there is another conformational change that allows an additional rearrangement of the Trp residues resulting in a total protection of these residues from exposure to the solvent. The oligomeric structure is membrane insertion competent since more than 96% of the Cry1Ab oligomer inserts into the membrane as a function of lipid:protein ratio, in contrast to the monomer of which only 5-10%, inserts into the membrane. Finally, analysis of the stability of monomeric, pre-pore and pore structures of Cry1Ab toxin after urea and thermal denaturation suggested that a more flexible conformation could be necessary for membrane insertion and this flexible structure is obtained by toxin oligomerization and by alkaline pH. Domain I is involved in the intermolecular interaction within the oligomeric Cry1Ab and this domain is inserted into the membrane in the membrane-inserted state.     

Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein.     

An ADAM metalloprotease is a Cry3Aa Bacillus thuringiensis toxin receptor

Bacillus thuringiensis insecticidal proteins toxic action relies on the interaction with receptor molecules on insect midgut target cells. Here, we describe an ADAM metalloprotease as a novel type of B. thuringiensis toxin receptor on the basis of the following data: (i) by ligand blot and N-terminal analysis, we detected a Colorado potato beetle Cry3Aa toxin binding molecule that shares homology with an ADAM10 metalloprotease; (ii) Colorado potato beetle brush border membrane vesicles display ADAM activity since it cleaves an ADAM fluorogenic substrate; (iii) Cry3Aa acts as a competitor of the cleavage of the ADAM fluorogenic substrate; (iv) Cry3Aa sequence contains the recognition motif R(345)FQPGYYGND(354) present in ADAM10 substrates. Accordingly, a peptide representative of the recognition motif localized within loop 1 of Cry3Aa domain II (Ac-F(341)HTRFQPGYYGNDSFN(358)-NH(2)) effectively prevented Cry3Aa proteolytic processing and nearly abolished pore formation, evidencing the functional significance of the Cry3Aa-ADAM interaction in relation to this toxin mode of action.     

Tobacco plants expressing the Cry1AbMod toxin suppress tolerance to Cry1Ab toxin of Manduca sexta cadherin-silenced larvae

Cry toxins produced by Bacillus thuringiensis bacteria are insecticidal proteins used worldwide in the control of different insect pests. Alterations in toxin-receptor interaction represent the most common mechanism to induce resistance to Cry toxins in lepidopteran insects. Cry toxins bind with high affinity to the cadherin protein present in the midgut cells and this interaction facilitates the proteolytic removal of helix ??-1 and pre-pore oligomer formation. Resistance to Cry toxins has been linked with mutations in the cadherin gene. One strategy effective to overcome larval resistance to Cry1A toxins is the production of Cry1AMod toxins that lack helix ??-1. Cry1AMod are able to form oligomeric structures without binding to cadherin receptor and were shown to be toxic to cadherin-silenced Manduca sexta larvae and Pectinophora gossypiella strain with resistance linked to mutations in a cadherin gene.We developed Cry1AbMod tobacco transgenic plants to analyze if Cry1AMod toxins can be expressed in transgenic crops, do not affect plant development and are able to control insect pests. Our results show that production of the Cry1AbMod toxin in transgenic plants does not affect plant development, since these plants exhibited healthy growth, produced abundant seeds, and were virtually undistinguishable from control plants. Most importantly, Cry1AbMod protein produced in tobacco plants retains its functional toxic activity against susceptible and tolerant M. sexta larvae due to the silencing of cadherin receptor by RNAi. These results suggest that CryMod toxins could potentially be expressed in other transgenic crops to protect them against both toxin-susceptible and resistant lepidopteran larvae affected in cadherin gene.     

Characterization of resistance to Bacillus thuringiensis toxin Cry1Ac in Plutella xylostella from China

A field population (SZ) of Plutella xylostella, collected from the cabbage field in Shenzhen, Guangdong Province of China in 2002, showed 2.3-fold resistance to Cry1Aa, 110-fold to Cry1Ab, 30-fold to Cry1Ac, 2.1-fold to Cry1F, 5.3-fold to Cry2Aa and 6-fold resistance to Bacillus thuringiensis var. kurstaki (Btk) compared with a susceptible strain (ROTH). The SZBT strain was derived from the SZ population through 20 generations of selection with activated Cry1Ac in the laboratory. While the SZBT strain developed 1200-fold resistance to Cry1Ac after selection, resistance to Cry1Aa, Cry1Ab, Cry1F, and Btk increased to 31-, 1900-,>33- and 17-fold compared with the ROTH strain. However, little or no cross-resistance was detected to Cry1B, Cry1C and Cry2Aa in the SZBT strain. Genetic cross analyses between the SZBT and ROTH strains revealed that Cry1Ac-resistance in the SZBT strain was controlled by a single, autosomal, incompletely recessive gene. Binding studies with ¹²⁵I-labeled Cry1Ac showed that the brush border membrane vesicles (BBMVs) of midguts from the resistant SZBT insects had lost binding to Cry1Ac. Allelic complementation tests demonstrated that the major Bt resistance locus in the SZBT strain was same as that in the Cry1Ac-R strain which has “mode 1” resistance to Bt. An F₁ screen of 120 single-pair families between the SZBT strain and three field populations collected in 2008 was carried out. Based on this approach, the estimated frequencies of Cry1Ac-resistance alleles were 0.156 in the Yuxi population from Yunnan province, and 0.375 and 0.472 respectively in the Guangzhou and Huizhou populations from Guangdong province.     

A membrane associated metalloprotease cleaves Cry3Aa Bacillus thuringiensis toxin reducing pore formation in Colorado potato beetle brush border membrane vesicles

Insect proteases are implicated in Bacillus thuringiensis insecticidal proteins mode of action determining toxin specificity and sensitivity. Few data are available on the involvement of proteases in the later steps of toxicity such as protease interaction with toxin-receptor complexes and the pore formation process. In this study, a Colorado potato beetle (CPB) midgut membrane metalloprotease was found to be involved in the proteolytic processing of Cry3Aa. Interaction of Cry3Aa with BBMV membrane proteases resulted in a distinct pattern of proteolysis. Cleavage was demonstrated to occur in protease accessible regions of domain III and was specifically inhibited by the metalloprotease inhibitors 1,10-phenanthroline and acetohydroxamic acid. Proteolytic inhibition by a peptide representing a segment of proteolysis in domain III and the metalloprotease inhibitor acetohydroxamic acid correlated with increased pore formation, evidencing that Cry3Aa is a specific target of a CPB membrane metalloprotease that degrades potentially active toxin.     

Binding analyses of Cry1Ab and Cry1Ac with membrane vesicles from Bacillus thuringiensis-resistant and -susceptible Ostrinia nubilalis

The binding properties of Bacillus thuringiensis toxins to brush border membrane vesicles of Dipel-resistant and -susceptible Ostrinia nubilalis larvae were compared using ligand-toxin immunoblot analysis, surface plasmon resonance (SPR), and radiolabeled toxin binding assays. In ligand-toxin immunoblot analysis, the number of Cry1Ab or Cry1Ac toxin binding proteins and the relative toxin binding intensity were similar in vesicles from resistant and susceptible larvae. Surface plasmon resonance with immobilized activated Cry1Ab toxin indicated that there were no significant differences in binding with fluid-phase vesicles from resistant and susceptible larvae. Homologous competition assays with radiolabeled Cry1Ab and Cry1Ac toxin and vesicles from resistant and susceptible larvae resulted in similar toxin dissociation constants and binding site concentrations. Heterologous competition binding assays indicated that Cry1Ab and Cry1Ac completely competed for binding, thus they share binding sites in the epithelium of the larval midguts of O. nubilalis. Overall, the binding analyses indicate that resistance to Cry1Ab and Cry1Ac in this Bt-resistant strain of O. nubilalis is not associated with a loss of toxin binding.