Bacillus thuringiensis Cry toxins are used worldwide as insecticides in agriculture, in forestry, and in the control of disease transmission vectors. In the lepidopteran Manduca sexta, cadherin (Bt-R 1 ) and aminopeptidase-N (APN) function as Cry1A toxin receptors. The interaction with Bt-R 1 promotes cleavage of the amino-terminal end, including helix ␣-1 and formation of prepore oligomer that binds to APN, leading to membrane insertion and pore formation. Loops of domain II of Cry1Ab toxin are involved in receptor interaction. Here we show that Cry1Ab mutants located in domain II loop 3 are affected in binding to both receptors and toxicity against Manduca sexta larvae. Interaction with both receptors depends on the oligomeric state of the toxin. Monomers of loop 3 mutants were affected in binding to APN and to a cadherin fragment corresponding to cadherin repeat 12 but not with a fragment comprising cadherin repeats 7-12. In contrast, the oligomers of loop 3 mutants were affected in binding to both Bt-R 1 fragments but not to APN. Toxicity assays showed that either monomeric or oligomeric structures of Cry1Ab loop 3 mutations were severely affected in insecticidal activity. These data suggest that loop 3 is differentially involved in the binding with both receptor molecules, depending on the oligomeric state of the toxin and also that possibly a "ping pong" binding mechanism with both receptors is involved in toxin action.Bacillus thuringiensis is a bacterium that produces crystalline inclusions formed by insecticidal proteins, called Cry toxins, during the sporulation phase of growth. Cry toxins are toxic to different insect orders as well as to other invertebrates, such as nematodes, mites, and protozoa (1). Cry toxins have been used worldwide in the control of insect pests in agriculture, either as transgenic crops or as spray formulations.The molecular mechanism proposed to describe the action of Cry1A toxins, which are active against different lepidopteran insect species, involves several steps. After larval ingestion of the crystalline inclusions, these are solubilized in midgut lumen and activated by proteases releasing a toxic 65-kDa fragment that binds, in a sequential manner, with at least two receptors located in midgut microvilli. The first interaction occurs with cadherin protein (Bt-R 1 2 in the case of Manduca sexta). This interaction promotes further proteolytic processing of the N-terminal end, including helix ␣-1 of the toxin, resulting in the formation of a prepore oligomeric structure (2). The oligomer has higher affinity to secondary receptors, which are anchored by glycosylphosphatidylinositol, such as aminopeptidase-N (APN) or alkaline phosphatase in the case of M. sexta or Heliothis virescens, respectively (3, 4). Glycosylphosphatidylinositol-anchored receptors are located in specific membrane regions called lipid rafts, where the oligomer inserts into the membrane-forming pores, disrupting the osmotic equilibrium and leading to cell death (1, 5). Although this mechanism of action is g...

The insecticidal Cry toxins are pore-forming toxins produced by the bacteria Bacillus thuringiensis that disrupt insect-midgut cells. In this work we analyzed the response of two different insect orders, the Lepidopteran Manduca sexta and Dipteran Aedes aegypti to highly specific Cry toxins, Cry1Ab and Cry11Aa, respectively. One pathway activated in different organisms in response to a variety of pore forming toxins is the mitogen activated protein kinase p38 pathway (MAPK p38) that activates a complex defense response. We analyzed the MAPK p38 activation by immunodetection of its phosphorylated isoform, and the induction of p38 by RT-PCR, real time-PCR quantitative assays and immunodection. We show that MAPK p38 is activated at postraductional level after Cry toxin intoxication in both insect orders. We detected the p38 induction at the transcriptional and traductional level, and observed a different response. In these three levels, we found that both insects respond to Cry toxin action but M sexta responses more strongly than A. aegypti. Gene silencing of MAPK p38 in vivo, resulted in both insect species becoming hypersensitive to Cry toxin action, suggesting that the MAPK p38 pathway is involved in insect defense against Bt Cry toxins. This finding may have biotechnological applications for enhancing the activity of some Bt Cry toxins against specific insect pests.

Cry1A toxins produced by Bacillus thuringiensis bind a cadherin receptor that mediates toxicity in different lepidopteran insect larvae. Insect cadherin receptors are modular proteins composed of three domains, the ectodomain formed by 9 to 12 cadherin repeats (CR), the transmembrane domain and the intracellular domain. Cry1A toxins interact with three regions of the Manduca sexta cadherin receptor that are located in CR7, CR11 and CR12 cadherin repeats. Binding of Cry1A toxin to cadherin induces oligomerization of the toxin, which is essential for membrane insertion. Also, it has been reported that cadherin fragments containing the CR12 region enhanced the insecticidal activity of Cry1Ab toxin to M. sexta and other lepidopteran larvae. Here we report that cadherin fragments corresponding to CR7 and CR11 regions also enhanced the activity of Cry1Ac and Cry1Ab toxin to M. sexta larvae, although not as efficient as the CR12 fragment. A single point mutation in the CR12 region (I1422R) affected Cry1Ac and Cry1Ab binding to the cadherin fragments and did not enhance the activity of Cry1Ab or Cry1Ac toxin in bioassays. Analysis of Cry1Ab in vitro oligomer formation in the presence of wild type and mutated cadherin fragments showed a correlation between enhancement of Cry1A toxin activity in bioassays and in vitro Cry1Ab-oligomer formation. Our data shows that formation of Cry1A toxin oligomer is in part responsible for the enhancement of Cry1A toxicity by cadherin fragments that is observed in vivo.

Glycosidases are associated with various human diseases. The development of efficient and specific inhibitors may provide powerful tools to modulate their activity. However, achieving high selectivity is a major challenge given that glycosidases with different functions can have similar enzymatic mechanisms and active-site architectures. As an alternative approach to small-chemical compounds, proteinaceous inhibitors might provide a better specificity by involving a larger surface area of interaction. We report here the design and characterization of proteinaceous inhibitors that specifically target endoglycosidases representative of the two major mechanistic classes; retaining and inverting glycosidases. These inhibitors consist of artificial affinity proteins, Affitins, selected against the thermophilic CelD from Clostridium thermocellum and lysozyme from hen egg. They were obtained from libraries of Sac7d variants, which involve either the randomization of a surface or the randomization of a surface and an artificially-extended loop. Glycosidase binders exhibited affinities in the nanomolar range with no cross-recognition, with efficient inhibition of lysozyme (Ki = 45 nM) and CelD (Ki = 95 and 111 nM), high expression yields in Escherichia coli, solubility, and thermal stabilities up to 81.1°C. The crystal structures of glycosidase-Affitin complexes validate our library designs. We observed that Affitins prevented substrate access by two modes of binding; covering or penetrating the catalytic site via the extended loop. In addition, Affitins formed salt-bridges with residues essential for enzymatic activity. These results lead us to propose the use of Affitins as versatile selective glycosidase inhibitors and, potentially, as enzymatic inhibitors in general.

Background Bacillus thuringiensis Cry toxins, that are used worldwide in insect control, kill insects by a mechanism that depends on their ability to form oligomeric pores that insert into the insect-midgut cells. These toxins are being used worldwide in transgenic plants or spray to control insect pests in agriculture. However, a major concern has been the possible effects of these insecticidal proteins on non-target organisms mainly in ecosystems adjacent to agricultural fields.Methodology/Principal FindingsWe isolated and characterized 11 non-toxic mutants of Cry1Ab toxin affected in different steps of the mechanism of action namely binding to receptors, oligomerization and pore-formation. These mutant toxins were analyzed for their capacity to block wild type toxin activity, presenting a dominant negative phenotype. The dominant negative phenotype was analyzed at two levels, in vivo by toxicity bioassays against susceptible Manduca sexta larvae and in vitro by pore formation activity in black lipid bilayers. We demonstrate that some mutations located in helix α-4 completely block the wild type toxin activity at sub-stoichiometric level confirming a dominant negative phenotype, thereby functioning as potent antitoxins.Conclusions/SignificanceThis is the first reported case of a Cry toxin dominant inhibitor. These data demonstrate that oligomerization is a fundamental step in Cry toxin action and represent a potential mechanism to protect special ecosystems from the possible effect of Cry toxins on non-target organisms.

The bacterium produces several insecticidal proteins, such as the crystal proteins (Cry) and the vegetative insecticidal proteins (Vip). In this work, we report that a specific interaction between two toxins creates insecticidal synergism and unravel the molecular basis of this interaction. When applied together, the three-domain Cry toxin Cry9Aa and the Vip Vip3Aa exhibited high insecticidal activity against an important insect pest, the Asiatic rice borer (). We found that these two proteins bind specifically to brush border membrane vesicles of and that they do not share binding sites because no binding competition was observed between them. Binding assays revealed that the Cry9Aa and Vip3Aa proteins interacted with high affinity. We mapped their specific interacting regions by analyzing binding of Cry9Aa to overlapping fragments of Vip3Aa and by analyzing binding of Vip3Aa to individual domains of Cry9Aa. Binding to peptide arrays helped narrow the binding sites to domain II loop-3 of Cry9Aa and toTKKMKTL in Vip3Aa. Site-directed mutagenesis confirmed that these binding regions participate in binding that directly correlates with the synergism between the two proteins. In summary, we show that the Cry9Aa and Vip3Aa toxins display potent synergy based on a specific interaction between them. Our results further our understanding of the complex synergistic activities among toxins and are highly relevant to the development of toxin combinations for effective insect control and for delaying development of insect resistance.

Bacillus thuringiensis insecticidal Cry toxins break down larval midgut-cells after forming pores. The 3D-structures of Cry4Ba and Cry5Ba revealed a trimeric-oligomer after cleavage of helices α-1 and α-2a, where helix α-3 is extended and made contacts with adjacent monomers. Molecular dynamic simulations of Cry1Ab-oligomer model based on Cry4Ba-coordinates showed that E101 forms a salt-bridge with R99 from neighbor monomer. An additional salt bridge was identified in the trimeric-Cry5Ba, located at the extended helix α-3 in the region corresponding to the α-2b and α-3 loop. Both salt-bridges were analyzed by site directed mutagenesis. Single-point mutations in the Lepidoptera-specific Cry1Ab and Cry1Fa toxins were affected in toxicity, while reversed double-point mutant partially recovered the phenotype, consistent with a critical role of these salt-bridges. The single-point mutations in the salt-bridge at the extended helix α-3 of the nematicidal Cry5Ba were also non-toxic. The incorporation of this additional salt bridge into the nontoxic Cry1Ab-R99E mutant partially restored oligomerization and toxicity, supporting that the loop between α-2b and α-3 forms part of an extended helix α-3 upon oligomerization of Cry1 toxins. Overall, these results highlight the role in toxicity of salt-bridge formation between helices α-3 of adjacent monomers supporting a conformational change in helix α-3.

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