Affinity maturation of Cry1Aa toxin to the Bombyx mori cadherin-like receptor by directed evolution based on phage display and biopanning selections of domain II loop 2 mutant toxins.
ABSTRACT: Directed evolution of a Cry1Aa toxin using phage display and biopanning was performed to generate an increased binding affinity to the Bombyx mori cadherin-like receptor (BtR175). Three mutant toxins (371 WGLA374 , 371 WPHH374 , 371 WRPQ374 25) with 16-, 16-, and 50-fold higher binding affinities, respectively, for BtR175 were selected from a phage library containing toxins with mutations in domain II loop 2. However, the observed toxicities of the three mutants against B. mori larvae and cultured cells expressing the BtR175 toxin-binding region did not increase, suggesting that increased binding affinity to cadherins does not contribute to the insecticidal activity. Affinity maturation of a Cry toxin to a receptor via directed evolution was relatively simple to achieve, and seems to have potential for generating a toxin with increased insecticidal activity.
Project description:Bacillus thuringiensis Cry1Aa insecticidal protein is the most active known B. thuringiensis toxin against the forest insect pest Lymantria dispar (gypsy moth), unfortunately it is also highly toxic against the non-target insect Bombyx mori (silk worm).Surface exposed hydrophobic residues over domains II and III were targeted for site-directed mutagenesis. Substitution of a phenylalanine residue (F328) by alanine reduced binding to the Bombyx mori cadherin by 23-fold, reduced biological activity against B. mori by 4-fold, while retaining activity against Lymantria dispar.The results identify a novel receptor-binding epitope and demonstrate that virtual elimination of binding to cadherin BR-175 does not completely remove toxicity in the case of B. mori.
Project description:Bacillus thuringiensis Cry toxins are insecticidal proteins used widely for pest control. They are lethal to a restricted range of insects via specific interactions with insect receptors such as the ABC transporter subfamily members C2 (ABCC2) and C3 (ABCC3). However, it is still unclear how these different receptors contribute to insect susceptibility to Cry1A toxins. Here, we investigated the differences between the silkworm (Bombyx mori) ABCC2 (BmABCC2_S) and ABCC3 (BmABCC3) receptors in mediating Cry toxicity. Compared with BmABCC2_S, BmABCC3 exhibited 80- and 267-fold lower binding affinities to Cry1Aa and Cry1Ab, respectively, and these decreased affinities correlated well with the lower receptor activities of BmABCC3 for these Cry1A toxins. To identify the amino acid residues responsible for these differences, we constructed BmABCC3 variants containing a partial amino acid replacement with extracellular loops (ECLs) from BmABCC2_S. Replacing three amino acids from ECL 1 or 3 increased BmABCC3 activity toward Cry1Aa and enabled its activity toward Cry1Ab. Meanwhile, BmABCC2_S and BmABCC3 exhibited no receptor activities for Cry1Ca, Cry1Da, and Cry3Bb, correlating with markedly lower binding affinities for these Cry toxins. ABCC2 from a Cry1Ab-resistant B. mori strain (BmABCC2_R), which has a tyrosine insertion in ECL 2, displayed 93-fold lower binding affinity to Cry1Ab compared with BmABCC2_S but maintained high binding affinity to Cry1Aa. These results indicate that the Cry toxin-binding affinities of ABCC transporters are largely linked to the level of Cry susceptibility of ABCC-expressing cells and that the ABCC ECL structures determine the specificities to Cry toxins.
Project description:The Bacillus thuringiensis ?-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted continuous evolution selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant Kd?=?11-41?nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.
Project description:The successful use of Bacillus thuringiensis insecticidal toxins to control agricultural pests could be undermined by the evolution of insect resistance. Under selection pressure in the laboratory, a number of insects have gained resistance to the toxins, and several cases of resistance in the diamondback moth have been reported from the field. The use of protein engineering to develop novel toxins active against resistant insects could offer a solution to this problem. The display of proteins on the surface of phages has been shown to be a powerful technology to search for proteins with new characteristics from combinatorial libraries. However, this potential of phage display to develop Cry toxins with new binding properties and new target specificities has hitherto not been realized because of the failure of displayed Cry toxins to bind their natural receptors. In this work we describe the construction of a display system in which the Cry1Ac toxin is fused to the amino terminus of the capsid protein D of bacteriophage lambda. The resultant phage was viable and infectious, and the displayed toxin interacted successfully with its natural receptor.
Project description:The bacterium Bacillus thuringiensis (Bt) produces protoxin proteins in parasporal crystals. Proteolysis of the protoxin generates an active toxin which is a potent microbial insecticide. Additionally, Bt toxin genes have been introduced into genetically modified crops to produce insecticidal toxins which protect crops from insect invasion. The insecticidal activity of Cry toxins is mediated by specific interaction between toxins and their respective cellular receptors. One such toxin (Cry1Ab) exerts toxicity by first targeting the 12th ectodomain region (EC12) of the moth cadherin receptor BT-R1. Binding promotes a highly regulated signaling cascade event that concludes in oncotic-like cell death. We previously determined that conserved sequence motifs near the N- and C-termini of EC12 are critical for toxin binding in insect cells. Here, we have established that Cry1Ab specifically binds to EC12 as a soluble heterodimeric complex with extremely high affinity (Kd = 19.5 ± 1.6 nM). Binding assays using Cry1Ab toxin and a fluorescently labeled EC12 revealed that the heterodimeric complex is highly specific in that no such formation occurs between EC12 and other Cry toxins active against beetle and mosquito. Disruption of one or both terminal sequence motifs in EC12 eliminates complex formation. Until now, comprehensive biophysical characterization of Cry1Ab recognition and binding by the BT-R1 receptor was unresolved. The findings presented here provide insight on the molecular determinants in the Cry family of toxins and should facilitate the assessment and advancement of their use as pesticidal agents.
Project description:The bacterium Bacillus thuringiensis 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 B. thuringiensis 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 (Chilo suppressalis). We found that these two proteins bind specifically to brush border membrane vesicles of C. suppressalis 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 to 428TKKMKTL434 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 B. thuringiensis 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 B. thuringiensis toxins and are highly relevant to the development of toxin combinations for effective insect control and for delaying development of insect resistance.
Project description:Activated forms of Bacillus thuringiensis insecticidal toxins have consistently been found to form insoluble and inactive precipitates when they are expressed in Escherichia coli. Genetic engineering of these proteins to improve their effectiveness as biological pesticides would be greatly facilitated by the ability to express them in E. coli, since the molecular biology tools available for Bacillus are limited. To this end, we show that activated B. thuringiensis toxin (Cry1Ac) can be expressed in E. coli as a translational fusion with the minor phage coat protein of filamentous phage. Phage particles displaying this fusion protein were viable, infectious, and as lethal as pure toxin on a molar basis when the phage particles were fed to insects susceptible to native Cry1Ac. Enzyme-linked immunosorbent assay and Western blot analysis showed the fusion protein to be antigenically equivalent to native toxin, and micropanning with anti-Cry1Ac antibody was positive for the toxin-expressing phage. Phage display of B. thuringiensis toxins has many advantages over previous expression systems for these proteins and should make it possible to construct large libraries of toxin variants for screening or biopanning.
Project description:The vegetative insecticidal proteins (Vips) secreted by Bacillus thuringiensis are regarded as the new generation of insecticidal toxins because they have different insecticidal properties compared with commonly applied insecticidal crystal proteins (Cry toxins). Vip3A toxin, representing the vast majority of Vips, has been used commercially in transgenic crops and bio-insecticides. However, the lack of both structural information on Vip3A and a clear understanding of its insecticidal mechanism at the molecular level limits its further development and broader application. Here we present the first crystal structure of the C-terminal fragment of Vip3A toxin (Vip3Aa11200-789). Since all members of this insecticidal protein family are highly conserved, the structure of Vip3A provides unique insight into the general domain architecture and protein fold of the Vip3A family of insecticidal toxins. Our structural analysis reveals a four-domain organization, featuring a potential membrane insertion region, a receptor binding domain, and two potential glycan binding domains of Vip3A. In addition, cytotoxicity assays and insect bioassays show that the purified C-terminal fragment of Vip3Aa toxin alone have no insecticidal activity. Taken together, these findings provide insights into the mode of action of the Vip3A family of insecticidal toxins and will boost the development of Vip3A into more efficient bio-insecticides.
Project description:<b>:</b> Cry toxins are insecticidal proteins produced by <i>Bacillus thuringiensis</i> (Bt). They are used commercially to control insect pests since they are very active in specific insects and are harmless to the environment and human health. The gene encoding ATP-binding cassette subfamily A member 2 (ABCA2) was identified in an analysis of Cry2A toxin resistance genes. However, we do not have direct evidence for the role of ABCA2 for Cry2A toxins or why Cry2A toxin resistance does not cross to other Cry toxins. Therefore, we performed two experiments. First, we edited the <i>ABCA2</i> sequence in <i>Bombyx mori</i> using transcription activator-like effector-nucleases (TALENs) and confirmed the susceptibility-determining ability in a diet overlay bioassay. Strains with C-terminal half-deleted BmABCA2 showed strong and specific resistance to Cry2A toxins; even strains carrying a deletion of 1 to 3 amino acids showed resistance. However, the C-terminal half-deleted strains did not show cross-resistance to other toxins. Second, we conducted a cell swelling assay and confirmed the specific ability of BmABCA2 to Cry2A toxins in HEK239 cells. Those demonstrated that BmABCA2 is a functional receptor for Cry2A toxins and that BmABCA2 deficiency-dependent Cry2A resistance does not confer cross-resistance to Cry1A, Cry1F, Cry1Ca, Cry1Da, or Cry9Aa toxins.
Project description:Bacillus thuringiensis (Bt) produces insecticidal proteins that have been used worldwide in the control of insect-pests in crops and vectors of human diseases. However, different insect species are poorly controlled by the available Bt toxins or have evolved resistance to these toxins. Evolution of Bt toxicity could provide novel toxins to control insect pests. To this aim, efficient display systems to select toxins with increased binding to insect membranes or midgut proteins involved in toxicity are likely to be helpful. Here we describe two display systems, phage display and ribosome display, that allow the efficient display of two non-structurally related Bt toxins, Cry1Ac and Cyt1Aa. Improved display of Cry1Ac and Cyt1Aa on M13 phages was achieved by changing the commonly used peptide leader sequence of the coat pIII-fusion protein, that relies on the Sec translocation pathway, for a peptide leader sequence that relies on the signal recognition particle pathway (SRP) and by using a modified M13 helper phage (Phaberge) that has an amber mutation in its pIII genomic sequence and preferentially assembles using the pIII-fusion protein. Also, both Cry1Ac and Cyt1Aa were efficiently displayed on ribosomes, which could allow the construction of large libraries of variants. Furthermore, Cry1Ac or Cyt1Aa displayed on M13 phages or ribosomes were specifically selected from a mixture of both toxins depending on which antigen was immobilized for binding selection. These improved systems may allow the selection of Cry toxin variants with improved insecticidal activities that could counter insect resistances.