Cyt1Aa toxin: crystal structure reveals implications for its membrane-perforating function

During sporulation, Bacillus thuringiensis subsp. israelensis produces a mosquito larvicidal protein complex containing several crystalline and cytolytic (Cyt) toxins. Here, the activated monomeric form of Cyt1Aa, the most toxic Cyt family member, was isolated and crystallized, and its structure was determined for the first time at 2.2 Å resolution.Cyt1Aa adopts a typical cytolysin fold containing a β-sheet held by two surrounding α-helical layers. The absence of a β-strand (between residues V26 and I37) in the dimeric structure of Cyt2Aa led us to deduce that this is the only essential segment for dimer formation and that activation of the toxin occurs by proteolytic processing of its N-terminus. Based on the Cyt1Aa structure, we suggest that the toxicity of Cyt1Aa and other nonrelated proteins, all sharing a cytolysin fold, is correlated with their ability to undergo conformational changes that are necessary prior to their membrane insertion and perforation. This fold allows the α-helical layers to swing away, exposing the β-sheet to insert into the membrane. The identification of a putative lipid binding pocket between the β-sheet and the helical layer of Cyt1Aa supports this mechanism. Sequence-based structural analysis of Cyt1Aa revealed that the lack of activity of Cyt1Ca may be related to the latter's inability to undergo this conformational change due to its lack of flexibility. The pattern of the hemolytic activity of Cyt1Aa presented here (resembling that of pore-forming agents), while differing from that imposed by ionic and nonionic detergents, further supports the pore-forming model by which conformational changes occur prior to membrane insertion and perforation.     

Structure-function relationships of a membrane pore forming toxin revealed by reversion mutagenesis

Cyt2Aa1 is a haemolytic membrane pore forming toxin produced by Bacillus thuringiensis subsp. kyushuensis. To investigate membrane pore formation by this toxin, second-site revertants of an inactive mutant toxin Cyt2Aa1-I150A were generated by random mutagenesis using error-prone PCR. The decrease in side chain length caused by the replacement of isoleucine by alanine at position 150 in the alphaD-beta4 loop results in the loss of important van der Waals contacts that exist in the native protein between I150 and K199 and L203 on alphaE. 28 independent revertants of I150A were obtained and their relative toxicity can be explained by the position of the residue in the structure and the effect of the mutation on side-chain interactions. Analysis of these revertants revealed that residues on alphaA, alphaB, alphaC, alphaD and the loops between alphaA and alphaB, alphaD and beta5, beta6 and beta7 are important in pore formation. These residues are on the surface of the molecule suggesting that they may participate in membrane binding and toxin oligomerization. Changing the properties of the amino acid side-chains of these residues could affect the conformational changes required to transform the water-soluble toxin into the membrane insertion competent state.     

Structure-function relationships of a membrane pore forming toxin revealed by reversion mutagenesis

Cyt2Aa1 is a haemolytic membrane pore forming toxin produced by Bacillus thuringiensis subsp. kyushuensis. To investigate membrane pore formation by this toxin, second-site revertants of an inactive mutant toxin Cyt2Aa1-I150A were generated by random mutagenesis using error-prone PCR. The decrease in side chain length caused by the replacement of isoleucine by alanine at position 150 in the αD-β4 loop results in the loss of important van der Waals contacts that exist in the native protein between I150 and K199 and L203 on αE. 28 independent revertants of I150A were obtained and their relative toxicity can be explained by the position of the residue in the structure and the effect of the mutation on side-chain interactions. Analysis of these revertants revealed that residues on αA, αB, αC, αD and the loops between αA and αB, αD and β5, β6 and β7 are important in pore formation. These residues are on the surface of the molecule suggesting that they may participate in membrane binding and toxin oligomerization. Changing the properties of the amino acid side-chains of these residues could affect the conformational changes required to transform the water-soluble toxin into the membrane insertion competent state.     

Amino acid substitutions in alphaA and alphaC of Cyt2Aa2 alter hemolytic activity and mosquito-larvicidal specificity

Cyt2Aa2 produced by Bacillus thuringiensis subsp. darmstadiensis exhibits in vitro cytolytic activity against broad range of cells but shows specific in vivo toxicity against larvae of Dipteran insects. To investigate the role of amino acids in alphaA and alphaC of this toxin, 3 single-point mutants (A61C, S108C and V109A) were generated. All 3 mutant proteins were highly produced as inclusion bodies that could be solubilized and activated by proteinase K similar to that of the wild type. Hemolytic activity of A61C and S108C mutants was significantly reduced whereas the V109A mutant showed comparable hemolytic activity to the wild type. Interestingly, the A61C mutant exhibited high larvicidal activity to both Aedes aegypti and Culex quinquefasciatus. S108C and V109A mutants showed low activity against C. quinquefasciatus but relatively high toxicity to A. aegypti. These results demonstrated for the first time that amino acids in alphaA and alphaC are involved in the selectivity of the Cyt toxin to the targeted organism.     

Alanine-162 of Bacillus thuringiensis Cyt2Aa2 toxin is essential for membrane binding and oligomerisation

Cyt2Aa2 is a cytolytic toxin produced by Bacillus thuringiensis subsp. darmstadiensis. It is specifically toxic to dipteran larvae in vivo and is also active against several cell types, such as erythrocytes. The active toxin is proposed to bind to the cell membrane, and membrane pore formation by toxin oligomerisation leads to cell lysis. This study aimed to characterise the role of residues (I139, S159, L160, S161, A162, D209 and V215) potentially involved in the membrane binding of Cyt2Aa2. All mutants, except I139A and V215A, showed similar characteristics to the wild-type toxin after proteinase K cleavage. Three mutants, S159A, L160A and S161A, showed high haemolytic activity but low toxicity against Aedes aegypti. Membrane interaction assays showed that these mutants could bind to rat red blood cells (rRBCs) and oligomerise. The mutant D209N had no haemolytic activity but was still mildly toxic to A. aegypti. The mutant A162V could not lyse rRBCs, even at high concentrations, and showed no toxicity against A. aegypti. Our data suggest that alanine 162 of the Cyt2Aa2 toxin is involved in membrane binding and oligomerisation. Substitution of this amino acid altered the conformation of the toxin and affected its biological activity.     

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.     

Functional characterizations of residues Arg-158 and Tyr-170 of the mosquito-larvicidal Bacillus thuringiensis Cry4Ba

The insecticidal activity of Bacillus thuringiensis (Bt) Cry toxins involves toxin stabilization, oligomerization, passage across the peritrophic membrane (PM), binding to midgut receptors and pore-formation. The residues Arg-158 and Tyr-170 have been shown to be crucial for the toxicity of Bt Cry4Ba. We characterized the biological function of these residues. In mosquito larvae, the mutants R158A/E/Q (R158) could hardly penetrate the PM due to a significantly reduced ability to alter PM permeability; the mutant Y170A, however, could pass through the PM, but degraded in the space between the PM and the midgut epithelium. Further characterization by oligomerization demonstrated that Arg-158 mutants failed to form correctly sized high-molecular weight oligomers. This is the first report that Arg-158 plays a role in the formation of Cry4Ba oligomers, which are essential for toxin passage across the PM. Tyr-170, meanwhile, is involved in toxin stabilization in the toxic mechanism of Cry4Ba in mosquito larvae. [BMB Reports 2014; 47(10): 546-551]     

球形芽孢杆菌和苏云金芽孢杆菌以色列亚种杀蚊毒素间的协同作用

本研究测定了分别表达苏云金芽孢杆菌Cry4Aa、Cry4Ba、Cry11Aa、Cyt1Aa和球形芽孢杆菌二元毒素Bin的转化菌株Bt B60 1、Bt B611、Bt B640、Bt U 30和Bt CW 3全发酵培养物两两或两两以上不同组合对抗性库蚊的毒力 ,分析了杀蚊毒素间的协同作用。结果表明 ,Bin和Cry4Aa、Bin和Cry 4Ba间有明显的协同作用 ,此外 ,Cry4Aa和Cry4Ba、Cry4Aa和Cry11Aa、Cyt1Aa和Cry4Aa之间也有明显的协同作用     

In vivo nanoscale analysis of the dynamic synergistic interaction of Bacillus thuringiensis Cry11Aa and Cyt1Aa toxins in Aedes aegypti

The insecticidal Cry11Aa and Cyt1Aa proteins are produced by Bacillus thuringiensis as crystal inclusions. They work synergistically inducing high toxicity against mosquito larvae. It was proposed that these crystal inclusions are rapidly solubilized and activated in the gut lumen, followed by pore formation in midgut cells killing the larvae. In addition, Cyt1Aa functions as a Cry11Aa binding receptor, inducing Cry11Aa oligomerization and membrane insertion. Here, we used fluorescent labeled crystals, protoxins or activated toxins for in vivo localization at nano-scale resolution. We show that after larvae were fed solubilized proteins, these proteins were not accumulated inside the gut and larvae were not killed. In contrast, if larvae were fed soluble non-toxic mutant proteins, these proteins were found inside the gut bound to gut-microvilli. Only feeding with crystal inclusions resulted in high larval mortality, suggesting that they have a role for an optimal intoxication process. At the macroscopic level, Cry11Aa completely degraded the gastric caeca structure and, in the presence of Cyt1Aa, this effect was observed at lower toxin-concentrations and at shorter periods. The labeled Cry11Aa crystal protein, after midgut processing, binds to the gastric caeca and posterior midgut regions, and also to anterior and medium regions where it is internalized in ordered “net like” structures, leading finally to cell break down. During synergism both Cry11Aa and Cyt1Aa toxins showed a dynamic layered array at the surface of apical microvilli, where Cry11Aa is localized in the lower layer closer to the cell cytoplasm, and Cyt1Aa is layered over Cry11Aa. This array depends on the pore formation activity of Cry11Aa, since the non-toxic mutant Cry11Aa-E97A, which is unable to oligomerize, inverted this array. Internalization of Cry11Aa was also observed during synergism. These data indicate that the mechanism of action of Cry11Aa is more complex than previously anticipated, and may involve additional steps besides pore-formation activity.     

The amino- and carboxyl-terminal fragments of the Bacillus thuringensis Cyt1Aa toxin have differential roles in toxin oligomerization and pore formation

The Cyt toxins produced by the bacteria Bacillus thuringiensis show insecticidal activity against some insects, mainly dipteran larvae, being able to kill mosquitoes and black flies. However, they also possess a general cytolytic activity in vitro, showing hemolytic activity in red blood cells. These proteins are composed of two outer layers of α-helix hairpins wrapped around a β-sheet. With regard to their mode of action, one model proposed that the two outer layers of α-helix hairpins swing away from the β-sheet, allowing insertion of β-strands into the membrane forming a pore after toxin oligomerization. The other model suggested a detergent-like mechanism of action of the toxin on the surface of the lipid bilayer. In this work, we cloned the N- and C-terminal domains form Cyt1Aa and analyzed their effects on Cyt1Aa toxin action. The N-terminal domain shows a dominant negative phenotype inhibiting the in vitro hemolytic activity of Cyt1Aa in red blood cells and the in vivo insecticidal activity of Cyt1Aa against Aedes aegypti larvae. In addition, the N-terminal region is able to induce aggregation of the Cyt1Aa toxin in solution. Finally, the C-terminal domain composed mainly of β-strands is able to bind to the SUV liposomes, suggesting that this region of the toxin is involved in membrane interaction. Overall, our data indicate that the two isolated domains of Cyt1Aa have different roles in toxin action. The N-terminal region is involved in toxin aggregation, while the C-terminal domain is involved in the interaction of the toxin with the lipid membrane.     

Oligomerization of Cry11Aa from Bacillus thuringiensis Has an Important Role in Toxicity against Aedes aegypti

Cry11Aa and Cyt1Aa of Bacillus thuringiensis are active against mosquitoes and show synergism. Cyt1Aa functions as a membrane receptor inducing Cry11Aa oligomerization. Here we characterized Cry11Aa helix α-3 mutants impaired in oligomerization and toxicity against Aedes aegypti, indicating that oligomerization of Cry11Aa is important for toxin action. Cyt1Aa did not recover the insecticidal activity of Cry11Aa mutants.Bacillus thuringiensis subsp. israelensis has been used worldwide for the control of different mosquitoes that are vectors of several human diseases (10, 11). This bacterium produces different toxins that individually show activity against mosquitoes, i.e., Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa (2). The toxicity of Cry11Aa and Cry4 toxins against Aedes aegypti is greatly increased in the presence of sublethal concentrations of Cyt1Aa (14). Also, Cyt1Aa overcomes the resistance of the Culex quinquefasciatus population to Cry11Aa (12, 13). Cyt1Aa synergizes the toxic activity of Cry11Aa by functioning as a Cry11Aa receptor, facilitating the oligomerization of Cry11Aa and its pore formation activity (7, 8). Oligomerization is a complex event that involves interaction with a toxin receptor and further proteolysis of helix α-1 (3). In the case of the Cry1Ab toxin, helix α-3 of domain I contains coiled-coil structures that are important for oligomerization (4). Some point mutations in helix α-3 do not affect interaction with receptors but severely affected oligomerization, influencing pore formation and toxicity against Manduca sexta larvae (4).Since binding with Cyt1Aa facilitates Cry11Aa oligomerization, we hypothesize that Cry11Aa mutants unable to oligomerize would be affected in synergism with Cyt1Aa and in toxicity. In this report, we analyzed the effect of point mutations in helix α-3 of Cry11Aa on oligomerization, synergism with Cyt1Aa, and toxicity against A. aegypti larvae.Helix α-3 of Cry11Aa potentially forms coiled-coil structures, as determined by the program COILS, which calculates the probability that a sequence will adopt a coiled-coil conformation (6). The coiled-coil structures are characterized by heptads of residues (abcdefg), where positions a and d are occupied mostly by apolar residues and g and e by charged residues. Here we mutagenized some residues located at positions g and a of the predicted coiled-coil (Fig. ​(Fig.1).1). Substitutions R90E, E97A, Y98E, V104E, and S105E were produced by site-directed mutagenesis (Quick Change; Stratagene, La Jolla, CA) using the pCG6 plasmid (1) as a template and appropriate mutagenic oligonucleotides. Point mutations were verified by automated DNA sequencing at Instituto de Biotecnología-UNAM and transformed into the acrystalliferous B. thuringiensis 407 strain. B. thuringiensis strains were grown in solid nutrient broth sporulation medium supplemented with 10 μg/ml erythromycin (5). Crystal inclusions were purified as described previously (8) and solubilized in 100 mM NaOH for 1 h at 4°C. After solubilization, the Cry11Aa protoxins were dialyzed for 12 h against 50 mM Na₂CO₃, pH 10.5. The pH was equilibrated at pH 8.6 with equal volumes of 1 M Tris-HCl, pH 8, and protoxins were activated with trypsin (1:50, wt/wt) for 2 h at 25°C. All mutants, with the exception of the V104E mutant, which was not analyzed further, produced crystal inclusions similar to those for the wild-type toxin, composed of a 70-kDa protoxin (Fig. ​(Fig.2A).2A). After trypsin activation, all mutants produced two polypeptides of 32 and 36 kDa, similarly to the Cry11Aa toxin, suggesting that these mutations did not cause a major structural disturbance (Fig. ​(Fig.2B).2B). The Cry11Aa and mutant activated toxins were analyzed by circular dichroism spectroscopy (Fig. ​(Fig.2C).2C). The activated toxins were dialyzed against 10 mM Na₂HPO₄, 50 mM NaF, pH 9, and then purified by anion-exchange chromatography with HiTrap Q-Sepharose (Pharmacia LKB Biotechnology) in the same buffer, using a linear NaF gradient from 50 to 400 mM. The similarities among the curves indicate that the mutant toxins have a structure similar to that of the wild-type toxin.Open in a separate windowFIG. 1.Schematic representation of the coiled-coil structures of the α-3 helices of Cry1Ab and Cry11Aa toxins. The positions of residues a, b, c, d, e, f, and g of the heptads are presented. The mutated residues in both toxins that affected oligomerization and toxicity are shown in boldface type (reference 4 and this work).Open in a separate windowFIG. 2.SDS-PAGE analysis and circular dichroism spectra of Cry11Aa mutant toxins. (A) The Cry11Aa protoxins were solubilized at pH 10.5 and analyzed by SDS-PAGE (15% acrylamide). (B) SDS-PAGE analysis (15% acrylamide) of the activated toxins with trypsin. Both SDS-polyacrylamide gels were stained with Coomassie blue. Lanes 1, Cry11Aa; lanes 2, E97A mutant; lanes 3, Y98E mutant; lanes 4, R90E mutant; lanes 5, S105E mutant. (C) Analysis of the secondary-structure compositions of the mutants and Cry11Aa activated toxins. Circular dichroism spectra were recorded with a Jasco model J-715 spectropolarimeter equipped with a Peltier temperature control supplied by Jasco. Spectra were collected from 190 to 250 nm. Eight replicate spectra were collected for each sample to improve the signal-to-noise ratios. The final purified-protein concentration was 0.3 mg/ml, and spectra were collected in a 0.1-cm-pathlength cell. The secondary-structure prediction was performed using the CDSSTR algorithm (1a, 11a). Solid black line, Cry11Aa; dotted black line, E97A mutant; dashed black line, Y98E mutant; solid gray line, R90E mutant; dotted gray line, S105E mutant; MRE, mean residue ellipticity; [θ], ellipticity.The toxicity of spore/crystal suspensions of Cry11Aa or the individual mutants (75 to 10,000 ng/ml) was analyzed with bioassays against 10 fourth-instar A. aegypti larvae reared at 28°C, 87% humidity, and 12:12 light-dark conditions in 100 ml dechlorinated water, and mortality was scored after 24 h (four independent assays). The Cry11Aa toxin showed a mean lethal concentration of 355 ng/ml, with 95% confidence limits of 265 to 446 (Probit analysis using Polo-PC LeOra Software). In contrast, the R90E, E97A, Y98E, and S105E mutants were severely affected in toxicity against A. aegypti larvae, since no mortality was observed at the highest concentration used (10,000 ng/ml).We then analyzed the oligomerization of Cry11Aa toxins as previously described (8). Small unilamelar vesicles (SUV), composed of egg yolk phosphatidyl choline, cholesterol (Avanti Polar Lipids, Alabaster, AL), and stearylamine (Sigma, St. Louis, MO) at a 10:3:1 proportion, respectively, were used (8). Cyt1Aa was purified from the 4Q7/pWF45 strain (14) grown as described above. Cyt1Aa inclusions were purified by sucrose gradients, solubilized in 50 mM Na₂CO₃, 10 mM dithiothreitol, pH 10.5 (2 h at 30°C), and activated with 1:100 proteinase K (Sigma-Aldrich Co.), wt/wt, for 20 min at 30°C.For oligomerization assays, 2.5 μg soluble Cry11Aa or mutant protoxin was incubated for 2 h at 37°C in a 100-μl final volume of 50 mM Na₂CO₃, pH 10.5, with 200 μM SUV, 1:50 trypsin (wt/wt), and 0.5 μg Cyt1Aa activated toxin. After 2 h of incubation, 1 mM phenylmethylsulfonyl fluoride was added to stop the reaction, and the membrane fraction was separated by centrifugation (1 h at 100,000 × g). The pellet was suspended in the same buffer solution. Oligomeric structures of Cry toxins are highly stable after boiling as well as after urea denaturation (9). The suspension was boiled for 4 min, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8% acrylamide), and electrotransferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The oligomeric and monomeric structures of Cry11Aa were detected using polyclonal anti-Cry11Aa antibody (1/15,000; 1 h) and a secondary antibody coupled with horseradish peroxidase (Sigma, St. Louis, MO) (1/5,000; 1 h) followed by luminol (ECL; Amersham Pharmacia Biotech) as described by the manufacturers. Figure ​Figure33 shows that only the Cry11Aa wild-type toxin was able to oligomerize, while the mutants were severely impaired in oligomerization.Open in a separate windowFIG. 3.Analysis of Cry11Aa oligomer formation. Soluble Cry11Aa protoxin was activated with trypsin for 2 h at 37°C in the presence of SUV and Cyt1Aa activated toxin. The membrane fraction was separated by ultracentrifugation, and the Cry11Aa protein was analyzed by Western blotting of the membrane pellet with polyclonal anti-Cry11A antibody. The sizes of the proteins were estimated from a molecular prestained plus standard, all blue (Bio-Rad). Lane 1, Cry11Aa; lane 2, R90E mutant; lane 3, Y98E mutant; lane 4, E97A mutant; lane 5, S105E mutant.Finally, the synergistic activity between Cyt1Aa and Cry11Aa was analyzed. A concentration of Cyt1Aa that produced 10% mortality was assayed in the presence of a protein concentration of wild-type Cry11A that produced 20% mortality. Larvae were examined 24 h after treatment, in three repetitions. This particular protein mixture produced a synergism factor of 8. Under these conditions, mortality was more than 80%, due to the synergistic activities of both toxins. Similar experiments were performed with the mutant toxins, using the same concentration of Cyt1Aa toxin and different concentrations (up to 6,000 ng/ml) of the mutant toxins. Cyt1A did not increase the toxicity of the Cry11Aa mutants, since only 10% mortality was observed, even at the highest concentration of the mutant toxins.Previously, helix α-3 of a lepidopteran-specific toxin (Cry1Ab) was subjected to mutagenesis. The R99E and Y107E mutants of the Cry1Ab toxin were severely impaired in oligomerization and toxicity, showing that oligomer formation is a necessary step to kill the larvae (4). The data presented here indicate that oligomer formation is also an essential step in the mechanism of toxicity of the mosquitocidal Cry11Aa toxin and that helix α-3 is involved in this process.     

Cyt1A of Bacillus thuringiensis Delays Evolution of Resistance to Cry11A in the Mosquito Culex quinquefasciatus

Insecticides based on Bacillus thuringiensis subsp. israelensis have been used for mosquito and blackfly control for more than 20 years, yet no resistance to this bacterium has been reported. Moreover, in contrast to B. thuringiensis subspecies toxic to coleopteran or lepidopteran larvae, only low levels of resistance to B. thuringiensis subsp. israelensis have been obtained in laboratory experiments where mosquito larvae were placed under heavy selection pressure for more than 30 generations. Selection of Culex quinquefasciatus with mutants of B. thuringiensis subsp. israelensis that contained different combinations of its Cry proteins and Cyt1Aa suggested that the latter protein delayed resistance. This hypothesis, however, has not been tested experimentally. Here we report experiments in which separate C. quinquefasciatus populations were selected for 20 generations to recombinant strains of B. thuringiensis that produced either Cyt1Aa, Cry11Aa, or a 1:3 mixture of these strains. At the end of selection, the resistance ratio was 1,237 in the Cry11Aa-selected population and 242 in the Cyt1Aa-selected population. The resistance ratio, however, was only 8 in the population selected with the 1:3 ratio of Cyt1Aa and Cry11Aa strains. When the resistant mosquito strain developed by selection to the Cyt1Aa-Cry11Aa combination was assayed against Cry11Aa after 48 generations, resistance to this protein was 9.3-fold. This indicates that in the presence of Cyt1Aa, resistance to Cry11Aa evolved, but at a much lower rate than when Cyt1Aa was absent. These results indicate that Cyt1Aa is the principal factor responsible for delaying the evolution and expression of resistance to mosquitocidal Cry proteins.     

球形芽孢杆菌Mtx1蛋白和苏云金杆菌Cyt1Aa晶体蛋白的协同作用

采用常规的生物测定方法确定了纯化的球形芽孢杆菌(Bacillus sphaericus)的缺失信号肽的97kDa营养期杀蚊毒素(Mosquitocidal toxin 1,Mtx1)蛋白和苏云金芽孢杆菌(Bacillus thuringiensis)27.3kDa的Cyt1Aa晶体蛋白对致倦库蚊(Culex quinquefasciatus)幼虫的杀虫活性。结果表明Mtx1和Cyt1Aa不同比例的混合物对致倦库蚊的毒力比单独毒素蛋白高,经统计分析表明两毒素蛋白对目标蚊幼虫具有明显的协同作用。在LC98处理浓度下,Mtx1和Cyt1Aa按3∶1混合的混合物LT50值比单独Mtx1的提前了6.36h。表明Cyt1Aa和Mtx1对致倦库蚊具有协同毒杀作用,提高对目标蚊虫的毒力、缩短半致死时间。该结果为深入研究Mtx1和Cyt1Aa的杀蚊作用方式奠定了基础,同时为其在蚊虫防治中的应用提供了新的思路和方法。     

Study of the irreversible binding of Bacillus thuringiensis Cry1Aa to brush border membrane vesicles from Bombyx mori midgut

The binding of Bacillus thuringiensis δ-endotoxin to brush border membrane vesicles (BBMVs) from the target insect larval midgut comprises with not only a reversible but also an irreversible component. The irreversible binding of δ-endotoxin is thought to be a pathologically important factor. Here, we studied the irreversible binding of Cry1Aa to the BBMVs of Bombyx mori. The ¹²⁵I-labeled Cry1Aa bound to the solubilized brush border membrane (BBM) through rapid dissociation only, unlike the binding to BBMVs, indicating that the toxin bound to the solubilized BBM through only a reversible process. Low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the toxin bound irreversibly to BBMVs formed an oligomer of 220 kDa, whereas that bound reversibly to the solubilized BBM did not oligomeraize. When the ¹²⁵I-labeled Cry1Aa bound irreversibly to the BBMVs was digested by proteinase K, approximately 40% of the toxin observed to be resistant to proteinase K. The molecular mass of the toxin resistant to proteinase K was 60 kDa, suggesting that the irreversible binding comprise two forms. These results support the notion that the irreversible binding of the toxin to BBMVs is due to the insertion of the toxin into the lipid bilayers and oligomerization to form channels.     

Single concentration tests show synergism among Bacillus thuringiensis subsp. israelensis toxins against the malaria vector mosquito Anophelesalbimanus

Bioassays of insecticidal proteins from Bacillus thuringiensis subsp. israelensis with larvae of the malaria vector mosquito Anophelesalbimanus showed that the cytolytic protein Cyt1Aa was not toxic alone, but it increased the toxicity of the crystalline proteins Cry4Ba and Cry11Aa. Synergism also occurred between Cry4Ba and Cry11Aa toxins. Whereas many previous analyses of synergism have been based on a series of toxin concentrations leading to comparisons between expected and observed values for the concentration killing 50% of insects tested (LC₅₀), we describe and apply a method here that enables testing for synergism based on single concentrations of toxins.     

The Introduction into Bacillus sphaericus of the Bacillus thuringiensis subsp. medellin cyt1Ab1 Gene Results in Higher Susceptibility of Resistant Mosquito Larva Populations to B. sphaericus

The fragment containing the gene encoding the cytolytic Cyt1Ab1 protein from Bacillus thuringiensis subsp. medellin and its flanking sequences (I. Thiery, A. Delécluse, M. C. Tamayo, and S. Orduz, Appl. Environ. Microbiol. 63:468–473, 1997) was introduced into Bacillus sphaericus toxic strains 2362, 2297, and Iab872 by electroporation with the shuttle vector pMK3. Only small amounts of the protein were produced in recombinant strains 2362 and Iab872. The protein was detected in these strains only by Western blotting and immunodetection with antibody raised against Cyt1Ab1 protein. Large amounts of Cyt1Ab1 protein were produced in B. sphaericus recombinant strain 2297, and there was an additional crystal, other than that of the binary toxin, within the exosporium. The production of the Cyt1Ab1 protein in addition to the binary toxin did not increase the larvicidal activity of the B. sphaericus recombinant strain against susceptible mosquito populations of Culex pipiens or Aedes aegypti. However, it partially restored (10 to 20 times) susceptibility of the resistant mosquito populations of C. pipiens (SPHAE) and Culex quinquefasciatus (GeoR) to the binary toxin. The Cyt1Ab1 protein produced in recombinant B. thuringiensis SPL407(pcyt1Ab1) was synthesized in two types of crystal—one round and with various dense areas, surrounded by an envelope, and the other a regular cuboid crystal, very similar to that found in the B. sphaericus recombinant strain.     

Cys31, Cys47, and Cys195 in BinA Are Essential for Toxicity of a Binary Toxin from Bacillus sphaericus

The mosquito larvicidal binary toxin produced by Bacillus sphaericus is composed of 2 proteins called BinA and BinB. While BinB acts as specificity determinant, BinA is expected to bind to BinB, translocates into cytosol, and exerts its activity via an unknown mechanism. To study the role of cysteine in BinA, 3 cysteine residues were substituted by alanine and serine. Substitution at Cys195 significantly reduced the toxin activity, whereas substitution at Cys31 and Cys47 abolished its toxicity. Intrinsic fluorescent analysis suggested that all mutant proteins should have similar tertiary structure to that of the wild type. Analysis of the mutant protein on sodium dodecyl sulfate–polyacrylamide gel electrophoresis with and without a reducing agent indicated that all 3 cysteine residues were not involved in disulfide bond formation within the BinA molecule. This is the first report to demonstrate that cysteine residues at 3 positions in BinA are required for full toxicity of the binary toxin. They may play a critical role during oligomerization or interaction between BinA and BinB to form the active complex.     

Identification of Bacillus thuringiensis Cry3Aa toxin domain II loop 1 as the binding site of Tenebrio molitor cadherin repeat CR12

Bacillus thuringiensis Cry toxins exert their toxic effect by specific recognition of larval midgut proteins leading to oligomerization of the toxin, membrane insertion and pore formation. The exposed domain II loop regions of Cry toxins have been shown to be involved in receptor binding. Insect cadherins have shown to be functionally involved in toxin binding facilitating toxin oligomerization. Here, we isolated a VHH (VHHA5) antibody by phage display that binds Cry3Aa loop 1 and competed with the binding of Cry3Aa to Tenebrio molitor brush border membranes. VHHA5 also competed with the binding of Cry3Aa to a cadherin fragment (CR12) that was previously shown to be involved in binding and toxicity of Cry3Aa, indicating that Cry3Aa binds CR12 through domain II loop 1. Moreover, we show that a loop 1 mutant, previously characterized to have increased toxicity to T. molitor, displayed a correlative enhanced binding affinity to T. molitor CR12 and to VHHA5. These results show that Cry3Aa domain II loop 1 is a binding site of CR12 T. molitor cadherin.     

Cyt1A of Bacillus thuringiensis delays evolution of resistance to Cry11A in the mosquito Culex quinquefasciatus

Insecticides based on Bacillus thuringiensis subsp. israelensis have been used for mosquito and blackfly control for more than 20 years, yet no resistance to this bacterium has been reported. Moreover, in contrast to B. thuringiensis subspecies toxic to coleopteran or lepidopteran larvae, only low levels of resistance to B. thuringiensis subsp. israelensis have been obtained in laboratory experiments where mosquito larvae were placed under heavy selection pressure for more than 30 generations. Selection of Culex quinquefasciatus with mutants of B. thuringiensis subsp. israelensis that contained different combinations of its Cry proteins and Cyt1Aa suggested that the latter protein delayed resistance. This hypothesis, however, has not been tested experimentally. Here we report experiments in which separate C. quinquefasciatus populations were selected for 20 generations to recombinant strains of B. thuringiensis that produced either Cyt1Aa, Cry11Aa, or a 1:3 mixture of these strains. At the end of selection, the resistance ratio was 1,237 in the Cry11Aa-selected population and 242 in the Cyt1Aa-selected population. The resistance ratio, however, was only 8 in the population selected with the 1:3 ratio of Cyt1Aa and Cry11Aa strains. When the resistant mosquito strain developed by selection to the Cyt1Aa-Cry11Aa combination was assayed against Cry11Aa after 48 generations, resistance to this protein was 9.3-fold. This indicates that in the presence of Cyt1Aa, resistance to Cry11Aa evolved, but at a much lower rate than when Cyt1Aa was absent. These results indicate that Cyt1Aa is the principal factor responsible for delaying the evolution and expression of resistance to mosquitocidal Cry proteins.     

Characterization of the mechanism of action of the genetically modified Cry1AbMod toxin that is active against Cry1Ab-resistant insects

Bacillus thuringiensis Cry toxins are used in the control of insect pests. They are pore-forming toxins with a complex mechanism that involves the sequential interaction with receptors. They are produced as protoxins, which are activated by midgut proteases. Activated toxin binds to cadherin receptor, inducing an extra cleavage including helix α-1, facilitating the formation of a pre-pore oligomer. The toxin oligomer binds to secondary receptors such as aminopeptidase and inserts into lipid rafts forming pores and causing larval death. The primary threat to efficacy of Bt-toxins is the evolution of insect resistance. Engineered Cry1AMod toxins, devoid of helix α-1, could be used for the control of resistance in lepidopterans by bypassing the altered cadherin receptor, killing resistant insects affected in this receptor. Here we analyzed the mechanism of action of Cry1AbMod. We found that alkaline pH and the presence of membrane lipids facilitates the oligomerization of Cry1AbMod. In addition, tryptophan fluorescence emission spectra, ELISA binding to pure aminopeptidase receptor, calcein release assay and analysis of ionic-conductance in planar lipid bilayers, indicated that the secondary steps in mode of action that take place after interaction with cadherin receptor such as oligomerization, receptor binding and pore formation are similar in the Cry1AbMod and in the wild type Cry1Ab. Finally, the membrane-associated structure of Cry1AbMod oligomer was analyzed by electron crystallography showing that it forms a complex with a trimeric organization.