In vitro assay for testing the toxicity of Bacillus thuringiensis subsp. israelensis delta endotoxin using blood agarose plates

An in vitro assay system that used erythrocytes was developed to assess the toxicity of delta endotoxin of Bacillus thuringiensis subsp. israelensis (Bti), a mosquito larvicidal agent. This endotoxin was activated at high alkaline pH without causing alkaline injury to the erythrocytes. The assay is carried out on blood agarose plates, prepared in MOPS-buffered saline (0·01 mol 1^-1), pH 7·0. The threshold dose for toxicity of Bti HD-567 was 8 ng protein and that of Bti H-14 and IPS-82 standard were 6 ng and 4 ng protein respectively. The assay is rapid, sensitive, quantitative and applicable to different Bti formulations to assess the toxicity of the products. Only the activated toxin is detected by this method and has good correlation with insect bioassay. The percentage toxicity dose of different Bti preparations agrees well with percentages of LC₅₀ values when the second instar larvae of Aedes aegypti were tested.     

Evaluation of synergism among Bacillus thuringiensis toxins.

A simple test for synergism among toxins is described and applied to previously reported data on independent and joint toxicities of insecticidal proteins from Bacillus thuringiensis. The analysis shows synergism between a 27-kDa (CytA) toxin and 130- or 65-kDa (CryIV) toxins from B. thuringiensis subsp. israelensis against Aedes aegypti larvae. No positive synergism between 130- and 65-kDa toxins or among three CryIA toxins tested against seven species of Lepidoptera occurred. Comparisons with the original interpretations of these data show one case in which synergism occurred but was reported previously as absent and two cases that were not synergistic but were reported previously as suggestive of synergism. These results show that lack of an appropriate test for synergism can produce misleading conclusions. The methods described here can be used to test for synergistic effects of any poisons.     

Evolution of Bacillus thuringiensis Cry toxins insecticidal activity

Insecticidal Cry proteins produced by Bacillus thuringiensis are use worldwide in transgenic crops for efficient pest control. Among the family of Cry toxins, the three domain Cry family is the better characterized regarding their natural evolution leading to a large number of Cry proteins with similar structure, mode of action but different insect specificity. Also, this group is the better characterized regarding the study of their mode of action and the molecular basis of insect specificity. In this review we discuss how Cry toxins have evolved insect specificity in nature and analyse several cases of improvement of Cry toxin action by genetic engineering, some of these examples are currently used in transgenic crops. We believe that the success in the improvement of insecticidal activity by genetic evolution of Cry toxins will depend on the knowledge of the rate‐limiting steps of Cry toxicity in different insect pests, the mapping of the specificity binding regions in the Cry toxins, as well as the improvement of mutagenesis strategies and selection procedures.     

Evaluation of synergism among Bacillus thuringiensis toxins.

A simple test for synergism among toxins is described and applied to previously reported data on independent and joint toxicities of insecticidal proteins from Bacillus thuringiensis. The analysis shows synergism between a 27-kDa (CytA) toxin and 130- or 65-kDa (CryIV) toxins from B. thuringiensis subsp. israelensis against Aedes aegypti larvae. No positive synergism between 130- and 65-kDa toxins or among three CryIA toxins tested against seven species of Lepidoptera occurred. Comparisons with the original interpretations of these data show one case in which synergism occurred but was reported previously as absent and two cases that were not synergistic but were reported previously as suggestive of synergism. These results show that lack of an appropriate test for synergism can produce misleading conclusions. The methods described here can be used to test for synergistic effects of any poisons.     

Effects and mechanisms of Bacillus thuringiensis crystal toxins for mosquito larvae

Bacillus thuringiensis is a Gram‐positive aerobic bacterium that produces insecticidal crystalline inclusions during sporulation phases of the mother cell. The virulence factor, known as parasporal crystals, is composed of Cry and Cyt toxins. Most Cry toxins display a common 3‐domain topology. Cry toxins exert intoxication through toxin activation, receptor binding and pore formation in a suitable larval gut environment. The mosquitocidal toxins of Bt subsp. israelensis (Bti) were found to be highly active against mosquito larvae and are widely used for vector control. Bt subsp. jegathesan is another strain which possesses high potency against broad range of mosquito larvae. The present review summarizes characterized receptors for Cry toxins in mosquito larvae, and will also discuss the diversity and effects of 3‐D mosquitocidal Cry toxin and the ongoing research for Cry toxin mechanisms generated from investigations of lepidopteran and dipteran larvae.     

Common features of Bacillus thuringiensis toxins specific for Diptera and Lepidoptera

The complete nucleotide sequence of a cloned gene encoding a 130-kDa crystal protein of Bacillus thuringiensis (B.t.) subspecies israelensis has been determined. The recombinant protein (Bt8) was purified and shown to be a mosquito-specific toxin with a LC50 value of 43 ng/ml to third-instar larvae of Aedes aegypti. Bt8 is processed by proteases or midgut extracts of mosquito larvae into toxic fragments of 68-78 kDa. Deletion mapping indicated that the active fragment of Bt8 is localized in the N-terminal half of the protoxin molecule. The deduced amino acid sequence of Bt8 has been compared with that of Bt2, a Lepidoptera-specific toxin, previously cloned from Bacillus thuringiensis berliner. Highly homologous amino acid stretches are present in the C-terminal half of the proteins. The N-terminal parts show much less sequence homology but they display a strikingly similar distribution of hydrophilic and hydrophobic amino acids. In addition, Bt8 and Bt2 show a significant immunological cross-reaction. The data indicate that although these B.t. delta endotoxins exhibit a different insect-host specificity, they are structurally related and might use a similar mechanism to interact with insect cell membranes.     

Transgenic crops expressing Bacillus thuringiensis toxins and biological control

The area devoted to growing transgenic plants expressing insecticidal Cry proteins derived from Bacillus thuringiensis (Bt) is increasing worldwide. A major concern with the adoption of Bt crops is their potential impact on nontarget organisms including biological control organisms. Regulatory frameworks should advocate a step-wise (tiered) approach to assess possible nontarget effects of Bt crops. Laboratory and glasshouse studies have revealed effects on natural enemies only when Bt-susceptible, sublethally damaged herbivores were used as prey or host, with no indication of direct toxic effects. Field studies have confirmed that the abundance and activity of parasitoids and predators are similar in Bt and non-Bt crops. In contrast, applications of conventional insecticides have usually resulted in negative impacts on biological control organisms. Because Bt-transgenic varieties can lead to substantial reductions in insecticide use in some crops, they can contribute to integrated pest management systems with a strong biological control component.     

Specificity domain localization of Bacillus thuringiensis insecticidal toxins is highly dependent on the bioassay system

The Bacillus thuringiensis cryIA(a) and cryIA(c) gene specificity regions were probed by creating and testing hybrid toxins both in vivo and in vitro against cultured insect cells or dissociated midgut epithelial cells. Toxin threshold dose determinations revealed that CryIA(c) is highly active against cultured Choristoneure fumiterana cells (CF-1) whereas CryIA(a) is nontoxic. In live insect bioassays, a reversed order of toxicity was observed. Hybrid analysis reversed that the CryIA(c) toxicity-determining region is located between codons 258 and 510. Two smaller subsections of this region (residues 258–358 and 450–510) were able to confer toxicity, although at lower levels, and one region (358–450) was present where progressive substitutions of CryIA(a) with cryIA(c) sequences had no effect. Exchanging the non-homologous N-terminal regions of CryIA(c) with CryIE suggested that the W-terminus does not play a role in specificity. One hybrid clone, MP80, displays a 99.3% homology to CryIA(b) but shows an 800-fold increase in toxicity to CF–1 cells relative to that shown by CryIA(b). Direct comparison between live Bombyx mori bioassays and a newly developed in vitro lawn assay using dissociated midgut epithelial cells from the same insect revealed striking differences in toxicity. The toxicity-determining region for B. mori larvae was determined to be between codons 283 and 450, although the 450–620 codon region may exert an influence on toxicity. In general, native or hybrid toxins showing little or no insect intoxication were very active against the epithelial cells, suggesting that factors other than toxin amino acid sequence play an important role in determining toxin specificity.     

Nitrogen-fixing cyanobacteria as gene delivery system for expressing mosquitocidal toxins of Bacillus thuringiensis ssp. israelensis

Classical biological control is the most successfuland promising way to replace chemical pesticides. Thesubspecies israelensis of Bacillusthuringiensis (Bti) is a safe and efficient agent tocontrol mosquito larvae and hence mosquito-bornediseases. One approach to overcome the low efficacyand short half-life in nature of current formulationsof Bti is by expressing the toxin genes in recombinantcyanobacteria as a delivery system. Attempts toexpress Bti toxin in cyanoabcteria have been carriedout during the last ten years. Toxicities of thetransgenic strains were however very low, even underregulation of strong promoters, too low to beeffective in vivo. Two Bti Cry proteins haverecently been co-expressed in the filamentousnitrogen-fixing cyanobacterium Anabaena PCC7120, resulting in clones with the highest toxicitiesand stabilities ever reached so far. However, toobtain a long-lasting preparation, it would be usefulto express Bti toxin genes in cyanobacterial strainsisolated from nature. This approach requiresdevelopment of a system for effective transformationinto such strains. Releasing such recombinant strainsto open environments is still a major obstacle inexploiting this biotechnology.     

Bacillus thuringiensis pulmonary infection: critical role for bacterial membrane-damaging toxins and host neutrophils

The occurrence of Bacillus thuringiensis bacteremia in a neutropenic patient suffering from severe pulmonary disease addressed the question of whether the aggressive behavior of B. thuringiensis depended on the host status and/or on the membrane-damaging toxins the isolate produced. After intratracheal injection, BALB/c mice developed pneumonia followed by fatal dissemination into deep organs, with mice rendered neutropenic by cyclophosphamide injection being extremely more susceptible to infection than normal animals. In animals infected with isogenic strains of B. thuringiensis progressively more defective in membrane-damaging toxins (407 Cry->IP2>MP02), an increase in the 50% lethal dose was registered (3.9x10(5), 1.1x10(6), 1.2x10(7)CFU). Consistently, after non-lethal dose application, only 407 Cry- replicated intrapulmonary, reaching a bacterial burden 4.7-fold and 40.9-fold higher than IP2 (P=0.018) and MP02 (P=0.008) at 48h post-inoculation. Notably, the time-course of infection was similar in animals infected with viable bacilli or spores, with neutropenic mice always being more susceptible to infection. The overall results indicate that B. thuringiensis may be responsible for opportunistic infections and strongly suggest that membrane-damaging toxins contribute to intrapulmonary bacterial persistence favoring dissemination.     

Different mechanisms of resistance to Bacillus thuringiensis toxins in the indianmeal moth

Susceptibility to protoxin and toxin forms of Cry1Ab and the binding of (125)I-labeled Cry1Ab and Cry1Ac has been examined in three Plodia interpunctella colonies, one susceptible (688(s)) and two resistant (198(r) and Dpl(r)) to Bacillus thuringiensis. Toxicological studies showed that the 198(r) colony was 11-fold more resistant to Cry1Ab protoxin than to Cry1Ab activated toxin, whereas the Dpl(r) colony was 4-fold more resistant to protoxin versus toxin. Binding results with (125)I-labeled toxins indicated the occurrence of two different binding sites for Cry1Ab in the susceptible insects, one of them shared with Cry1Ac. Cry1Ab binding was found to be altered in insects from both resistant colonies, though in different ways. Compared with the susceptible colony, insects from the Dpl(r) colony showed a drastic reduction in binding affinity (60-fold higher K(d)), although they had similar concentrations of binding sites. Insects from the 198(r) colony showed a slight reduction in both binding affinity and binding site concentration (five-fold-higher K(d) and ca. three-fold-lower R(t) compared with the 688(s) colony). No major difference in Cry1Ac binding was found among the three colonies. The fact that the 198(r) colony also has a protease-mediated mechanism of resistance (B. Oppert, R. Hammel, J. E. Throne, and K. J. Kramer, J. Biol. Chem. 272:23473-23476, 1997) is in agreement with our toxicological data in which this colony has a different susceptibility to the protoxin and toxin forms of Cry1Ab. It is noteworthy that the three colonies used in this work derived originally from ca. 100 insects, which reflects the high variability and high frequency of B. thuringiensis resistance genes occurring in natural populations.     

Bacillus thuringiensis insecticidal crystal toxins: gene structure and mode of action

Thanks to the techniques of recombinant DNA, there is now abundant sequence information on several endotoxin genes of Bacillus thuringiensis. The task of correlating this sequence information with the economically important aspects of the toxins such as insect specificity, LD(50) and speed of kill is now under worldwide investigation. Progress has also been made on understanding the mechanism of action of the toxins and on identifying the parts of the protoxin which are important in toxicity. Taken together, the mechanistic data and the sequence information allow the first attempts at rational design of mutant endotoxin genes and greatly facilitate the transfer of those genes to other organisms such as plants. More information is still needed, however, as to the nature of the binding site of the toxin and on the three-dimensional structure of the activated toxins.     

Susceptibility of Spodoptera exigua to 9 toxins from Bacillus thuringiensis

Nine of the most common lepidopteran active Cry proteins from Bacillus thuringiensis have been tested for activity against Spodoptera exigua. Because of possible intraspecific variability, three laboratory strains (FRA, HOL, and MUR) have been used. Mortality assays were performed with the three strains. LC₅₀ values for the active toxins were determined to the FRA and the HOL strains, whereas susceptibility of the MUR strain was assessed using only two concentrations. The results showed that Cry1Ca, Cry1Da, and Cry1Fa were the most effective toxins with all strains. Cry1Ab was found effective for the HOL strain, but very little effective against FRA (6.5-fold) and MUR strains. Cry1Aa and Cry1Ac were marginally toxic to all strains, whereas the rest of the toxins tested (Cry1Ba, Cry2Aa, and Cry2Ab) were non toxic. Significant differences in susceptibility among strains were also found for Cry1Da, being the FRA strain 25-fold more susceptible than the HOL strain. Growth inhibition, as an additional susceptibility parameter, was determined in the FRA strain with the 9 toxins. The toxicity profile obtained differed from that observed in mortality assays. Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, Cry1Da, and Cry1Fa toxins produced a similar larval growth inhibition. Cry2Aa had a lower but clear effect on larval growth inhibition, whereas Cry1Ba and Cry2Ab did not have any effect.     

Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis

Bacillus thuringiensis Cry toxins have been widely used in the control of insect pests either as spray products or expressed in transgenic crops. These proteins are pore-forming toxins with a complex mechanism of action that involves the sequential interaction with several toxin-receptors. Cry toxins are specific against susceptible larvae and although they are often highly effective, some insect pests are not affected by them or show low susceptibility. In addition, the development of resistance threatens their effectiveness, so strategies to cope with all these problems are necessary. In this review we will discuss and compare the different strategies that have been used to improve insecticidal activity of Cry toxins. The activity of Cry toxins can be enhanced by using additional proteins in the bioassay like serine protease inhibitors, chitinases, Cyt toxins, or a fragment of cadherin receptor containing a toxin-binding site. On the other hand, different modifications performed in the toxin gene such as site-directed mutagenesis, introduction of cleavage sites in specific regions of the protein, and deletion of small fragments from the amino-terminal region lead to improved toxicity or overcome resistance, representing interesting alternatives for insect pest control.