No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Mutagenic analysis of putative domain II and surface residues in mosquitocidal Bacillus thuringiensis Cry19Aa toxin
Abstract
The mosquitocidal crystal protein, Cry19Aa, from Bacillus thuringiensis ssp. jegathesan, has high toxicity to Anopheles stephensi and Culex pipiens but is less toxic to Aedes aegypti. To study the functional role of putative domain II and surface residues in mosquito toxicity, 16 alanine substitution mutations were introduced into Cry19Aa. All mutant constructs were expressed as 65-kDa protoxins and subsequently digested by trypsin to produce further fragmented polypeptides of 40 and 25 kDa. With chymotrypsin, however, most protoxins were digested to 60 kDa and minor bands. The circular dichroism spectra of the chymotrypsin-activated toxins of Cry19Aa and muteins, Y324A, W357A, Y412A, Y414A, W416A, D418A and F485A indicated that there was no significant variation in their structure. In mosquito bioassays, Y324A, W357A, Y410A, W416A, D418A and F485A muteins showed substantial reductions in mosquitocidal activity toward A. aegypti and C. pipiens. These muteins also showed reduced competition with wild-type fluorescein 5-isothiocyanate-labeled Cry19Aa for binding to C. pipiens brush border membrane vesicles. These data suggest that the reduction of toxicity was a result of the reduced binding affinity. From these studies we have identified loop residues of domain II that are important in toxicity and receptor binding to Culex larval midgut.
... Alanine mutagenesis, achieved in various loops of Cry19Aa domain II, revealed critical residues for Culex toxicity (Roh, Nair, Liu & Dean, 2009). ...
... Culex pipiens and Anopheles stephensi, was investigated (Roh, Nair, Liu, & Dean, 2009 ). An array of insect specificity and toxicity exists among the Cry2 family of proteins . ...
... Three-domain structure of Cry2Aa crystal structure and Cry2Ab modelled structure. (Roh, et al., 2009). Cry2Ab mutants; V307I, N309S, F311I, A314T, N318I ...
... Involvement of the loops (loops 1, 2, and 3) in domain II in the toxin-receptor interaction has been reported for many Cry toxins (3,25,28,29,37). For example, in Cry3Aa, replacement of residues N353 and D354 in loop 1 with alanine results in loss of receptor binding ability, as well as toxicity against larvae of Tenebrio molitor and Leptinotarsa decemlineata (37). ...
... For example, in Cry3Aa, replacement of residues N353 and D354 in loop 1 with alanine results in loss of receptor binding ability, as well as toxicity against larvae of Tenebrio molitor and Leptinotarsa decemlineata (37). Similarly, replacement of W357 in loop 1 of Cry19Aa with alanine results in significant loss of toxicity against C. pipiens (29). The alanine substitution mutations Y410A, W416A, and D418A in loop 2 of Cry19Aa result in reduced toxicity against C. pipiens (Ͼ130fold) and Aedes aegypti (4-fold) (29). ...
... Similarly, replacement of W357 in loop 1 of Cry19Aa with alanine results in significant loss of toxicity against C. pipiens (29). The alanine substitution mutations Y410A, W416A, and D418A in loop 2 of Cry19Aa result in reduced toxicity against C. pipiens (Ͼ130fold) and Aedes aegypti (4-fold) (29). Deletion of residues 365 to 371 in loop 2 of Cry1Aa or replacement of these residues with alanine eliminates nearly all toxicity against Bombyx mori (25). ...
Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is of great interest for developing a bioinsecticide to control mosquitoes. Therefore, it is very important to characterize the functional motif of Cry4Aa that is responsible for its mosquitocidal activity. In this study, to characterize a potential receptor binding site, namely, loops 1, 2, and 3 in domain II, we constructed a series of Cry4Aa mutants in which a residue in these three loops was replaced with alanine. A bioassay using Culex pipiens larvae revealed that replacement of some residues affected the mosquitocidal activity of Cry4Aa, but the effect was limited. This finding was partially inconsistent with previous results which suggested that replacement of the Cry4Aa loop 2 results in a significant loss of mosquitocidal activity. Therefore, we constructed additional mutants in which multiple (five or six) residues in loop 2 were replaced with alanine. Although the replacement of multiple residues also resulted in some decrease in mosquitocidal activity, the mutants still showed relatively high activity. Since the insecticidal spectrum of Cry4Aa is specific, Cry4Aa must have a specific receptor on the surface of the target tissue, and loss of binding to the receptor should result in a complete loss of mosquitocidal activity. Our results suggested that, unlike the receptor binding site of the well-characterized molecule Cry1, the receptor binding site of Cry4Aa is different from loops 1, 2, and 3 or that there are multiple binding sites that work cooperatively for receptor binding.
... Domains II and III are rich in βsheets and resemble lectins of the β-prism fold and jellyroll topology families, respectively (16). Domains II and III have been implicated in receptor binding (6,(17)(18)(19)(20)(21)(22), and domain III also modulates pore activity (23). ...
... Cry5B is structurally even more divergent than Cry2Aa, with the largest divergence between Cry5B and the insecticidal Cry proteins occurring in domain II. This domain has been implicated in receptor specificity in other Cry proteins (17,(19)(20)(21)(22), and in Cry5B this activity would fit its divergent organismal specificity. In contrast, domain I is highly conserved between Cry5B and the insecticidal Cry proteins, which likely reflects its conserved role in pore formation (8). ...
Crystal (Cry) proteins are globally used in agriculture as proteinaceous insecticides. They have also been recently recognized to have great potential as anthelmintic agents in targeting parasitic roundworms (e.g. hookworms). The most extensively characterized of the anthelmintic Cry proteins is Cry5B. We report here the 2.3 Å resolution structure of the proteolytically activated form of Cry5B. This structure, which is the first for a nematicidal Cry protein, shows the familiar three-domain arrangement seen in insecticidal Cry proteins. However, domain II is unusual in that it more closely resembles a banana lectin than it does other Cry proteins. This result is consistent with the fact that the receptor for Cry5B consists of a set of invertebrate-specific glycans (attached to lipids), and also suggests that domain II is important for receptor binding. We found that not only is galactose an efficient competitor for binding between Cry5B and glycolipids, but so too is N-acetylgalactosamine (GalNAc). GalNAc is one of the core arthroseries tetrasaccharides of the Cry5B receptor, and galactose an antennary sugar that emanates from this core. These and prior data suggest that the minimal binding determinant for Cry5B consists of a core GalNAc and two antennary galactoses. Lastly, the protoxin form of Cry5B was found to bind nematode glycolipids with equal specificity as activated Cry5B, but with lower affinity. This suggests that the initial binding of Cry5B protoxin to glycolipids can be stabilized at the nematode cell surface by proteolysis. These results lay the groundwork for the design of effective Cry5B-based anthelmintics.
... Finally, Cry1Ai-h-loop2&3, a mutant of Cry1Ai with decreased toxicity against B. mori and increased activity against H. armigera (Zhou et al., 2017), was characterized. As mentioned above, binding with specific receptors is a crucial step in the mode of action and reduction in binding ability with receptors and BBMVs could result in a reduction in insecticidal activity (Atsumi et al., 2008;Rajamohan et al., 1996;Roh et al., 2009). Cry1Ai-h-loop2& 3 exhibited a 3-fold decrease in the binding affinity with BBMVs from B. mori compared with Cry1Ai. ...
Bacillus thuringiensis Cry1Ai belongs to three-domain Cry toxins and only shows growth inhibition effects against the agricultural pest Helicoverpa armigera, although it exhibits high toxicity against the non-target insect Bombyx mori. In previous studies, loop2 and loop3 on domain II from Cry1Ah were found to be related to binding and high toxicity against H. armigera. However, toxicity for B. mori of Cry1Ai-h-loop2, obtained by replacing loop 2 from Cry1Ah into Cry1Ai, was not modified. In this study, to further characterize the role of loop2 and loop3 in Cry1Ai, all of the amino acids in these two loops were substituted with the same amount of alanine residues. The Cry1Ai-loop3 mutant exhibited significantly lower toxicity against B. mori, but the toxicity of the loop2 mutant was not significantly changed. Furthermore, the double-exchange mutant Cry1Ai-h-loop2&3, replacing loop2 and loop3 from Cry1Ah into Cry1Ai, showed decreased toxicity against B. mori related to Cry1Ai. In addition, we found that the binding affinity of Cry1Ai-h-loop2&3 with brush border membrane vesicles (BBMVs) from the midgut of B. mori was lower than that of Cry1Ai, which correlates with the reduced toxicity.
... In Cry1A toxins, binding with BBMV is a crucial step in the toxicology. Loss or reduction of toxicity for these toxins has most often been shown to be due to decreased binding to BBMV or receptors (Lu et al. 1994;Rajamohan et al. 1996b;Atsumi et al. 2008;Roh et al. 2009); however, in some cases, binding and toxicity were shown to be unrelated (Howlader et al. 2010;Adegawa et al. 2017). In this case, the dual-mutant G373A and N375A only exhibited a decrease in binding affinity for BBMV from H. armigera, not a complete loss of binding. ...
Cry1Ai-h-loop 2 is a mutant of Cry1Ai constructed by exchanging loop 2 from Cry1Ah protein and shows insecticidal activity against Helicoverpa armigera. The toxicity of Cry1Ai-h-loop 2, in contrast to the very low toxicity of Cry1Ai, is closely associated with the eleven residues in the loop 2 region. To characterize the key sites of loop 2 in Cry1Ai-h-loop 2, alanine-substituted mutants were generated. The toxicity of these mutants against H. armigera indicated that dual-mutant on Gly ³⁷³ and Asn ³⁷⁵ caused a significant decrease in toxic activity. ELISA binding and competition binding assays demonstrated that the reduction of toxicity in the mutant of interest was correlated with decreased binding affinity.
... Directed mutagenesis of Cry1A toxins denotes that amino acid residues in domain II loop 3 are involved in initial interactions between the toxin and the midgut membrane (Rajamohan et al., 1996c;Pacheco et al., 2009a), while residues in loop 2 are important for irreversible toxin binding (Rajamohan et al., 1996b). Similar observations have been reported for domain II loops of mosquitocidal toxins, such as Cry19Aa (Roh et al., 2009b), Cry11Aa (Fernández et al., 2005), or Cry4Aa (Howlader et al., 2009). Diverse domain II loops may determine specificity to diverse insects, as binding of Cry1C to midgut proteins from Ae. aegypti depends on loop 2 while loop 3 is important for binding and toxicity in S. littoralis larvae (Abdul-Rauf and Ellar, 1999). ...
Bacterial entomopathogens and/or their toxins must be ingested and enter an insect's alimentary tract, where they multiply or are activated to initiate disease. Bacterial toxins and enzymes target midgut cells, disrupt the epithelial barrier and break through to the body cavity. Bacterial proliferation in the hemocoel causes septicemia, killing the host. Most microbial insect-control products are based on Gram-positive, spore-forming bacteria in the genus Bacillus . The most successful microbial pesticide, Bacillus thuringiensis (Bt), has dominated microbial control of insect pests. Toxin genes from Bt are cloned and transformed into plants to develop transgenic Bt crops, which have revolutionized pest control. Bt crops are protected from insect attack by constitutive production of Bt toxins. Alternative entomopathogenic bacteria include Gram-positive Bacillus sphaericus and Gram-negative Serratia bacteria. Research into novel pathogens and modified toxins aims to increase the efficacy and range of microbial insect-control technologies. This chapter reviews bacterial entomopathogens and discusses their taxonomy, genetics, pathology, and implications for insect control.
... Structural stability of Cry2Ab mutants was explored by comparatively evaluating the protease digestion pattern of mutants to Cry2Ab wild type (WT) (Roh et al., 2009). Cry2Ab mutants; V307I, N309S, F311I, A314T, N318I and A334S, yielded toxin size bands within only 2 min of chymotrypsin digestion (Fig. 3). ...
Cry2Aa exhibits dual activity to Lepidoptera and Diptera. Cry2Ab differs in amino acid sequence from Cry2Aa by 13% and has shown significant lepidopteran activity, but no mosquitocidal activity. Previous studies implicate 23 Cry2Aa specificity-conferring residues of domain II, which differ in Cry2Ab. Nine residues are putatively involved in conferring Cry2Aa dipteran specificity. To explore Cry2Ab dipteran toxicity, site-directed mutagenesis was employed to exchange Cry2Ab residues with Cry2Aa D (dipteran) block residues. Cry2Ab wild type demonstrated high toxicity (LC(50) of 540 ng mL(-1)) to Anopheles gambiae, but not to Aedes or Culex, within a 24-h time period. Cry2Ab should be reclassified as a dual active Cry toxin. Cry2Ab mutagenesis revealed critical residues for Cry2Ab protein function, as well as enhanced activity against the malarial mosquito, An. gambiae.
Cry39Aa from Bacillus thuringiensis serovar aizawai BUN1-14 is highly toxic to the larvae of Anopheles stephensi mosquitoes, which transmit malarial parasites. We constructed a homology model of Cry39Aa toxin using the reported structure of Cry4Ba toxin (PDB file 1W99) as a template, and we identified 349KYAYWR354 as the putative loop 1 in domain II. Many studies of various Cry toxins have shown that surface-exposed loops of domain II are critical for toxicity and receptor binding. To investigate the functional role of the putative loop 1 of Cry39Aa toxin, we performed site-directed mutagenesis in the loop. The results of alanine substitutions revealed that the entire structure of loop 1, and aromatic amino acids Y350 and Y352 in particular, are essential for larvicidal activity. In contrast, the toxicities of Cry39Aa mutants with phenylalanine substitutions at these two tyrosine residues did not differ markedly from the toxicity of wild-type Cry39Aa. A competitive binding assay involving A. stephensi brush border membrane vesicles revealed that the alanine mutants showed reduced competition with wild-type Cry39Aa toxin. Our results suggest that the molecular structure of loop 1 in domain II of Cry39Aa toxin is important for toxicity and receptor binding in the midgut of A. stephensi larvae.
The lepidopteran-specific, insecticidal crystal proteins of Bacillus thuringiensis vary in toxicity to different species of lepidopteran larvae. We report studies of CryIA(a) and CryIA(c), two related proteins that have different degrees of toxicity to Heliothis virescens yet very similar degrees of toxicity to Manduca sexta. The amino acid differences between these proteins are located primarily between residues 280 and 722. We have constructed a series of chimeric proteins and determined their toxicities to both insects. The most significant findings arise from the replacement of three segments of the cryIA(c) gene with homologous portions of the cryIA(a) gene: codons 332-428, 429-447, and 448-722. Each of these segments contributed substantially and largely additively toward efficacy for H. virescens. However, replacement of the 429-447 segment of cryIA(c) gene with the cryIA(a) sequence resulted in a 27-50-fold reduction in toxicity toward M. sexta whereas the reduction in toxicity to H. virescens was only 3-4-fold. Subdivision of the 429-447 segment and replacements involving residues within this segment reduced toxicity to M. sexta by 5- to more than 2000-fold whereas toxicity to H. virescens was only reduced 3-10-fold. These observations indicate that: 1) different but overlapping regions of the cryIA(c) gene determine specificity to each of the two test insects; 2) some of the examined gene segments interact in determining specificity; and 3) different sequences in the cryIA(a) and cryIA(c) genes are required for maximal toxicity to M. sexta.
The identification, cloning, and characterization of protein toxins from various species of bacilli have demonstrated the existence of mosquitocidal toxins with different structures, mechanisms of action, and host ranges. A start has been made in understanding the polypeptide determinants of toxicity and insecticidal activity, and the purification of toxins from recombinant organisms may lead to the elucidation of their X-ray crystal structures and the cloning of brush border membrane receptors. The results of cloning mosquitocidal toxins in heterologous microorganisms show the potential of expanding the range of susceptible mosquito species by combining several toxins of different host specificity in one cell. Toxins have been expressed in new microorganisms with the potential for increasing potency by persisting at the larval feeding zone. The powerful tools of bacterial genetics are being applied to engineer genetically stable, persistent toxin expression and expand the insecticidal host ranges of Bacillus sphaericus and Bacillus thuringiensis strains. These techniques, together with modern formulation technology, should eventually lead to the construction of mosquitocidal microorganisms which are effective enough to have a real impact on mosquito-borne diseases.
Forty homolog-scanning (double-reciprocal-crossover) mutant proteins of two Bacillus thuringiensis delta-endotoxin genes (cryIAa and cryIAc) were examined for potential structural alterations by a series of proteolytic assays. Three groups of mutants could be identified. Group 1, consisting of 13 mutants, showed no delta-endotoxin present during overexpression conditions in Escherichia coli (48 h at 37 degrees C, with a ptac promoter). These mutants produced full-sized delta-endotoxin detectable by polyacrylamide gel electrophoresis with Coomassie blue staining or Western immunoanalysis after 24 h of growth but not after 48 h, suggesting sensitivity to intracellular proteases. Group 2 consisted of 13 mutants that produced stable delta-endotoxins that were completely digested by 2% bovine trypsin. In contrast, native delta-endotoxin produces a 65,000-Da trypsin-resistant peptide, which is the active toxin. Group 3 mutants expressed delta-endotoxin and trypsin-stable toxins, similar to the wild type. In this study, 12 group 3 mutant toxins were compared with wild type toxins by thermolysin digestion at a range of temperatures. The two wild-type toxins exhibited significant differences in thermolysin digestion midpoints. Among the group 3 mutants, most possessed significantly different protein stabilities relative to their parental toxins. Two of the group 3 mutants were observed to have exchanged the thermolysin sensitivity properties of the parental toxins.
A gene, designated cry11B, encoding a 81,293-Da crystal protein of Bacillus thuringiensis subsp. jegathesan was cloned by using a gene-specific oligonucleotide probe. The sequence of the Cry11B protein, as deduced from the sequence of the cry11B gene, contains large regions of similarity with the Cry11A toxin (previously CryIVD) from B. thuringiensis subsp. israelensis. The Cry11B protein was immunologically related to both Cry11A and Cry4A proteins. The cry11B gene was expressed in a nontoxic strain of B. thuringiensis, in which Cry11B was produced in large amounts during sporulation and accumulated as inclusions. Purified Cry11B inclusions were highly toxic for mosquito larvae of the species Aedes aegypti, Culex pipiens, and Anopheles stephensi. The activity of Cry11B toxin was higher than that of Cry11A and similar to that of the native crystals from B. thuringiensis subsp. jegathesan, which contain at least seven polypeptides.
Four strains belonging to Bacillus thuringiensis serovars thompsoni, malaysiensis, canadensis, jegathesan and two auto-agglutinating B.t. strains were identified as being highly toxic to the mosquito larvae of the species Aedes aegypti, Anopheles stephensi, and Culex pipiens. Their larvicidal and hemolytic activities were determined and compared with those of strains known to be highly mosquitocidal and/or cytolytic from serovars of B.t. israelensis, morrisoni, darmstadiensis, medellin, kyushuensis, and fukuokaensis. The electrophoretic protein profiles of purified crystals and immunological relationships with B.t.i. polypeptides were studied. Five out of the six new strains showed the same larvicidal and hemolytic activities and the same crystal proteins and toxin genes as B.t.i. One strain, B.t. jegathesan 367, presented a novel pattern of larvicidal activity and a protein profile different from those of other strains.
We have evaluated the binding of Bacillus thuringiensis Cry toxins to aminopeptidase N (APN) purified from Lymantria dispar (gypsy moth) brush border membrane vesicle (BBMV). CryIAc toxin bound strongly to APN, while either the structurally related CryIAa and CryIAb toxins or CryIC, CryIIA, and CryIIIA toxins showed weak binding to APN. An in vitro competition binding study demonstrated that the binding of CryIAc to L. dispar BBMV was inhibited by APN. Inhibition of short circuit current for CryIAc, measured by voltage clamping of whole L. dispar midgut, was substantially reduced by addition of phosphatidylinositol-specific phospholipase C, which is known to release APN from the midgut membrane. In contrast, addition of phosphatidylinositol-specific phospholipase C had only a marginal effect on the inhibition of short circuit current for CryIAa. These data suggest that APN is the major functional receptor for CryIAc in L. dispar BBMV. A ligand blotting experiment demonstrated that CryIAc recognized a 120-kDa peptide (APN), while CryIAa and CryIAb recognized a 210-kDa molecule in L. dispar BBMV. In contrast, CryIAa and CryIAb bound to both the 120- and 210-kDa molecules in Manduca sexta BBMV, while CryIAc recognized only the 120-kDa peptide. The 120-kDa peptide (APN) in L. dispar BBMV reacted with soybean agglutinin, indicating that N-acetylgalactosamine is a component of this glycoprotein.
The crystal proteins of Bacillus thuringiensis have been extensively studied because of their pesticidal properties and their high natural levels of production. The increasingly rapid characterization of new crystal protein genes, triggered by an effort to discover proteins with new pesticidal properties, has resulted in a variety of sequences and activities that no longer fit the original nomenclature system proposed in 1989. Bacillus thuringiensis pesticidal crystal protein (Cry and Cyt) nomenclature was initially based on insecticidal activity for the primary ranking criterion. Many exceptions to this systematic arrangement have become apparent, however, making the nomenclature system inconsistent. Additionally, the original nomenclature, with four activity-based primary ranks for 13 genes, did not anticipate the current 73 holotype sequences that form many more than the original four subgroups. A new nomenclature, based on hierarchical clustering using amino acid sequence identity, is proposed. Roman numerals have been exchanged for Arabic numerals in the primary rank (e.g., Cry1Aa) to better accommodate the large number of expected new sequences. In this proposal, 133 crystal proteins comprising 24 primary ranks are systematically arranged.
Alanine substitution mutations in the Cry1Ac domain III region, from amino acid residues 503 to 525, were constructed to study the functional role of domain III in the toxicity and receptor binding of the protein to Lymantria dispar, Manduca sexta, and Heliothis virescens. Five sets of alanine block mutants were generated at the residues (503)SS(504), (506)NNI(508), (509)QNR(511), (522)ST(523), and (524)ST(525). Single alanine substitutions were made at the residues (509)Q, (510)N, (511)R, and (513)Y. All mutant proteins produced stable toxic fragments as judged by trypsin digestion, midgut enzyme digestion, and circular dichroism spectrum analysis. The mutations, (503)SS(504)-AA, (506)NNI(508)-AAA, (522)ST(523)-AA, (524)ST(525)-AA, and (510)N-A affected neither the protein's toxicity nor its binding to brush border membrane vesicles (BBMV) prepared from these insects. Toward L. dispar and M. sexta, the (509)QNR(511)-AAA, (509)Q-A, (511)R-A, and (513)Y-A mutant toxins showed 4- to 10-fold reductions in binding affinities to BBMV, with 2- to 3-fold reductions in toxicity. Toward H. virescens, the (509)QNR(511)-AAA, (509)Q-A, (511)R-A, and (513)Y-mutant toxins showed 8- to 22-fold reductions in binding affinities, but only (509)QNR(511)-AAA and (511)R-A mutant toxins reduced toxicity by approximately three to four times. In the present study, greater loss in binding affinity relative to toxicity has been observed. These data suggest that the residues (509)Q, (511)R, and (513)Y in domain III might be only involved in initial binding to the receptor and that the initial binding step becomes rate limiting only when it is reduced more than fivefold.
Culex quinquefasciatus mosquitoes with high levels of resistance to single or multiple toxins from Bacillus thuringiensis subsp. israelensis were tested for cross-resistance to the Bacillus thuringiensis subsp.jegathesan polypeptide Cry19A. No cross-resistance was detected in mosquitoes that had been selected with the Cry11A, Cry4A and Cry4B,
or Cry4A, Cry4B, Cry11A, and CytA toxins. A low but statistically significant level of cross-resistance, three to fourfold,
was detected in the colony selected with Cry4A, Cry4B, and Cry11A. This cross-resistance was similar to that previously detected
with B. thuringiensis subsp.jegathesan in the same colony. These data help explain the toxicity of B. thuringiensis subsp.jegathesan against the resistant colonies and indicate that the Cry19A polypeptide might be useful in managing resistance and/or as
a component of synthetic combinations of mosquitocidal toxins.
Bacillus sphaericus is a spore-forming aerobic bacterium, several strains of which are pathogenic for mosquito larvae. During sporulation, the most active strains produce a crystal toxin with a high degree of larvicidal activity. The toxin is composed of two proteins of 51.4 and 41.9 kDa, which are encoded by highly conserved chromosomal genes. After B. sphaericus is ingested, these proteins are released in the larva's midgut, and, in susceptible mosquito species, bind to a specific receptor present on midgut brush-border membranes. The resulting damages to the midgut cells leads to the mosquitoes' death. During vegetative growth, some B. sphaericus strains also synthesize mosquito larvicidal proteins of 100 and 30.8 kDa (Mtx toxins), the mode of action of which is still unknown. The mechanism of acquisition of the recessive mosquito resistance to the crystal toxin varies with selection conditions.
Improvements in the mosquitocidal activity of Bacillus thuringiensis Cry19Aa were achieved by protein engineering of putative surface loop residues in domain II through rational design. The
improvement of Aedes toxicity in Cry19Aa was 42,000-fold and did not affect its toxicity against Anopheles or Culex.
The impending widespread use of transgenic crop plants encoding a single insecticidal toxin protein of Bacillus thuringiensis has focused attention on the perceived risk of rapid selection of resistance in target insects. We have used Bacillus thuringiensis subsp. israelensis toxins as a model system and determined the speed and magnitude of evolution of resistance in colonies of the mosquito Culex quinquefasciatus during selection for 28 consecutive generations with single or multiple toxins. The parental strain was synthesized by combining approximately 500 larvae from each of 19 field collections obtained from the states of California, Oregon, Louisiana, and Tennessee. At least 10,000 larvae were selected in each generation of each line at an average mortality level of 84%. The susceptibilities of the parental and selected lines were compared in parallel tests in every third generation by using fresh suspensions of toxin powders. The normal toxin complement of B. thuringiensis subsp. israelensis consists of four toxins, CryIVA, CryIVB, CryIVD, and CytA. Resistance became evident first in the line that was selected with a single toxin (CryIVD), attaining the highest level (resistance ratio [RR], >913 at 95% lethal concentration) by generation F(inf28) when the study was completed. Resistance evolved more slowly and to a lower level (RR, >122 by F(inf25)) in the line selected with two toxins (CryIVA+CryIVB) and lower still (RR, 91 by F(inf28)) in the line selected with three toxins (CryIVA+CryIVB+ CryIVD). Resistance was remarkably low (RR, 3.2) in the line selected with all four toxins. The results reveal the importance of the full complement of toxins found in natural populations of B. thuringiensis subsp. israelensis as an effective approach to resistance management.
Bacillus thuringiensis mosquitocidal toxin Cry4Ba has no significant natural activity against Culex quinquefasciatus or Culex pipiens (50% lethal concentrations [LC50], >80,000 and >20,000 ng/ml, respectively). We introduced amino acid substitutions in three putative loops of domain II of
Cry4Ba. The mutant proteins were tested on four different species of mosquitoes, Aedes aegypti, Anopheles quadrimaculatus, C. quinquefasciatus, and C. pipiens. Putative loop 1 and 2 exchanges eliminated activity towards A. aegypti and A. quadrimaculatus. Mutations in a putative loop 3 resulted in a final increase in toxicity of >700-fold and >285-fold against C. quinquefasciatus (LC50 ≅ 114 ng/ml) and C. pipiens (LC50 ⩬ 37 ng/ml), respectively. The enhanced protein (mutein) has very little negative effect on the activity against Anopheles or Aedes. These results suggest that the introduction of short variable sequences of the loop regions from one toxin into another
might provide a general rational design approach to enhancing B. thuringiensis Cry toxins.
Inclusions consisting of CryIVA, CryIVB, or CryIVD proteins were purified from recombinant strains of Bacillus thuringiensis subsp. israelensis and used in bioassay tests against Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae. Two- and three-component mixtures were also tested. Synergy among the three polypeptides was clearly evidenced. In particular, there were synergistic interactions between CryIVA and both CryIVB and CryIVD, and simple additive effects between CryIVB and CryIVD. However, other crystal polypeptides presumably contribute to the full toxicity of wild-type B. thuringiensis subsp. israelensis crystals.
The synthesis and properties of l-lysine p-nitroanilide dihydrobromide and benzoyl dl-arginine p-nitroanilide hydrochloride are described. Both compounds are hydrolyzed by trypsin, the latter being a substrate at least as sensitive as benzoyl l-argininamide. Their hydrolysis can be followed conveniently by colorimetric procedures, since one of the products, p-nitroaniline, is yellow. Values of Km and k3 for their reaction with trypsin were determined, as well as the K4 of benzoyl d-arginine p-nitroanilide, which was isolated from a tryptic digest of the dl isomer. The pH-activity curves were also determined, that of l-LPA being in a more alkaline region than normally found for trypsin substrates. The possible significance of this shift is discussed.Preliminary studies indicate that benzoyl dl-arginine p-nitroanilide hydrochloride is also hydrolyzed by papain.
Analysis of the three surface loops in domain II ofBacillus thuringiensisCryIIIA δ-endotoxin has been carried out to assess their role in receptor binding and toxicity. Site-directed mutagenesis was used to convert loop residues to alanine and the mutant proteins were analyzed for structural stability, toxicity to beetle larvae (Tenebrio molitor), binding to receptors onT. molitorbrush border membrane vesicles (Tm-BBMV) and insertion into BBMV, as measured by irreversible membrane receptor binding. This study demonstrates the functional significance of loops for binding and insertion. Alanine replacements in loop I resulted in disruption of receptor binding or structural instability. The double mutation, Y350A,Y351A, could be suppressed by replacing a nearby R345with alanine, and the resultant mutant protein also showed reduced receptor binding. Substitution of N353and D354in loop I with alanine residues caused the loss of binding ability and toxicity. A loop II double mutant, P412A,S413A, had no effect on binding or toxicity. A block mutation of loop III residues to alanine had the effect of reducing receptor binding while concomitantly increasing toxicity by 2.4-fold. We compared this up-mutant to wild-type toxin in each step of physiological processing of protoxin: solubility, proteolytic activation, and insertion into the Tm-BBMV. The loop III block mutant showed increased membrane insertion, but was similar to wild-type toxin in other parameters. These results reveal that loop I and loop III in domain II of CryIIIA δ-endotoxin are involved in receptor binding. In addition, the direct correlation between toxicity and irreversible binding of the loop III block mutant (despite the indirect relationship to reversible binding) suggests that loop III may play a role in membrane insertion.
A strategy, called alanine-scanning mutagenesis, was used to identify specific side chains in human growth hormone (hGH) that
strongly modulate binding to the hGH receptor cloned from human liver. Single alanine mutations (62 in total) were introduced
at every residue contained within the three discontinuous segments of hGH (residues 2 to 19, 54 to 74, and 167 to 191) that
have been implicated in receptor recognition. The alanine scan revealed a cluster of a dozen large side chains that when mutated
to alanine each showed more than a four times lower binding affinity to the hGH receptor. Many of these residues that promote
binding to the hGH receptor are altered in homologs of hGH (such as placental lactogens and prolactins) that do not bind tightly
to the hGH receptor. The overall folding of these mutant proteins was indistinguishable from that of the wild-type hGH, as
determined by strong cross-reactivities with seven different conformationally sensitive monoclonal antibodies. The alanine
scan also identified at least one side chain, Glu174, that hindered binding because when it was mutated to alanine the receptor
affinity increased by more than a factor of four.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
A novel Bacillus thuringiensis strain highly toxic to mosquitoes was isolated from soil samples in Malaysia. This strain was shown to display a new subfraction of the H-28 flagellar antigen determining a new serovar H28a28c, which was designated serovar jegathesan. Bioassays indicated that Culex quinquefasciatus larvae are the most susceptible to this new isolate, whereas toxicity to Anopheles maculatus and Aedes aegypti larvae was 10 times lower. The potency of this new serotype is also comparable to most of the Malaysian B. thuringiensis H-14 isolates.
A larval population of Culex quinquefasciatus Say from an urban area (Coque) of Recife, Brazil, submitted to selection with Bacillus sphaericus Neide for a 26-mo trial, was 10 times less susceptible to B. sphaericus and slightly more susceptible to Bacillus thuringiensis israelensis, compared with untreated populations. The low-level resistance to B. sphaericus was unstable in the absence of selection pressure. The LC50 of B. sphaericus to the Coque population declined gradually and attained a susceptibility level similar to that observed in a laboratory control colony 16 mo after the treatment period was interrupted. In parallel to the recovery of B. sphaericus susceptibility. Coque larvae also were as susceptible as laboratory larvae to B. thuringiensis israelensis.
Two new crystal protein genes, cry19A and orf2, isolated from Bacillus thuringiensis subsp. jegathesan were cloned and characterized. The cry19A gene encodes a 74.7-kDa protein, and the orf2 gene encodes a 60-kDa protein. Cry19A contains the five conserved blocks present in most B. thuringiensis delta-endotoxins. The ORF2 amino acid sequence is similar to that of the carboxy terminus of Cry4 proteins. The cry 19A gene was expressed independently or in combination with orf2 in a crystal-negative B. thuringiensis host. The proteins accumulated as inclusions. Purified inclusions containing either Cry19A alone or Cry19A and ORF2 together were toxic to Anopheles stephensi and Culex pipiens mosquito larvae. They were more toxic to C. pipiens than to A. stephensi. However, inclusions containing Cry19A and ORF2 together were more toxic than inclusions of Cry19A alone but less toxic than the wild-type inclusions of B. thuringiensis subsp. jegathesan.
Studies of the binding interactions of dipteran-specific Bacillus thuringiensis delta-endotoxins are hindered by the lengthy midgut dissection procedure needed for preparation of brush border membrane vesicles. In an attempt to resolve this problem, brush border membrane vesicles were isolated from homogenates of whole Aedes aegypti larvae by a modification of the method of MacIntosh et al. (1994). These preparations were found to resolve well on SDS-PAGE and appeared as spherical vesicles of various sizes under electron microscopic examination. Specific activities of the brush border membrane marker enzymes alkaline phosphatase and leucine amino acid arylamidase were enriched 10.9- and 10.7-fold, respectively. Direct binding experiments using 35S-labeled B. thuringiensis CryIC toxin revealed a single class of high-affinity binding sites with a dissociation constant (Kd) of 27 +/- 0.6 nM and a maximum binding capacity (Bmax) of approximately 27 +/- 1.2 pmol/mg BBMV protein. These binding parameters are similar to those of vesicles prepared from isolated midguts, indicating that whole larval brush border membrane vesicles are suitable for in vitro membrane binding studies.
Sporulating cells of Bacillus thuringiensis (Bt) produce insecticidal toxins that are sequestered as protoxins in a crystalline inclusion, hence the name insecticidal crystal proteins, or ICPs.1 The ICPs are classified by amino acid sequence homology into two main families, the Cry and the Cyt toxins.2 Here, we are concerned with Cry toxins. Following ingestion by insects, the ICPs are solubilised in the midgut and activated by gut proteases. The activated Cry toxin then binds to specific receptors on midgut epithelial cells. For example, two types of receptors for the Lepidoptera-specific Cry1A toxins have been characterised, the aminopeptidases N (APN) and the cadherin-like proteins, which are all glycoproteins.3, 4, 5, 6 and 7 The specificity-determining group on the APN receptor for the Cry1Ac toxin was identified as N-acetyl galactosamine (GalNAc).8 and 9 It has been demonstrated that the GalNAc-mediated reversible binding to APN is followed rapidly by a rate-limiting, GalNAc-independent event that promotes irreversible toxin insertion into the lipid.10 Binding to receptors induces a conformational change in the toxin necessary for membrane insertion. Binding probably also enhances the localisation of the toxin at the membrane surface, leading to the subsequent oligomerisation or membrane penetration. After insertion into the membrane, the toxin forms transmembrane leakage pores or channels that cause cell swelling and disruption by colloid osmotic lysis.11
The Bacillus thuringiensis crystal protein Cry1Aa is normally selectively active to caterpillar larvae. Through rational design, toxicity (microg/ml) to the mosquito Culex pipiens was introduced by selected deletions and substitutions of the loop residues of domain II. Toxicity to its natural target Manduca sexta was concomitantly abolished. The successful grafting of the alternate mosquito toxicity onto the original lepidopteran Cry1Aa toxin demonstrates the possibility of designing and engineering a desired toxicity into any toxin of a common scaffold by reshaping the receptor binding region with desired specificities.
Use of Bacillus thuringiensis israelensis against mosquitoes and blackflies. Bacillus thuringiensis an Environmental Biopesticide: Theory and Practice
- Becker
- J Margalit
- Becker
- J Margalit
Reversal of low-level resistance
- Silva-Filha
Silva-Filha MH & Regis L (1997) Reversal of low-level resistance
Use of Bacillus thuringiensis israelensis against mosquitoes and blackflies
- N Becker
- J Margalit
- PF Entwistle
- PF Cory
- MJ Bailey
- S Higgs
Becker N & Margalit J (1993) Use of Bacillus thuringiensis
israelensis against mosquitoes and blackflies. Bacillus
thuringiensis an Environmental Biopesticide: Theory and
Practice (Entwistle PF, Cory PF, Bailey MJ & Higgs S, eds), pp.
145-170. John Wiley & Sons, New York.