RESUMO
The Brazilian Bacillus thuringiensis serovar japonensis strain S725 was selected for its toxicity to the velvetbean caterpillar, Anticarsia gemmatalis. This strain produces spherical crystals harbouring a major protein of about 130 kDa which yields fragments of between 50 and 70 kDa upon trypsin activation. The protein showed a high level of identity and immunoafinity to the Cry9 class of delta-endotoxins. The cloned cry9-like gene sequence contains a 3492 bp ORF, which encodes a polypeptide of 1163 amino acids, with a predicted molecular mass of 131.4 kDa. The deduced amino acid sequence is unique and shows 73% identity to Cry9Ba, 64% identity to Cry9Ea, 63% identity to Cry9Da, and 59% identity to Cry9Ca proteins. The novel delta-endotoxin was assigned to a new subclass, Cry9Bb, by the Bt Toxin Nomenclature Committee. The Cry9Bb protein was expressed in an acrystalliferous Bt strain, and exhibited activity against the tobacco hornworm, Manduca sexta, and the velvetbean caterpillar, A. gemmatalis. The biological effect of an amino acid residue change, A84P, was investigated. The LC(50) for the Cry9Bb crystals against M. sexta neonate larvae was 6.84 microg/cm(2), while the LC(50) for the mutant's Cry9Bb crystals was 0.78 microg/cm(2). PCR screening revealed that in addition to cry9Bb, Bt strain S725 also contains cry1I and vip3 genes. Transcription analysis, using RT-PCR, showed that the cry1I gene was transcribed at T(2) and T(5) stages of sporulation.
Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Inseticidas/metabolismo , Controle Biológico de Vetores , Animais , Bacillus thuringiensis/patogenicidade , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clonagem Molecular , Endotoxinas/química , Endotoxinas/genética , Genes Bacterianos , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Inseticidas/química , Larva/microbiologia , Manduca/microbiologia , Análise de Sequência de ProteínaRESUMO
The insecticidal Cry toxins produced by the bacterium Bacillus thuringiensis are comprised of three structural domains. Domain I, a seven-helix bundle, is thought to penetrate the insect epithelial cell plasma membrane through a hairpin composed of alpha-helices 4 and 5, followed by the oligomerization of four hairpin monomers. The alpha-helix 4 has been proposed to line the lumen of the pore, whereas some residues in alpha-helix 5 have been shown to be responsible for oligomerization. Mutation of the Cry1Ac1 alpha-helix 4 amino acid Asn135 to Gln resulted in the loss of toxicity to Manduca sexta, yet binding was still observed. In this study, the equivalent mutation was made in the Cry1Ab5 toxin, and the properties of both wild-type and mutant toxin counterparts were analyzed. Both mutants appeared to bind to M. sexta membrane vesicles, but they were not able to form pores. The ability of both N135Q mutants to oligomerize was also disrupted, providing the first evidence that a residue in alpha-helix 4 can contribute to toxin oligomerization.
Assuntos
Bacillus thuringiensis/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Mutação , Animais , Bacillus thuringiensis/química , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Endotoxinas/química , Endotoxinas/genética , Endotoxinas/metabolismo , Endotoxinas/toxicidade , Proteínas Hemolisinas , Manduca/efeitos dos fármacosRESUMO
Cry1Ac from Bacillus thuringiensis ssp. kurstaki HD-73 is a pore-forming protein specifically toxic to lepidopteran insect larvae. It binds to the cell-surface receptor aminopeptidase N in Manduca sexta midgut via the sugar N-acetyl-D-galactosamine (GalNAc). By using 1,3-diaminopropane (DAP) as the buffer throughout protoxin activation and chromatography on Q-Sepharose at pH 10.3, trypsin-activated Cry1Ac has been purified in a monomeric state, which was crucial to obtaining single crystals of Cry1Ac and of the Cry1Ac-GalNAc complex. Crystals of Cry1Ac alone are triclinic, with unit-cell parameters a = 51.78, b = 113.23, c = 123.41 A, alpha = 113.11, beta = 91.49, gamma = 100.46 degrees; those of the Cry1Ac-GalNAc complex show P2(1) symmetry, with unit-cell parameters a = 121.36, b = 51.19, c = 210.56 A, beta = 105.75 degrees. Data sets collected to 2.36 and 2.95 A resolution, respectively, show that both crystal forms contain four molecules of the 66 kDa toxin in the asymmetric unit and have related packing arrangements. The deaggregating effect of DAP may be explained by its capacity for bivalent hydrogen bonding and hydrophobic interactions at protein interfaces.
Assuntos
Acetilgalactosamina/química , Bacillus thuringiensis/química , Proteínas de Bactérias/química , Toxinas Bacterianas , Endotoxinas/química , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Endotoxinas/isolamento & purificação , Proteínas Hemolisinas , Microvilosidades/metabolismo , Modelos Moleculares , Conformação ProteicaRESUMO
Crystal structures combined with biochemical data show that the delta-endotoxins from Bacillus thuringiensis are structurally poised towards large-scale, irreversible conformational changes that transform them from the soluble protein bound at the cell surface into a membrane-embedded form causing lysis of susceptible insect cells. Cry delta-endotoxins are made of a helix bundle, a beta-prism and a beta-sandwich. The conformational change involves an umbrella-like opening between the helix-4,5-hairpin and the remaining helices, and between the helical domain and the two sheet domains. Comparison of Cry1Ac structures with and without the bound receptor ligand GalNAc associates occupation of the high-affinity site on the beta-sandwich with an increase of temperature factors in the helical, pore-forming domain, which may indicate how receptor binding could trigger the required major conformational change. The structure of Cyt delta-endotoxins indicates that the surface helix hairpins must peel away to expose the beta-strands for membrane attack. Single amino acid substitutions in hinge residues or the core can restore activity following an inhibitory mutation.
Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas , Endotoxinas/química , Endotoxinas/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas Hemolisinas , Modelos Moleculares , Mutagênese , Mutagênese Sítio-Dirigida , Filogenia , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , SolubilidadeRESUMO
Bacillus thuringiensis protein delta-endotoxins are toxic to a variety of different insect species. Larvicidal potency depends on the completion of a number of steps in the mode of action of the toxin. Here, we investigated the role of proteolytic processing in determining the potency of the B. thuringiensis Cry1Ac delta-endotoxin towards Pieris brassicae (family: Pieridae) and Mamestra brassicae (family: Noctuidae). In bioassays, Cry1Ac was over 2,000 times more active against P. brassicae than against M. brassicae larvae. Using gut juice purified from both insects, we processed Cry1Ac to soluble forms that had the same N terminus and the same apparent molecular weight. However, extended proteolysis of Cry1Ac in vitro with proteases from both insects resulted in the formation of an insoluble aggregate. With proteases from P. brassicae, the Cry1Ac-susceptible insect, Cry1Ac was processed to an insoluble product with a molecular mass of approximately 56 kDa, whereas proteases from M. brassicae, the non-susceptible insect, generated products with molecular masses of approximately 58, approximately 40, and approximately 20 kDa. N-terminal sequencing of the insoluble products revealed that both insects cleaved Cry1Ac within domain I, but M. brassicae proteases also cleaved the toxin at Arg423 in domain II. A similar pattern of processing was observed in vivo. When Arg423 was replaced with Gln or Ser, the resulting mutant toxins resisted degradation by M. brassicae proteases. However, this mutation had little effect on toxicity to M. brassicae. Differential processing of membrane-bound Cry1Ac was also observed in qualitative binding experiments performed with brush border membrane vesicles from the two insects and in midguts isolated from toxin-treated insects.
Assuntos
Proteínas de Bactérias/farmacologia , Toxinas Bacterianas , Endotoxinas/farmacologia , Sequência de Aminoácidos , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sequência de Bases , Borboletas/efeitos dos fármacos , Borboletas/enzimologia , Primers do DNA/genética , Endopeptidases/metabolismo , Endotoxinas/genética , Endotoxinas/isolamento & purificação , Endotoxinas/metabolismo , Proteínas Hemolisinas , Larva/efeitos dos fármacos , Larva/enzimologia , Microvilosidades/enzimologia , Peso Molecular , Mariposas/efeitos dos fármacos , Mariposas/enzimologia , Mutagênese Sítio-Dirigida , Controle Biológico de Vetores , Especificidade da EspécieRESUMO
We report here the first Bacillus thuringiensis (Bt) toxin which is toxic to insects from three insect orders (Diptera, Coleoptera, and Lepidoptera). An oligonucleotide probe based on the delta-endotoxin N-terminal sequence was used to detect the gene. A 23-kb BamHI fragment containing the intact gene was identified and cloned from Bt strain YBT-226 plasmid DNA into the vector pBluescript II. Through a series of DNA manipulations the size of this fragment was reduced and the gene sequenced. The deduced amino acid sequence gave a predicted molecular mass of 137 kDa and was identical to a cry1Ba protein from Bt subsp. thuringiensis HD-2, which is now designated as Cry1Ba1 under a new classification scheme. This protein also showed 81.6% similarity with the Cry1B protein (Cry1Bb1) from Bt strain EG 5847. When the YBT-226 cry1Ba1 gene was expressed in an acrystalliferous Bt subsp. israelensis strain it produced irregular bipyramidal crystals during sporulation, which reacted specifically with anti-Cry1Ba antiserum. Bioassays using these crystals after purification resulted in significant mortality at low to moderate concentrations to larvae of the house fly (Musca domestica, Diptera), cottonwood leaf beetle (Chrysomela scripta, Coleoptera), and tobacco hornworm (Manduca sexta, Lepidoptera). This broad-spectrum toxicity was not dependent on presolubilization. In assays with insect cell lines not derived from midgut cells, the soluble toxin killed CH1t (Manduca sexta cells) but was inactive against CF1 (Choristoneura fumiferana cells), Aa(s) (Aedes aegypti), and C2 (Culex quinquefasciatus) mosquito cells.
Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Toxinas Bacterianas , Endotoxinas/genética , Endotoxinas/farmacologia , Inseticidas/farmacologia , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Sequência de Bases/genética , DNA/genética , Expressão Gênica , Proteínas Hemolisinas , Dados de Sequência MolecularRESUMO
To investigate the membrane pore structure of Cyt2Aa1 toxin from Bacillus thuringiensis, 14 single-cysteine substitutions of the toxin were constructed. Five of these mutants (L172C, V186C, L189C, E214C and L220C) yielded characteristic products when processed by proteinase K; other mutants were degraded by this enzyme. Mutants that yielded characteristic proteolysed products and wild-type toxin were labelled with polarity-sensitive acrylodan (6-acryloyl-2-dimethylaminonaphthalene) at the thiol group of cysteine residues. A green-blue shift in the emission spectra was observed with all labelled toxins on transfer from an aqueous solution into a solution containing membranes or liposomes from red blood cells. These results suggested that the label moved into the hydrophobic environment of the membrane or became buried within hydrophobic regions of the protein oligomers. Digestion of membrane-bound labelled toxin with proteinase K did not cause a significant decrease in emission intensity from any of the labelled mutants. This suggests that L172C, V186C, L189C, E214C and L220C are inserted into the membrane and are therefore protected from proteolysis. In contrast, a marked decrease in emission intensity was observed when membrane-bound labelled wild-type toxin was digested with proteinase K. This suggests that Cys-19 does not insert into the membrane. Fluorimetric analysis of delipidated pore complexes suggests that L172C, V186C, L189C and E214C point towards the lipid in the membrane, whereas L220C is either within the hydrophobic environment of the protein oligomers or exposed to the membrane lipids. Most of the Cys-19 from wild-type molecules is enclosed within the hydrophobic pockets of the protein oligomers.
Assuntos
2-Naftilamina/análogos & derivados , Bacillus thuringiensis/química , Toxinas Bacterianas/química , 2-Naftilamina/química , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/farmacologia , Sequência de Bases , Primers do DNA , Hemólise/efeitos dos fármacos , Hidrólise , Mutagênese Sítio-Dirigida , Processamento de Proteína Pós-TraducionalRESUMO
Sixteen Bacillus thuringiensis, four Bacillus cereus and three Bacillus anthracis isolates were screened for a selection of known and putative B. thuringiensis virulence factors. PCR primers were designed to detect genes for phosphatidylcholine specific phospholipase C, phosphatidylinositol specific phospholipase C, immune inhibitor A, vegetative insecticidal protein 3A, a protein proposed to be involved in capsule synthesis, a newly identified Ser/Thr kinase homologue and enterotoxin entS. Motility, the presence of flagella, haemolysis, chitinase and lecithinase production were also evaluated. The widely varying profiles of the 23 strains from the complex provide a pool of different genotypes that can help to identify factors involved in pathogenicity.
Assuntos
Bacillus cereus/patogenicidade , Bacillus thuringiensis/patogenicidade , Bacillus anthracis/classificação , Bacillus anthracis/genética , Bacillus anthracis/patogenicidade , Bacillus cereus/classificação , Bacillus cereus/genética , Bacillus thuringiensis/classificação , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Genótipo , Humanos , Fenótipo , Reação em Cadeia da Polimerase , Virulência/genéticaRESUMO
The olive fly, Bactrocera oleae, is the key pest on olives in the Mediterranean area. The pest can destroy, in some cases, up to 70% of the olive production. Its control relies mainly on chemical treatments, sometimes applied by aircraft over vast areas, with their subsequent ecological and toxicological side effects. Bacillus thuringiensis is a spore-forming soil bacterium which produces a protein crystal toxic to some insects, including the orders of Lepidoptera, Diptera, and Coleoptera and other invertebrates. The aim of this study was to search for isolates toxic to B. oleae. Several hundred B. thuringiensis isolates were obtained from olive groves and olive presses in different areas of Greece, Sardinia (Italy), and Spain and from cooperating scientists throughout the world. Some isolates were found toxic only to adults or larvae and some to both stages of the olive fly. In addition, the most toxic isolates were assayed on Opius concolor Szepl. (Hym. Braconidae), the most important parasitoid of the olive fruit fly. Only 3 isolates out of 14 gave significant mortality against this parasitoid. Several of the most toxic crystalliferous isolates may contain novel toxins since they gave no PCR products when probed with primers specified for 39 known toxin genes.
Assuntos
Bacillus thuringiensis , Dípteros/microbiologia , Larva/microbiologia , Animais , Bacillus thuringiensis/isolamento & purificação , Proteínas de Bactérias/genética , Primers do DNA , Reação em Cadeia da Polimerase , TemperaturaRESUMO
Site-directed mutagenesis was used to examine the role of predicted loops 2 (374QPWP377) and 3 (436QRSGTPF442) in domain II of the Bacillus thuringiensis Cry1C delta endotoxin for insecticidal specificity and receptor binding. Q374E, S438F, and G439A substitutions resulted in near or complete loss of toxicity toward both Spodoptera littoralis and Aedes aegypti. R437K, R437I, and G439V mutants exhibited significantly reduced toxicity to S. littoralis and A. aegypti, while mutations of T440, P441, and F442 showed only slight reductions in toxicity to both insects. Loop 2 mutations Q374N, P375A, W376Y, and P377A did not significantly affect S. littoralis toxicity but exhibited reduced activity to A. aegypti. In contrast, the loop 3 mutations Q436K, Q436E, and S438Y had no effect on A. aegypti toxicity, but showed significantly decreased S. littoralis activity. Heterologous competition binding assays with brush border membrane vesicles (BBMV) from both insects correlated well with the toxicity data with the exception of the R437 mutants, where steps other than initial receptor binding appear to be involved. Overall we conclude that, while loops 2 and 3 play an important role in binding and toxicity to both insects, loop 2 appears to play the greater role in A. aegypti activity, while loop 3 is more important for S. littoralis toxicity.
Assuntos
Aedes , Bacillus thuringiensis/metabolismo , Proteínas de Bactérias , Toxinas Bacterianas , Endotoxinas , Mutagênese Sítio-Dirigida , Controle Biológico de Vetores , Spodoptera , Aedes/metabolismo , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Ligação Competitiva , Endotoxinas/química , Endotoxinas/genética , Endotoxinas/metabolismo , Endotoxinas/toxicidade , Proteínas Hemolisinas , Plasmídeos/genética , Spodoptera/metabolismo , Transformação BacterianaRESUMO
Binding of the insecticidal Bacillus thuringiensis Cry1Ac toxin to the putative receptor aminopeptidase N is specifically inhibited by N-acetylgalactosamine (GalNAc), suggesting that this toxin recognises GalNAc on the receptor. A possible structural basis for involvement of domain III of the toxin in carbohydrate-mediated receptor recognition was noted in the similarity between the domain III fold of the related toxin Cry3A and a carbohydrate-binding domain in the 1,4-beta-glucanase from Cellulomonas fimi. This possibility was investigated by making selected mutations in domain III of the Cry1Ac delta-endotoxin. Mutagenesis of residues Asn506, Gln509 or Tyr513 resulted in toxins with reduced binding and a slower rate of pore formation in Manduca sexta midgut membrane vesicles compared to the wild-type Cry1Ac. These mutants also showed reduced binding to the 120 kDa Cry1Ac putative receptor aminopeptidase N. Unlike the wild-type toxin, binding of the triple mutant N506D,Q509E,Y513A (Tmut) to M. sexta midgut membrane vesicles could not be inhibited by GalNAc. These data indicate that GalNAc binding is located on domain III of Cry1Ac and therefore support a lectin-like role for this domain. A preliminary analysis of the Cry1Ac crystal structure locates Asn506, Gln509 and Tyr513 in a region on and adjacent to beta-16 in domain III, which has a unique conformation compared to the other known Cry structures. These residues are in a favourable position to interact with either soluble or protein-bound carbohydrate.
Assuntos
Acetilglucosamina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Antígenos CD13/química , Antígenos CD13/metabolismo , Endotoxinas/química , Endotoxinas/metabolismo , Acetilglucosamina/química , Sítio Alostérico , Sequência de Aminoácidos , Animais , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Sítios de Ligação , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sistema Digestório/metabolismo , Sistema Digestório/microbiologia , Endotoxinas/genética , Endotoxinas/toxicidade , Proteínas Hemolisinas , Interações Hospedeiro-Parasita , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Inseticidas/química , Inseticidas/metabolismo , Lectinas/química , Manduca/microbiologia , Dados de Sequência Molecular , Mutação , Permeabilidade , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The interaction of two Bacillus thuringiensis cytolytic toxins, CytA and CytB, with a phospholipid bilayer and their structure in the membrane-bound state were investigated by proteolysis using phospholipid vesicles as a model system. A toxin conformational change upon membrane binding was detected by comparing the proteolytic profile of membrane-bound toxin and saline-solubilized toxin. When membrane-bound toxin was exposed to protease K or trypsin, novel cleavage sites were found between the alpha-helical N-terminal half and beta-strand C-terminal half of the structure at K154 and N155 in CytA and at I150 and G141 in CytB. N-terminal sequencing of membrane-protected fragments showed that the C-terminal half of the toxin structure comprising mainly beta-strands was inserted into the membrane, whereas the N-terminal half comprising mainly alpha-helices was exposed on the outside of the liposomes and could be removed when liposomes with bound toxin were washed extensively after proteolysis. The C-termini of the membrane-inserted proteolytic fragments were also located by a combination of N-terminal sequencing and measurement of the molecular masses of the fragments by electrospray MS. Using a liposome glucose-release assay, the membrane-inserted structure was seen to retain its function as a membrane pore even after removal of exposed N-terminal segments by proteolysis. These data strongly suggest that the pores for glucose release are assembled from the three major beta-strands (beta-5, beta-6 and beta-7) in the C-terminal half of the toxin.
Assuntos
Bacillus thuringiensis/química , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Endotoxinas/química , Bicamadas Lipídicas/química , Fosfolipídeos/química , Sequência de Aminoácidos , Animais , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Endopeptidase K/química , Endotoxinas/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Hidrólise , Bicamadas Lipídicas/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosfolipídeos/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Coelhos , SolubilidadeRESUMO
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.
Assuntos
Aedes/anatomia & histologia , Animais , Larva , Microvilosidades/ultraestruturaRESUMO
This study describes an investigation into the relationship among toxicity, specificity, and binding of CryIC delta-endotoxin from Bacillus thuringiensis subspecies aizawai HD-229. In vivo bioassays of larvae using inclusions (Aedes aegypti) or solubilized toxin (Spodoptera littoralis, Spodoptera exigua, Spodoptera frugiperda, and Manduca sexta) revealed that CryIC was toxic to both dipteran and lepidopteran larvae. In vitro saturation binding assays with CryIC toxin labeled in vivo with [35S]methionine revealed specific and saturable binding to brush border membrane vesicles from both resistant and susceptible insect species. In contrast, dissociation binding experiments showed that the relative toxicities and specificities of CryIC to the insect species tested were correlated with the extent of irreversible binding to the insects' midgut brush border membrane vesicles.
Assuntos
Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas , Endotoxinas/metabolismo , Inseticidas , Aedes , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/toxicidade , Bioensaio , Endotoxinas/toxicidade , Proteínas Hemolisinas , Metionina , Microvilosidades , Spodoptera , Radioisótopos de EnxofreRESUMO
The toxicity of Bacillus thuringiensis Cyt1Aa protein to sheep blowfly larvae depends on its solubilization and proteolytic activation. Cyt1Aa crystals were not toxic. Full-length and trypsin-digested Cyt1Aa proteins were toxic to larvae of three species of sheep blowfly. Neither full-length nor trypsin-digested Cyt2A soluble crystal proteins were toxic.
Assuntos
Bacillus thuringiensis , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Dípteros , Endotoxinas/toxicidade , Ovinos/parasitologia , Animais , Toxinas de Bacillus thuringiensis , Proteínas Hemolisinas , Larva , Controle Biológico de VetoresRESUMO
Methods for the formation of supported lipid monolayers on top of a hydrophobic self assembled monolayer in a surface plasmon resonance instrument are described. Small unilamellar vesicles absorb spontaneously to the surface of the hydrophobic self-assembled monolayer to form a surface which resembles the surface of a cellular membrane. Lipophilic ligands, such as small acylated peptides or glycosylphosphatidylinositol-anchored proteins, were inserted into the absorbed lipid and binding of analytes to these ligands was analysed by surface plasmon resonance. Conditions for the formation of lipid monolayers have been optimised with respect to lipid type, chemical and buffer compatibility, ligand stability and reproducibility.
Assuntos
Toxinas Bacterianas , Lipídeos/química , Análise Espectral/métodos , Antibacterianos/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Glicopeptídeos , Glicosilfosfatidilinositóis/química , Glicosilfosfatidilinositóis/metabolismo , Proteínas Hemolisinas , Metabolismo dos Lipídeos , Espectroscopia de Ressonância Magnética , Ligação Proteica , Reprodutibilidade dos TestesRESUMO
The Bacillus thuringiensis Cry1Ac delta-endotoxin was shown to bind in a biphasic manner to Manduca sexta aminopeptidase N (APN) present in a novel model membrane. Surface plasmon resonance analysis allowed the quantification of toxin binding to M. sexta APN in a supported lipid monolayer. The initial binding was rapid and reversible, with an affinity constant of 110 nM. The second phase was slower and resulted in an overall affinity constant of 3.0 nM. Reagents used to disrupt protein-protein interactions did not dissociate the toxin after high-affinity binding was attained. The initial association between Cry1Ac and APN was inhibited by the sugar GalNAc, but the higher-affinity state was resistant to GalNAc-induced dissociation. The results suggest that after binding to M. sexta APN, the Cry1Ac toxin undergoes a rate-limiting step leading to a high-affinity state. A site-directed Cry1Ac mutant, N135Q, exhibited a similar initial binding affinity for APN but did not show the second slower phase. This inability to form an irreversible association with the APN-lipid monolayer helps explain the lack of toxicity of this protein towards M. sexta larvae and its deficient membrane-permeabilizing activity on M. sexta midgut brush border membrane vesicles.
Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Antígenos CD13/metabolismo , Endotoxinas/metabolismo , Endotoxinas/toxicidade , Manduca/enzimologia , Animais , Toxinas de Bacillus thuringiensis , Técnicas Biossensoriais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Interações Medicamentosas , Proteínas Hemolisinas , Cinética , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Análise Espectral/métodosRESUMO
The Cry3A delta-endotoxin protein inclusion synthesized by Bacillus thuringiensis subsp. tenebrionis is soluble in alkaline and acid buffer solutions but the toxin precipitates when returned to neutral pH conditions. The midgut pH of susceptible beetle larvae is neutral to slightly acidic, a pH environment in which the Cry3A toxin is insoluble. To investigate this paradox we studied the Cry3A toxin after various proteolytic treatments. In many cases the toxin was cleaved into polypeptides that remained associated under non-denaturing conditions. Interestingly a chymotrypsinized Cry3A product was soluble under neutral pH conditions, retained full activity against susceptible beetle larvae, and exhibited specific binding to Leptinotarsa decemlineata midgut membranes.
Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Besouros/efeitos dos fármacos , Endotoxinas/química , Inseticidas/química , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/toxicidade , Endotoxinas/isolamento & purificação , Endotoxinas/toxicidade , Proteínas Hemolisinas , Hidrólise , Inseticidas/isolamento & purificaçãoRESUMO
An osmotic swelling assay utilising carboxyfluorescein self-quenching to measure intravesicular volume changes was adapted to investigate permeability changes induced by the Bacillus thuringiensis Cry1Ac delta-endotoxin in Manduca sexta midgut-brush-border-membrane vesicles (BBMV). This assay provides a more quantitative analysis of Cry-toxin-induced BBMV permeability changes, extending our previously published protocol which employed a light-scattering signal to monitor delta-endotoxin activity [Carroll, J. & Ellar, D. J. (1993) Eur. J. Biochem. 214, 771-778]. The fluorescence signal changes, supported by electron microscopy of the BBMV, demonstrated that Cry1Ac altered the membrane permeability for large non-electrolyte solutes. With this approach Cry1Ac was observed to induce or form pores freely permeant for raffinose (1.14 nm diameter) and using non-electrolytes of increasing size the pores were estimated to have a limiting diameter of approximately 2.4-2.6 nm under alkaline pH conditions.
Assuntos
Bacillus thuringiensis/metabolismo , Toxinas Bacterianas/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Inseticidas/farmacologia , Microvilosidades/efeitos dos fármacos , Animais , Intestinos/ultraestrutura , Manduca/ultraestruturaRESUMO
The spontaneous insertion of Bacillus thuringiensis Cry delta-endotoxins into planar lipid bilayers to form discrete channels in the absence of receptors is the subject of conflicting reports in the literature. Because these proteins are synthesized as protoxins requiring proteolytic activation for conversion to the active form, differences in the in-vitro protocol used for this activation could be responsible for the contradictory results. To investigate this, CrylA(c) toxin was activated by different procedures, and its ability to release glucose entrapped within liposomes and to form channels in planar lipid bilayers assessed. The toxin preparations exhibited widely differing activities on the lipid membranes; SDS-PAGE and immunoblot analysis suggested that variations in the protein profile of the activated samples could be responsible. These findings raise important practical considerations for further in-vitro studies into the mechanism of action of these toxins.