Cyt1Aa from Bacillus thuringiensis subsp. israelensis is toxic to the diamondback moth, Plutella xylostella, and synergizes the activity of Cry1Ac towards a resistant strain.

The Bacillus thuringiensis subsp. israelensis cytolytic protein Cyt1Aa was found to be toxic to an insecticide-susceptible laboratory population of Plutella xylostella. Cry1Ac-resistant populations of P. xylostella showed various degrees of resistance to Cyt1Aa. Cyt1Aa/Cry1Ac mixtures showed a marked level of synergism in the Cry1Ac-resistant populations.     

Genetic and Biochemical Characterization of Field-Evolved Resistance to Bacillus thuringiensis Toxin Cry1Ac in the Diamondback Moth, Plutella xylostella

The long-term usefulness of Bacillus thuringiensis Cry toxins, either in sprays or in transgenic crops, may be compromised by the evolution of resistance in target insects. Managing the evolution of resistance to B. thuringiensis toxins requires extensive knowledge about the mechanisms, genetics, and ecology of resistance genes. To date, laboratory-selected populations have provided information on the diverse genetics and mechanisms of resistance to B. thuringiensis, highly resistant field populations being rare. However, the selection pressures on field and laboratory populations are very different and may produce resistance genes with distinct characteristics. In order to better understand the genetics, biochemical mechanisms, and ecology of field-evolved resistance, a diamondback moth (Plutella xylostella) field population (Karak) which had been exposed to intensive spraying with B. thuringiensis subsp. kurstaki was collected from Malaysia. We detected a very high level of resistance to Cry1Ac; high levels of resistance to B. thuringiensis subsp. kurstaki Cry1Aa, Cry1Ab, and Cry1Fa; and a moderate level of resistance to Cry1Ca. The toxicity of Cry1Ja to the Karak population was not significantly different from that to a standard laboratory population (LAB-UK). Notable features of the Karak population were that field-selected resistance to B. thuringiensis subsp. kurstaki did not decline at all in unselected populations over 11 generations in laboratory microcosm experiments and that resistance to Cry1Ac declined only threefold over the same period. This finding may be due to a lack of fitness costs expressed by resistance strains, since such costs can be environmentally dependent and may not occur under ordinary laboratory culture conditions. Alternatively, resistance in the Karak population may have been near fixation, leading to a very slow increase in heterozygosity. Reciprocal genetic crosses between Karak and LAB-UK populations indicated that resistance was autosomal and recessive. At the highest dose of Cry1Ac tested, resistance was completely recessive, while at the lowest dose, it was incompletely dominant. A direct test of monogenic inheritance based on a backcross of F₁ progeny with the Karak population suggested that resistance to Cry1Ac was controlled by a single locus. Binding studies with ¹²⁵I-labeled Cry1Ab and Cry1Ac revealed greatly reduced binding to brush border membrane vesicles prepared from this field population.     

Cytolytic Peptide Fragments of Cyt1Aa from Bacillus thuringiensis subsp. israelensis

Cyt1Aa is the major and most active component of the parasporal crystal of the Gram-positive soil entomopathogenic bacterium Bacillus thuringiensis subsp. israelensis. The Cyt1Aa protoxin exhibits some hemolytic and cytolytic activity. However, highly active 22–25 kDa toxins are obtained after proteolysis of Cyt1Aa from both the N- and the C-termini. As shown in this study, preliminary binding of the protoxin to polylamellary liposomes or partial denaturation of Cyt1Aa and further processing by several exogenous proteases yielded short 4.9–11.5 kDa cytolytic peptide fragments of Cyt1Aa. The shortest 51 amino acid peptide was obtained after pre-incubation of Cyt1Aa with SDS and proteolysis with proteinase K. This peptide was purified, identified as the Ile87–Asp137 fragment of Cyt1Aa and was shown to exhibit more than 30 % hemolysis of rabbit erythrocytes.     

Binding of Bacillus thuringiensis Cry1Ac Toxin to Aminopeptidase in Susceptible and Resistant Diamondback Moths (Plutella xylostella)

Bacillus thuringiensis Cry1Ac toxin bound to a 120-kDa protein isolated from the brush border membranes of both susceptible and resistant larvae of Plutella xylostella, the diamondback moth. The 120-kDa protein was purified by Cry1Ac toxin affinity chromatography. Like Cry1Ac-binding aminopeptidase N (EC 3.4.11.2) from other insects, this protein was eluted from the affinity column with 200 mM N-acetylgalactosamine. The purified protein had aminopeptidase activity and bound Cry1Ac toxin on ligand blots. Purified aminopeptidase was recognized by antibodies to the cross-reacting determinant found on phosphatidylinositol-specific phospholipase C-solubilized proteins. The results show that the presence of Cry1Ac-binding aminopeptidase in the brush border membrane is not sufficient to confer susceptibility to Cry1Ac. Furthermore, the results do not support the hypothesis that resistance to Cry1Ac was caused by lack of a Cry1Ac-binding aminopeptidase.     

Phage Displayed Bacillus thuringiensis Cry1Ba4 Toxin Is Toxic to Plutella xylostella

We constructed recombinant phage particles displaying the Bacillus thuringiensis Cry1Ba4 active toxin using the pfUSE5 and pComb3X phagemid vectors. The recombinant phage particles were screened and evaluated for displayed biologically active Cry1Ba4 toxin against the target insect larvae. Concurrent expression of Cry1Ba4 protoxin was carried out using the pETBlue^TM-2 plasmid expression vector in Escherichia coli Tuner^TM(DE3)pLacI and the protoxin was successfully expressed at a size of 129 kDa. In the bioassay, 3.30 mg crude extract of Cry1Ba4 protoxin, 9.35 × 10⁹ TU and 7.70 × 10⁹ TU of induced recombinant phage particles carrying Cry1Ba4 active toxin displayed on pComb3X and pFUSE5, respectively, demonstrated mortality of greater than 85% against Plutella xylostella (third-instar) within 48 hours. Thus, we have successfully displayed the Cry1Ba4 activated toxin on the surface of a phage and demonstrated toxicity towards larvae.     

Toxicity of Bacillus thuringiensis Spore and Crystal Protein to Resistant Diamondback Moth (Plutella xylostella)

A colony of Plutella xylostella from crucifer fields in Florida was used in mortality bioassays with HD-1 spore, CryIA(a), CryIA(b), CryIA(c), CryIB, CryIC, CryID, CryIE, or CryIIA. The data revealed high levels of field-evolved resistance to HD-1 spore and all CryIA protoxins and no resistance to CryIB, CryIC, or CryID. CryIE and CryIIA were essentially not toxic. When HD-1 spore was combined 1:1 with protoxin and fed to susceptible larvae, spore synergized the activity of CryIA and CryIC 5- to 8-fold and 1.7-fold, respectively, and did not synergize the mortality of CryIIA. When fed to Florida larvae, spore failed to synergize the activity of all three CryIA protoxins, synergized the activity of CryIC 5.3-fold, and did not synergize the mortality for CryIIA. Binding studies with CryIA(b), CryIB, and CryIC were performed to determine possible mechanisms of resistance. The two techniques used were (i) binding of biotinylated toxin to tissue sections of larval midguts and (ii) binding of biotinylated toxin to brush border membrane vesicles prepared from whole larvae. Both showed dramatically reduced binding of CryIA(b) in resistant larvae compared with that in susceptible larvae but no differences in binding of CryIB or CryIC.     

Genetic and Biochemical Approach for Characterization of Resistance to Bacillus thuringiensis Toxin Cry1Ac in a Field Population of the Diamondback Moth, Plutella xylostella

Four subpopulations of a Plutella xylostella (L.) strain from Malaysia (F₄ to F₈) were selected with Bacillus thuringiensis subsp. kurstaki HD-1, Bacillus thuringiensis subsp. aizawai, Cry1Ab, and Cry1Ac, respectively, while a fifth subpopulation was left as unselected (UNSEL-MEL). Bioassays at F₉ found that selection with Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai gave resistance ratios of >95, 10, 7, and 3, respectively, compared with UNSEL-MEL (>10,500, 500, >100, and 26, respectively, compared with a susceptible population, ROTH). Resistance to Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai in UNSEL-MEL declined significantly by F₉. The Cry1Ac-selected population showed very little cross-resistance to Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai (5-, 1-, and 4-fold compared with UNSEL-MEL), whereas the Cry1Ab-, B. thuringiensis subsp. kurstaki-, and B. thuringiensis subsp. aizawai-selected populations showed high cross-resistance to Cry1Ac (60-, 100-, and 70-fold). The Cry1Ac-selected population was reselected (F₉ to F₁₃) to give a resistance ratio of >2,400 compared with UNSEL-MEL. Binding studies with ¹²⁵I-labeled Cry1Ab and Cry1Ac revealed complete lack of binding to brush border membrane vesicles prepared from Cry1Ac-selected larvae (F₁₅). Binding was also reduced, although less drastically, in the revertant population, which indicates that a modification in the common binding site of these two toxins was involved in the resistance mechanism in the original population. Reciprocal genetic crosses between Cry1Ac-reselected and ROTH insects indicated that resistance was autosomal and showed incomplete dominance. At the highest dose of Cry1Ac tested, resistance was recessive while at the lowest dose it was almost completely dominant. The F₂ progeny from a backcross of F₁ progeny with ROTH was tested with a concentration of Cry1Ac which would kill 100% of ROTH moths. Eight of the 12 families tested had 60 to 90% mortality, which indicated that more than one allele on separate loci was responsible for resistance to Cry1Ac.     

Differential Activity and Activation of Bacillus thuringiensis Insecticidal Proteins in Diamondback Moth, Plutella xylostella

Whole-crystal preparations from strains HD-1 and HD-133, activated Cry1Ab and Cry1C toxins as well as Cry1Aa, Cry1Ac, Cry1D, and Cry2Aa protoxins were tested for toxicity to 2nd-instar larvae of the diamondback moth, Plutella xylostella. Mortality data recorded after 2 and 5 days provided different results that were related to differential rates of solubilization, activation, and degradation of insecticidal crystal proteins. The two most active proteins are Cry1Ab and Cry1C, which are both present in HD-133. The Cry1Ab protoxin is activated within 2 days, whereas activation of the Cry1C protoxin occurs between 2 and 5 days. HD-133 is more active than HD-1 immediately after infection and remains toxic over 5 days owing to the sequential activation of its crystal components. Solubility properties of crystals and rates of activation of protoxins influence the overall toxicity of HD-1 and HD-133 to the diamondback moth. Received: 30 March 1999 / Accepted: 3 May 1999     

Cross-Resistance to Bacillus thuringiensis Toxin CryIF in the Diamondback Moth (Plutella xylostella)

Selection with Bacillus thuringiensis subsp. kurstaki, which contains CryIA and CryII toxins, caused a >200-fold cross-resistance to CryIF toxin from B. thuringiensis subsp. aizawai in the diamondback moth, Plutella xylostella. CryIE was not toxic, but CryIB was highly toxic to both selected and unselected larvae. The results show that extremely high levels of cross-resistance can be conferred across classes of CryI toxins of B. thuringiensis.     

Cyt1A from Bacillus thuringiensis Lacks Toxicity to Susceptible and Resistant Larvae of Diamondback Moth (Plutella xylostella) and Pink Bollworm (Pectinophora gossypiella)

We tested Cyt1Aa, a cytolytic endotoxin of Bacillus thuringiensis, against susceptible and Cry1A-resistant larvae of two lepidopteran pests, diamondback moth (Plutella xylostella) and pink bollworm (Pectinophora gossypiella). Unlike previous results obtained with mosquito and beetle larvae, Cyt1Aa alone or in combination with Cry toxins was not highly toxic to the lepidopteran larvae that we examined.     

Common, but Complex, Mode of Resistance of Plutella xylostella to Bacillus thuringiensis Toxins Cry1Ab and Cry1Ac

A field collected population of Plutella xylostella (SERD4) was selected in the laboratory with Bacillus thuringiensis endotoxins Cry1Ac (Cry1Ac-SEL) and Cry1Ab (Cry1Ab-SEL). Both subpopulations showed similar phenotypes: high resistance to the Cry1A toxins and little cross-resistance to Cry1Ca or Cry1D. A previous analysis of the Cry1Ac-SEL showed incompletely dominant resistance to Cry1Ac with more than one factor, at least one of which was sex influenced. In the present study reciprocal mass crosses between Cry1Ab-SEL and a laboratory susceptible population (ROTH) provided evidence that Cry1Ab resistance was also inherited as incompletely dominant trait with more than one factor, and at least one of the factors was sex influenced. Analysis of single pair mating indicated that Cry1Ab-SEL was still heterogeneous for Cry1Ab resistance genes, showing genes with different degrees of dominance. Binding studies showed a large reduction of specific binding of Cry1Ab and Cry1Ac to midgut membrane vesicles of the Cry1Ab-SEL subpopulation. Cry1Ab-SEL was found to be more susceptible to trypsin-activated Cry1Ab toxin than protoxin, although no defect in toxin activation was found. Present and previous results indicate a common basis of resistance to both Cry1Ab and Cry1Ac in selected subpopulations and suggest that a similar set of resistance genes are responsible for resistance to Cry1Ab and Cry1Ac and are selected whichever toxin was used. The possibility of an incompletely dominant trait of resistant to these toxins should be taken into account when considering refuge resistance management strategies.     

Susceptibility of a Field-Derived, Bacillus thuringiensis-Resistant Strain of Diamondback Moth to In Vitro-Activated Cry1Ac Toxin

Resistant and susceptible populations of the diamondback moth (Plutella xylostella) were tested with crystalline, solubilized, and partially and fully activated forms of the Bacillus thuringiensis Cry1Ac δ-endotoxin. Fully activated toxin greatly reduced the resistance ratio (ratio of the 50% lethal concentration for the resistant population to that for the susceptible population) of the resistant population, suggesting that a defect in toxin activation is a major resistance mechanism.     

Parasporal Body Formation via Overexpression of the Cry10Aa Toxin of Bacillus thuringiensis subsp. israelensis,and Cry10Aa-Cyt1Aa Synergism

Bacillus thuringiensis subsp. israelensis is the most widely used microbial control agent against mosquitoes and blackflies. Its insecticidal success is based on an arsenal of toxins, such as Cry4A, Cry4B, Cry11A, and Cyt1A, harbored in the parasporal crystal of the bacterium. A fifth toxin, Cry10Aa, is synthesized at very low levels; previous attempts to clone and express Cry10Aa were limited, and no parasporal body was formed. By using a new strategy, the whole Cry10A operon was cloned in the pSTAB vector, where both open reading frames ORF1 and ORF2 (and the gap between the two) were located, under the control of the cyt1A operon and the STAB-SD stabilizer sequence characteristic of this vector. Once the acrystalliferous mutant 4Q7 of B. thuringiensis subsp. israelensis was transformed with this construct, parasporal bodies were observed by phase-contrast microscopy and transmission electron microscopy. Discrete, ca. 0.9-μm amorphous parasporal bodies were observed in the mature sporangia, which were readily purified by gradient centrifugation once autolysis had occurred. Pure parasporal bodies showed two major bands of ca. 68 and 56 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These bands were further characterized by N-terminal sequencing of tryptic fragments using matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, which identified both bands as the products of ORF1 and ORF2, respectively. Bioassays against fourth-instar larvae of Aedes aegypti of spore-crystal complex and pure crystals of Cry10Aa gave estimated 50% lethal concentrations of 2,061 ng/ml and 239 ng/ml, respectively. Additionally, synergism was clearly detected between Cry10A and Cyt1A, as the synergistic levels (potentiation rates) were estimated at 13.3 for the mixture of Cyt1A crystals and Cry10Aa spore-crystal complex and 12.6 for the combination of Cyt1A and Cry10Aa pure crystals.The subspecies Bacillus thuringiensis subsp. israelensis (serotype H-14) was discovered by Goldberg and Margalit in 1977 (11). To date, its insecticidal potential has not been overcome by any other bacterium (or any biological control agent) as an effective control measure against mosquito and blackfly larvae (8). Recently, its toxicity spectrum has been expanded to a coleopteran pest, the coffee berry borer (Hypothenemus hampei) (23), indicating that this strain may have potential versatility. Also, the so-called pBtoxis megaplasmid harbored in this strain, containing all the endotoxin-encoding genes found in its parasporal crystal, including cry4A, cry4B, cry10A, cry11A, and cyt1A, was recently sequenced (1). Among many other interesting aspects of this serotype, the occurrence of this mosquitocidal arsenal in one strain and their synergistic interaction make this bacterium scientifically and technologically attractive.The parasporal crystal of B. thuringiensis subsp. israelensis contains large amounts of Cry4A, Cry4B, Cry11A, and Cyt1A toxins (14), and consequently, most of the knowledge about the toxicity of this strain has been focused on these proteins, acting either as a complex (31) or tested separately (6). Although the cry10Aa gene was originally cloned in 1986 (known then as cryIVC) (30), to date, little is known about cry10Aa and the protein it encodes, mostly due to its very low levels of expression (10) in B. thuringiensis subsp. israelensis. Interestingly, cry10Aa is an operon as it includes two open reading frames (ORFs), previously reported as pBt047 and pBt048 (hereafter referred to only as ORF1 and ORF2, respectively), separated by a 48-bp untranslated gap (1). ORF1 contains the complete δ-endotoxin sequence (active toxin), with a coding capacity for a 78-kDa protein. Interestingly, ORF2 shows high identity with the coding sequence of the C-terminal half of Cry4-type proteins, with a coding capacity for a 56-kDa protein. Therefore, it is believed that a putative ancestral cry10Aa gene is similar in size to the cry4-type genes (ca. 4 kbp), but either a small sequence had been inserted in the middle of the coding sequence or site mutations produced end codons (two end codons flank the gap) in this region (1).Previous attempts to clone and express the cry10Aa gene included ORF1 and only part of ORF2 (7, 10, 30). This was a reasonable strategy, as most of the so-called “complete” protoxins are partially digested to become active toxins (δ-endotoxins) (28), and ORF1 included the complete sequence to code the Cry10Aa δ-endotoxin. However, in all these cases, the expression levels were very low, and no parasporal body was formed. Similar results were obtained when the promoter was changed and a stabilizing sequence was added to the construction (13). The low expression levels achieved in these cases led to conclusions that assumed low toxic levels of Cry10Aa when tested against mosquito larvae (30). In spite of the low toxicity of Cry10Aa found against mosquito larvae, a synergistic effect was reported between Cry10Aa and Cry4Ba toxins in Culex (7). Obtaining high levels of expression and crystallization of Cry10Aa are required to properly characterize and understand the toxic spectrum of this protein.In this report, we show the formation of parasporal bodies of Cry10Aa, achieved by cloning the whole Cry10Aa operon under the control of the cyt1A promoter and the STAB-SD sequence. We also show that Cry10Aa is as toxic as most of the other B. thuringiensis subsp. israelensis toxins acting separately, and in synergism with the Cyt1A toxin.     

苏云金芽孢杆菌以色列亚种cyt1 Aa基因在球形芽孢杆菌中表达

将编码cyt1Aa基因和 p2 0蛋白基因的DNA片段分别克隆连接于两个不同的穿梭载体 pBU 4和pMK 3上 ,构建了重组质粒 pBA 30和 pMA 6,通过电击法 ,将重组质粒分别转化 B .s野生株2 2 97,获得了转化菌株Bs 97 30和Bs 97 6。SDS PAGE和Westernblot分析证实了cyt1Aa基因在转化菌株Bs 97 30中获得了表达 ,而在转化菌株Bs 97 6中未检测到cyt1Aa基因表达的蛋白。转化菌株Bs 97 30中 ,cyt1Aa基因与B .s二元毒素基因同步于菌体生长的对数期起始表达 ,并持续至芽孢形成。生测结果表明 ,转化菌株Bs 97 30中cyt1Aa基因的表达并未明显增强其对敏感和抗性致倦库蚊幼虫的毒力。其原因可能是弱毒性的 cyt1Aa蛋白在转化菌株中的表达量不高。     

Common, but complex, mode of resistance of Plutella xylostella to Bacillus thuringiensis toxins Cry1Ab and Cry1Ac

A field collected population of Plutella xylostella (SERD4) was selected in the laboratory with Bacillus thuringiensis endotoxins Cry1Ac (Cry1Ac-SEL) and Cry1Ab (Cry1Ab-SEL). Both subpopulations showed similar phenotypes: high resistance to the Cry1A toxins and little cross-resistance to Cry1Ca or Cry1D. A previous analysis of the Cry1Ac-SEL showed incompletely dominant resistance to Cry1Ac with more than one factor, at least one of which was sex influenced. In the present study reciprocal mass crosses between Cry1Ab-SEL and a laboratory susceptible population (ROTH) provided evidence that Cry1Ab resistance was also inherited as incompletely dominant trait with more than one factor, and at least one of the factors was sex influenced. Analysis of single pair mating indicated that Cry1Ab-SEL was still heterogeneous for Cry1Ab resistance genes, showing genes with different degrees of dominance. Binding studies showed a large reduction of specific binding of Cry1Ab and Cry1Ac to midgut membrane vesicles of the Cry1Ab-SEL subpopulation. Cry1Ab-SEL was found to be more susceptible to trypsin-activated Cry1Ab toxin than protoxin, although no defect in toxin activation was found. Present and previous results indicate a common basis of resistance to both Cry1Ab and Cry1Ac in selected subpopulations and suggest that a similar set of resistance genes are responsible for resistance to Cry1Ab and Cry1Ac and are selected whichever toxin was used. The possibility of an incompletely dominant trait of resistant to these toxins should be taken into account when considering refuge resistance management strategies.     

Evidence of the Importance of the Met115 for Bacillus thuringiensis subsp. israelensis Cyt1Aa Protein Cytolytic Activity in Escherichia coli

Cyt1Aa is a cytolytic toxin, found together with the delta-endotoxins in Bacillus thuringiensis subsp. israelensis parasporal insecticidal crystals. The latter are used as an environmental friendly insecticide against mosquitoes and black flies. Contrary to Cry delta-endotoxin, the mode of action of Cyt1Aa is not completely understood. In the absence of direct structural data, a novel mutated cyt1Aa gene was used to obtain indirect informations on Cyt1Aa conformation changes in the lipid membrane environment. A mutated cyt1Aa gene named cyt1A97 has been isolated from a B. thuringiensis israelensis strain named BUPM97. The nucleotide sequence predicted a protein of 249 amino acids residues with a calculated molecular mass of 27 kDa. Both nucleotide and amino acid sequences similarity analysis revealed that cyt1A97 presents one amino acid different from the native cyt1Aa gene. This mutation was located in the helix α C corresponding to a substitution of Met¹¹⁵ by a Thr. The heterologous expression of the cyt1A97 and another cyt1Aa-type gene called cyt1A98, not affected by such mutation used as control, was performed in Escherichia coli. It revealed that the mutated Cyt1A97 protein was over produced as inclusion bodies showing a very weak toxicity to E. coli contrarily to Cyt1A98 that stopped E. coli growth. Hence, hydrophobic residue Met at position 115 of Cyt1Aa should play a very important role for the maintenance of the structure and cytolytic functions of Cyt1Aa.     

Lack of Cross-Resistance to Cry19A from Bacillus thuringiensis subsp. jegathesan in Culex quinquefasciatus (Diptera: Culicidae) Resistant to Cry Toxins from Bacillus thuringiensis subsp. israelensis

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.     

Specific targeting to murine myeloma cells of Cyt1Aa toxin from Bacillus thuringiensis subspecies israelensis

Multiple myeloma is currently an incurable cancer of plasma B cells often characterized by overproduction of abnormally high quantities of a patient-specific, clonotypic immunoglobulin "M-protein." The M-protein is expressed on the cell membrane and secreted into the blood. We previously showed that ligand-toxin conjugates (LTC) incorporating the ribosome-inactivating Ricin-A toxin were very effective in specific cytolysis of the anti-ligand antibody-bearing target cells used as models for multiple myeloma. Here, we report on the incorporation of the membrane-disruptive Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis into LTCs targeted to murine myeloma cells. Proteolytically activated Cyt1Aa was conjugated chemically or genetically through either its amino or carboxyl termini to the major peptidic epitope VHFFKNIVTPRTP (p87-99) of the myelin basic protein. The recombinant fusion-encoding genes were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis through the shuttle vector pHT315. Both chemically conjugated and genetically fused LTCs were toxic to anti-myelin basic protein-expressing murine hybridoma cells, but the recombinant conjugates were more active. LTCs comprising the Cyt1Aa toxin might be useful anticancer agents. As a membrane-acting toxin, Cyt1Aa is not likely to induce development of resistant cell lines.     

Bacillus thuringiensis ssp. israelensis Cyt1Aa enhances activity of Cry11Aa toxin by facilitating the formation of a pre-pore oligomeric structure

Bacillus thuringiensis ssp. israelensis (Bti) has been used worldwide for the control of dipteran insect pests. This bacterium produces several Cry and Cyt toxins that individually show activity against mosquitoes but together show synergistic effect. Previous work demonstrated that Cyt1Aa synergizes the toxic activity of Cry11Aa by functioning as a membrane-bound receptor. In the case of Cry toxins active against lepidopteran insects, receptor interaction triggers the formation of a pre-pore oligomer that is responsible for pore formation and toxicity. In this work we report that binding of Cry11Aa to Cyt1Aa facilitates the formation of a Cry11Aa pre-pore oligomeric structure that is capable of forming pores in membrane vesicles. Cry11Aa and Cyt1A point mutants affected in binding and in synergism had a correlative effect on the formation of Cry11Aa pre-pore oligomer and on pore-formation activity of Cry11Aa. These data further support that Cyt1Aa interacts with Cry11Aa and demonstrate the molecular mechanism by which Cyt1Aa synergizes or suppresses resistance to Cry11Aa, by providing a binding site for Cry11Aa that will result in an efficient formation of Cry11Aa pre-pore that inserts into membranes and forms ionic pores.