Biological Control by Bacillus thuringiensis subsp. israelensis

Margalith, Yoel; Ben‐Dov, Eitan

doi:10.1201/9781439822685.ch8

Cited by 47 publications

(60 citation statements)

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“…13, FL, and U136 cells were treated for 48 h with different concentrations of conjugates 1 or 2 (Cyt1Aa cleaved at both termini and chemically cross-linked to the carboxyl or amino terminus of MBPp, respectively). Both conjugates caused decreased viability (MTT test) at doses above 3 g ml…”

Section: Resultsmentioning

confidence: 99%

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Specific Targeting to Murine Myeloma Cells of Cyt1Aa Toxin from Bacillus thuringiensis Subspecies israelensis

Cohen

Cahan

Ben‐Dov

et al. 2007

Journal of Biological Chemistry

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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.Chemotherapy has for many years been an important component of the cancer therapeutic arsenal, but lack of target cell specificity of most anticancer drugs leads to concentration-dependent toxicity of normal cells. The resultant use of suboptimal therapeutic doses precludes the elimination of all the cancer cells, a situation that encourages clinical recurrence of the tumor (1) as well as emergence of resistant clones (2). This scenario has stimulated development of a variety of targeted drug delivery systems, the advantages of which include, aside from target cell specificity, reduced drug dosage and increased cytotoxic efficacy (reviewed in Refs. 3, 4).As components of the targeting system, delivery moieties with a wide range of biological functions have been employed, such as monoclonal antibodies, receptor binding peptides, liposomes, and nucleotide aptamers (4 -6). Most of the cytotoxic components currently used are designed to interfere with intracellular biochemical processes (7-10). For instance, we recently demonstrated that delivery of the ribosome-inactivating protein Ricin-A efficiently destroys murine myeloma cells (11) and that targeted, chemiluminescent activation of the photodynamic process results in endomembrane destruction (12). The activity of these types of cytotoxic systems requires endocytosis, and this process often induces intracellular activation of resistance mechanisms (2). There is therefore a need to expand the portfolio of cytotoxic agents, for instance to those whose mode of action is endocytosis-independent.Cyt1Aa is a plasma membrane-acting cytotoxin isolated from the paracrystalline ␦-endotoxin of the bacterium Bacillus thurin...

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Section: Resultsmentioning

confidence: 99%

“…Cyt1Aa is a plasma membrane-acting cytotoxin isolated from the paracrystalline ␦-endotoxin of the bacterium Bacillus thuringiensis israelensis (Bti) 3 (13). It is a major component (45-50%) of the mosquito-larvicidal crystal proteins of Bti, displaying relatively low larvicidal activity per se but high synergy with other Bti toxins (14 -20).…”

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confidence: 99%

Specific Targeting to Murine Myeloma Cells of Cyt1Aa Toxin from Bacillus thuringiensis Subspecies israelensis

Cohen

Cahan

Ben‐Dov

et al. 2007

Journal of Biological Chemistry

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“…3 To achieve a real biological control, the vector must multiply in the same niche as its target. Photo-synthetic cyanobacteria have several additional features that render them excellent candidates to control mosquito larvae:…”

Section: Cyanobacteria To Deliver Bti Toxins Against Mosquitoesmentioning

confidence: 99%

“…3 Most importantly, no resistance has been observed in nature after about 30 years of extensive use worldwide. 4 Different activities and modes of action of its four major toxins form a lethal combination against larvae of all mosquito species tested.…”

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Transgenic organisms expressing genes fromBacillus thuringiensisto combat insect pests

et al. 2010

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Bti is highly efficient and specific against mosquito and black fly larvae. 3 Most importantly, no resistance has been observed in nature after about 30 years of extensive use worldwide. 4 Different activities and modes of action of its four major toxins form a lethal combination against larvae of all mosquito species tested. 5 Resistance is not selected due to synergy among Bti components, mostly the lowtoxic, non-specific Cyt1Aa. High synergy levels affected by Cyt1Aa were observed by Crickmore 6 and Wirth, 7 the latter also demonstrated that Cyt1Aa prevented selection of resistant mosquitoes. 8The question raised was whether a combination of antiLepidopteran toxins with Cyt1Aa imitates this rare advantage of Bti. To partially answer this question, two genes were cloned for expression in Escherichia coli, cry1Ac (from Bt ssp. kurstaki) and cry1Ca (from Bt ssp. aizawai), with and without cyt1Aa, and tested against three pests, Helicoverpa armigera, Pectinophora gossypiella and Spodoptera littoralis.9 Co-expression of all three genes, and with p20 encoding an accessory protein (for reasons beyond Various subspecies (ssp.) of Bacillus thuringiensis (Bt) are considered the best agents known so far to control insects, being highly specific and safe, easily mass produced and with long shelf life.1 The para-crystalline body that is produced during sporulation in the exosporium includes polypeptides named d-endotoxins, each killing a specific set of insects. The different entomopathogenic toxins of various Bt ssp. can be manipulated genetically in an educated way to construct more efficient transgenic bacteria or plants that express combinations of toxin genes to control pests.2 Joint research projects in our respective laboratories during the last decade demonstrate what can be done by implementing certain ideas using molecular biology with Bt ssp. israelensis (Bti) as a model system. Here, we describe our progress achieved with Gram-negative bacterial species, including cyanobacteria, and some preliminary experiments to form transgenic plants, mainly to control mosquitoes (Diptera), but also a particular Lepidopteran and Coleopteran pest species. In addition, a system is described by which environment-damaging genes can be removed from the recombinants thus alleviating procedures for obtaining permits to release them in nature.

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“…The genes encoding different protein toxins are normally associated with large plasmids in B. thuringiensis subsp. israelensis (20,26) and are named cry4A, cry4B, cry11A, and cytA. cry4A and cry4B code for the 135-kDa and 125-kDa protoxins, respectively, which are activated by the gut proteases in the alkaline conditions of the insect gut.…”

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Loss of Catabolite Repression Function of HPr, the Phosphocarrier Protein of the Bacterial Phosphotransferase System, Affects Expression of the cry4A Toxin Gene in Bacillus thuringiensis subsp. israelensis

Khan

Banerjee-Bhatnagar

2002

J Bacteriol

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HPr, the phosphocarrier protein of the bacterial phosphotransferase system, mediates catabolite repression of a number of operons in gram-positive bacteria. In order to participate in the regulatory process, HPr is activated by phosphorylation of a conserved serine-46 residue. To study the potential role of HPr in the regulation of Cry4A protoxin synthesis in Bacillus thuringiensis subsp. israelensis, we produced a catabolite repression-negative mutant by replacing the wild-type copy of the ptsH gene with a mutated copy in which the conserved serine residue of HPr was replaced with an alanine. HPr isolated from the mutant strain was not phosphorylated at Ser-45 by HPr kinase, but phosphorylation at His-14 was found to occur normally. The enzyme I and HPr kinase activities of the mutant were not affected. Analysis of the B. thuringiensis subsp. israelensis mutant harboring ptsH-S45A in the chromosome showed that cry4A expression was derepressed from the inhibitory effect of glucose. The mutant strain produced both cry4A and 35 gene transcripts 4 h ahead of the parent strain, but there was no effect on 28 synthesis. In wild-type B. thuringiensis subsp. israelensis cells, cry4A mRNA was observed from 12 h onwards, while in the mutant it appeared at 8 h and was produced for a longer period. The total amount of cry4A transcripts produced by the mutant was higher than by the parent strain. There was a 60 to 70% reduction in the sporulation efficiency of the mutant B. thuringiensis subsp. israelensis strain compared to the wild-type strain.Biological control of dipteran pests in general and mosquitoes in particular has been a subject of primary importance for many years. Bacillus thuringiensis subsp. israelensis has been found to be the most effective microbial strain to date, possessing all the desirable properties of an ideal biocontrol agent. B. thuringiensis subsp. israelensis produces larvicidal crystal proteins in the stationary phase, concomitant with sporulation. The larvicidal activity of the parasporal crystals is attributed to the ␦-endotoxin, composed of at least four major polypeptide species of about 27, 72, 128, and 135 kDa, which act synergistically in the manifestation of toxicity (27,32). The high levels of toxin accumulation are controlled by a variety of mechanisms at the transcriptional, posttranscriptional, and posttranslational levels (2). The genes encoding different protein toxins are normally associated with large plasmids in B. thuringiensis subsp. israelensis (20,26) and are named cry4A, cry4B, cry11A, and cytA. cry4A and cry4B code for the 135-kDa and 125-kDa protoxins, respectively, which are activated by the gut proteases in the alkaline conditions of the insect gut.In sporulating cells of B. thuringiensis, the accumulation of large amounts of toxin proteins is achieved by expression from strong promoters associated with the gene. As in Bacillus subtilis, the developmental process is temporally and spatially regulated at the transcriptional level in B. thuringiensis by successive activati...

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Biological Control by Bacillus thuringiensis subsp. israelensis

Cited by 47 publications

References 250 publications

Specific Targeting to Murine Myeloma Cells of Cyt1Aa Toxin from Bacillus thuringiensis Subspecies israelensis

Specific Targeting to Murine Myeloma Cells of Cyt1Aa Toxin from Bacillus thuringiensis Subspecies israelensis

Transgenic organisms expressing genes fromBacillus thuringiensisto combat insect pests

Loss of Catabolite Repression Function of HPr, the Phosphocarrier Protein of the Bacterial Phosphotransferase System, Affects Expression of the cry4A Toxin Gene in Bacillus thuringiensis subsp. israelensis

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