Toxins

Research

14 pages, 483 KiB

Open AccessArticle

Evaluation of Cytotoxicity, Genotoxicity and Hematotoxicity of the Recombinant Spore-Crystal Complexes Cry1Ia, Cry10Aa and Cry1Ba6 from Bacillus thuringiensis in Swiss Mice

by Ingrid De Souza Freire, Ana Luisa Miranda-Vilela, Lilian Carla Pereira Barbosa, Erica Soares Martins, Rose Gomes Monnerat and Cesar Koppe Grisolia

Toxins 2014, 6(10), 2872-2885; https://doi.org/10.3390/toxins6102872 - 29 Sep 2014

Cited by 9 | Viewed by 6081

Abstract

The insecticidal properties of Cry-endotoxins from Bacillus thuringiensis (Bt) have long been used as spore-crystals in commercial spray formulations for insect control. Recently, some Bt-endotoxin genes have been cloned in many different plants. Toxicological evaluations of three spore-crystal endotoxins, BtCry1Ia, BtCry10Aa and BtCry1Ba6 [...] Read more.

The insecticidal properties of Cry-endotoxins from Bacillus thuringiensis (Bt) have long been used as spore-crystals in commercial spray formulations for insect control. Recently, some Bt-endotoxin genes have been cloned in many different plants. Toxicological evaluations of three spore-crystal endotoxins, BtCry1Ia, BtCry10Aa and BtCry1Ba6 from B. thuringiensis, were carried out on mice to understand their adverse effects on hematological systems and on genetic material. These three spore-crystals have shown toxic activity to the boll weevil, which is one of the most aggressive pests of the cotton crop. Cry1Ia, Cry10Aa and Cry1Ba6 did not increase the micronucleus frequency in the peripheral erythrocytes of mice and did not cause changes in the frequency of polychromatic erythrocytes. However, some hematologic disburbances were observed, specifically related to Cry1Ia and Cry1Ba6, respectively, for the erythroid and lymphoid lineage. Thus, although the profile of such adverse side effects can be related to their high level of exposure, which is not commonly found in the environment, results showed that these Bt spore-crystals were not harmless to mice, indicating that each spore-crystal endotoxin presents a characteristic profile of toxicity and might be investigated individually. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

14 pages, 572 KiB

Open AccessArticle

Inheritance Patterns, Dominance and Cross-Resistance of Cry1Ab- and Cry1Ac-Selected Ostrinia furnacalis (Guenée)

by Tiantao Zhang, Mingxia He, Angharad M. R. Gatehouse, Zhenying Wang, Martin G. Edwards, Qing Li and Kanglai He

Toxins 2014, 6(9), 2694-2707; https://doi.org/10.3390/toxins6092694 - 11 Sep 2014

Cited by 47 | Viewed by 7898

Abstract

Two colonies of Asian corn borer, Ostrinia furnacalis (Guenée), artificially selected from a Bt-susceptible colony (ACB-BtS) for resistance to Cry1Ab (ACB-AbR) and Cry1Ac (ACB-AcR) toxins, were used to analyze inheritance patterns of resistance to Cry1 toxins. ACB-AbR and ACB-AcR evolved significant levels of [...] Read more.

Two colonies of Asian corn borer, Ostrinia furnacalis (Guenée), artificially selected from a Bt-susceptible colony (ACB-BtS) for resistance to Cry1Ab (ACB-AbR) and Cry1Ac (ACB-AcR) toxins, were used to analyze inheritance patterns of resistance to Cry1 toxins. ACB-AbR and ACB-AcR evolved significant levels of resistance, with resistance ratios (RR) of 39-fold and 78.8-fold to Cry1Ab and Cry1Ac, respectively. The susceptibility of ACB-AbR larvae to Cry1Ac and Cry1F toxins, which had not previously been exposed, were significantly reduced, being >113-fold and 48-fold, respectively. Similarly, susceptibility of ACB-AcR larvae to Cry1Ab and Cry1F were also significantly reduced (RR > nine-fold, RR > 18-fold, respectively), indicating cross-resistance among Cry1Ab, Cry1Ac, and Cry1F toxins. However, ACB-AbR and ACB-AcR larvae were equally susceptible to Cry1Ie as were ACB-BtS larvae, indicating no cross-resistance between Cry1Ie and Cry1Ab or Cry1Ac toxins; this may provide considerable benefits in preventing or delaying the evolution of resistance in ACB to Cry1Ab and Cry1Ac toxins. Backcrossing studies indicated that resistance to Cry1Ab toxin was polygenic in ACB-AbR, but monogenic in ACB-AcR, whilst resistance to Cry1Ac toxin was primarily monogenic in both ACB-AbR and ACB-AcR, but polygenic as resistance increased. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

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18 pages, 732 KiB

Open AccessArticle

Downregulation and Mutation of a Cadherin Gene Associated with Cry1Ac Resistance in the Asian Corn Borer, Ostrinia furnacalis (Guenée)

by Tingting Jin, Xue Chang, Angharad M. R. Gatehouse, Zhenying Wang, Martin G. Edwards and Kanglai He

Toxins 2014, 6(9), 2676-2693; https://doi.org/10.3390/toxins6092676 - 11 Sep 2014

Cited by 33 | Viewed by 7975

Abstract

Development of resistance in target pests is a major threat to long-term use of transgenic crops expressing Bacillus thuringiensis (Bt) Cry toxins. To manage and/or delay the evolution of resistance in target insects through the implementation of effective strategies, it is [...] Read more.

Development of resistance in target pests is a major threat to long-term use of transgenic crops expressing Bacillus thuringiensis (Bt) Cry toxins. To manage and/or delay the evolution of resistance in target insects through the implementation of effective strategies, it is essential to understand the basis of resistance. One of the most important mechanisms of insect resistance to Bt crops is the alteration of the interactions between Cry toxins and their receptors in the midgut. A Cry1Ac-selected strain of Asian corn borer (ACB), Ostrinia furnacalis, a key pest of maize in China, evolved three mutant alleles of a cadherin-like protein (OfCAD) (MPR-r1, MPR-r2 and MPR-r3), which mapped within the toxin-binding region (TBR). Each of the three mutant alleles possessed two or three amino acid substitutions in this region, especially Thr¹⁴⁵⁷→Ser. In highly resistant larvae (ACB-Ac200), MPR-r2 had a 26-amino acid residue deletion in the TBR, which resulted in reduced binding of Cry1Ac compared to the MPR from the susceptible strain, suggesting that the number of amino acid deletions influences the level of resistance. Furthermore, downregulation of OfCAD gene (ofcad) transcription was observed in the Cry1Ac resistant strain, ACB-Ac24, suggesting that Cry1Ac resistance in ACB is associated with the downregulation of the transcript levels of the cadherin-like protein gene. The OfCAD identified from ACB exhibited a high degree of similarity to other members of the cadherin super-family in lepidopteran species. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

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18 pages, 971 KiB

Open AccessArticle

The Correlation of the Presence and Expression Levels of cry Genes with the Insecticidal Activities against Plutella xylostella for Bacillus thuringiensis Strains

by Ming-Lun Chen, Pin-Hsin Chen, Jen-Chieh Pang, Chia-Wei Lin, Chin-Fa Hwang and Hau-Yang Tsen

Toxins 2014, 6(8), 2453-2470; https://doi.org/10.3390/toxins6082453 - 19 Aug 2014

Cited by 6 | Viewed by 5864

Abstract

The use of Bacillus thuringiensis (Bt) strains with high insecticidal activity is essential for the preparation of bioinsecticide. In this study, for 60 Bt strains isolated in Taiwan, their genotypes and the correlation of some cry genes as well as the [...] Read more.

The use of Bacillus thuringiensis (Bt) strains with high insecticidal activity is essential for the preparation of bioinsecticide. In this study, for 60 Bt strains isolated in Taiwan, their genotypes and the correlation of some cry genes as well as the expression levels of cry1 genes, with their insecticidal activities against Plutella xylostella, were investigated. Pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) results revealed that the genotypes of these Bt strains are highly diversified. Also, a considerable number of the Bt strains isolated in Taiwan were found to have high insecticidal activities. Since strains that showed individual combined patterns of PFGE and RAPD exhibited distinct insecticidal activities against P. xylostella, thus, these genotypes may be useful for the identification of the new Bt strains and those which have been used in bioinsecticides. In addition, although the presence of cry2Aa1 may have a greater effect on the insecticidal activity of Bt strains in bioassay than other cry genes, only high expression level of cry1 genes plays a key role to determine the insecticidal activity of Bt strains. In conclusion, both RAPD and PFGE are effective in the differentiation of Bt strains. The presence of cry2Aa1 and, especially, the expression level of cry1 genes are useful for the prediction of the insecticidal activities of Bt strains against P. xylostella. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

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12 pages, 1126 KiB

Open AccessArticle

Toxicity of Parasporin-4 and Health Effects of Pro-parasporin-4 Diet in Mice

by Shiro Okumura, Hironori Koga, Kuniyo Inouye and Eiichi Mizuki

Toxins 2014, 6(7), 2115-2126; https://doi.org/10.3390/toxins6072115 - 16 Jul 2014

Cited by 4 | Viewed by 14409

Abstract

Parasporin-4 (PS4) is an aerolysin-type β-pore-forming toxin produced by Bacillus thuringiensis strain A1470. It exhibits specific cytotoxicity against human cancer cell lines; therefore, it is expected to be useful for the diagnosis and treatment of particular types of cancer cells. We examined the [...] Read more.

Parasporin-4 (PS4) is an aerolysin-type β-pore-forming toxin produced by Bacillus thuringiensis strain A1470. It exhibits specific cytotoxicity against human cancer cell lines; therefore, it is expected to be useful for the diagnosis and treatment of particular types of cancer cells. We examined the acute toxicity of PS4 on ICR mice. The LD₅₀ value was 160 μg/kg by a subcutaneous route. Potassium, ammonium, magnesium ion, creatinine, and urea nitrogen decreased in urine by the injection of PS4. Simultaneously, creatinine and urea nitrogen in mice serum increased. These results imply that PS4 impairs kidney function in mice. PS4 is obtained from Pro-parasporin-4 (ProPS4) by processing, and ProPS4 is produced by recombinant Escherichia coli as the inclusion body. The inclusion body of ProPS4 can be solubilized in a weak acid solution and activated by pepsin, implying that it would be solubilized and activated in the stomach of mammals after oral administration. Thus, the influence of the oral administration of it by C57BL/6J mice was examined. Although ProPS4 was activated to PS4 in the mouse digestive tract, any serious health hazard was not observed and there was no significant difference in body weight change. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

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14 pages, 1116 KiB

Open AccessArticle

Identification of Distinct Bacillus thuringiensis 4A4 Nematicidal Factors Using the Model Nematodes Pristionchus pacificus and Caenorhabditis elegans

by Igor Iatsenko, Angel Nikolov and Ralf J. Sommer

Toxins 2014, 6(7), 2050-2063; https://doi.org/10.3390/toxins6072050 - 14 Jul 2014

Cited by 22 | Viewed by 12280

Abstract

Bacillus thuringiensis has been extensively used for the biological control of insect pests. Nematicidal B. thuringiensis strains have also been identified; however, virulence factors of such strains are poorly investigated. Here, we describe virulence factors of the nematicidal B. thuringiensis 4A4 strain, using [...] Read more.

Bacillus thuringiensis has been extensively used for the biological control of insect pests. Nematicidal B. thuringiensis strains have also been identified; however, virulence factors of such strains are poorly investigated. Here, we describe virulence factors of the nematicidal B. thuringiensis 4A4 strain, using the model nematodes Pristionchus pacificus and Caenorhabditis elegans. We show that B. thuringiensis 4A4 kills both nematodes via intestinal damage. Whole genome sequencing of B. thuringiensis 4A4 identified Cry21Ha, Cry1Ba, Vip1/Vip2 and β-exotoxin as potential nematicidal factors. Only Cry21Ha showed toxicity to C. elegans, while neither Cry nor Vip toxins were active against P. pacificus, when expressed in E. coli. Purified crystals also failed to intoxicate P. pacificus, while autoclaved spore-crystal mixture of B. thuringiensis 4A4 retained toxicity, suggesting that primary β-exotoxin is responsible for P. pacificus killing. In support of this, we found that a β-exotoxin-deficient variant of B. thuringiensis 4A4, generated by plasmid curing lost virulence to the nematodes. Thus, using two model nematodes we revealed virulence factors of the nematicidal strain B. thuringiensis 4A4 and showed the multifactorial nature of its virulence. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

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14 pages, 382 KiB

Open AccessArticle

Cloning and Characterization of a Unique Cytotoxic Protein Parasporin-5 Produced by Bacillus thuringiensis A1100 Strain

by Keisuke Ekino, Shiro Okumura, Tomoyuki Ishikawa, Sakae Kitada, Hiroyuki Saitoh, Tetsuyuki Akao, Takuji Oka, Yoshiyuki Nomura, Michio Ohba, Takashi Shin and Eiichi Mizuki

Toxins 2014, 6(6), 1882-1895; https://doi.org/10.3390/toxins6061882 - 18 Jun 2014

Cited by 24 | Viewed by 9495

Abstract

Parasporin is the cytocidal protein present in the parasporal inclusion of the non-insecticidal Bacillus thuringiensis strains, which has no hemolytic activity but has cytocidal activities, preferentially killing cancer cells. In this study, we characterized a cytocidal protein that belongs to this category, which [...] Read more.

Parasporin is the cytocidal protein present in the parasporal inclusion of the non-insecticidal Bacillus thuringiensis strains, which has no hemolytic activity but has cytocidal activities, preferentially killing cancer cells. In this study, we characterized a cytocidal protein that belongs to this category, which was designated parasporin-5 (PS5). PS5 was purified from B. thuringiensis serovar tohokuensis strain A1100 based on its cytocidal activity against human leukemic T cells (MOLT-4). The 50% effective concentration (EC₅₀) of PS5 to MOLT-4 cells was approximately 0.075 μg/mL. PS5 was expressed as a 33.8-kDa inactive precursor protein and exhibited cytocidal activity only when degraded by protease at the C-terminal into smaller molecules of 29.8 kDa. Although PS5 showed no significant homology with other known parasporins, a Position Specific Iterative-Basic Local Alignment Search Tool (PSI-BLAST) search revealed that the protein showed slight homology to, not only some B. thuringiensis Cry toxins, but also to aerolysin-type β-pore-forming toxins (β-PFTs). The recombinant PS5 protein could be obtained as an active protein only when it was expressed in a precursor followed by processing with proteinase K. The cytotoxic activities of the protein against various mammalian cell lines were evaluated. PS5 showed strong cytocidal activity to seven of 18 mammalian cell lines tested, and low to no cytotoxicity to the others. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

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17 pages, 358 KiB

Open AccessArticle

Requirement of Simultaneous Assessment of Crystal- and Supernatant-Related Entomotoxic Activities of Bacillus thuringiensis Strains for Biocontrol-Product Development

by Ronaldo Costa Argôlo-Filho, Robson Luz Costa, Daniele Heloisa Pinheiro, Fábio Mathias Corrêa, Fernando Hercos Valicente, Alan William Vilela Pomella and Leandro Lopes Loguercio

Toxins 2014, 6(5), 1598-1614; https://doi.org/10.3390/toxins6051598 - 20 May 2014

Cited by 5 | Viewed by 5999

Abstract

Bioinsecticides with lower concentrations of endospores/crystals and without loss of efficiency are economically advantageous for pest biocontrol. In addition to Cry proteins, other Bacillus thuringiensis (Bt) toxins in culture supernatants (SN) have biocontrol potential (e.g., Vip3A, Cry1I, Sip1), whereas others are [...] Read more.

Bioinsecticides with lower concentrations of endospores/crystals and without loss of efficiency are economically advantageous for pest biocontrol. In addition to Cry proteins, other Bacillus thuringiensis (Bt) toxins in culture supernatants (SN) have biocontrol potential (e.g., Vip3A, Cry1I, Sip1), whereas others are unwanted (β-exotoxins), as they display widespread toxicity across taxa. A strain simultaneously providing distinct toxin activities in crystals and SN would be desirable for bioinsecticides development; however, strains secreting β-exotoxins should be discarded, independently of other useful entomotoxins. Entomotoxicity of crystals and SN from a Brazilian Bt tolworthi strain (Btt01) was tested against Spodoptera frugiperda to assess the potential for biocontrol-product development based on more than one type of toxin/activity. Tests showed that 10⁷ endospores mL⁻¹ caused >80% of larvae mortality, suggesting Btt01 may be used in similar concentrations as those of other Bt-based biopesticides. When it was applied to cornfields, a significant 60% reduction of larvae infestation was observed. However, bioassays with Btt01 SN revealed a thermostable toxic activity. Physicochemical characterization strongly suggests the presence of unwanted β-exotoxins, with isolate-specific temporal variation in its secretion. Knowledge of the temporal pattern of secretion/activity in culture for all forms of toxins produced by a single strain is required to both detect useful activities and avoid the potential lack of identification of undesirable toxins. These findings are discussed in the contexts of commercial Bt product development, advantages of multiple-activity strains, and care and handling recommended for large-scale fermentation systems. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

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15 pages, 767 KiB

Open AccessArticle

Draft Genome Sequences of Two Bacillus thuringiensis Strains and Characterization of a Putative 41.9-kDa Insecticidal Toxin

by Leopoldo Palma, Delia Muñoz, Colin Berry, Jesús Murillo and Primitivo Caballero

Toxins 2014, 6(5), 1490-1504; https://doi.org/10.3390/toxins6051490 - 30 Apr 2014

Cited by 24 | Viewed by 8973

Abstract

In this work, we report the genome sequencing of two Bacillus thuringiensis strains using Illumina next-generation sequencing technology (NGS). Strain Hu4-2, toxic to many lepidopteran pest species and to some mosquitoes, encoded genes for two insecticidal crystal (Cry) proteins, cry1Ia and cry9Ea, [...] Read more.

In this work, we report the genome sequencing of two Bacillus thuringiensis strains using Illumina next-generation sequencing technology (NGS). Strain Hu4-2, toxic to many lepidopteran pest species and to some mosquitoes, encoded genes for two insecticidal crystal (Cry) proteins, cry1Ia and cry9Ea, and a vegetative insecticidal protein (Vip) gene, vip3Ca2. Strain Leapi01 contained genes coding for seven Cry proteins (cry1Aa, cry1Ca, cry1Da, cry2Ab, cry9Ea and two cry1Ia gene variants) and a vip3 gene (vip3Aa10). A putative novel insecticidal protein gene 1143 bp long was found in both strains, whose sequences exhibited 100% nucleotide identity. The predicted protein showed 57 and 100% pairwise identity to protein sequence 72 from a patented Bt strain (US8318900) and to a putative 41.9-kDa insecticidal toxin from Bacillus cereus, respectively. The 41.9-kDa protein, containing a C-terminal 6× HisTag fusion, was expressed in Escherichia coli and tested for the first time against four lepidopteran species (Mamestra brassicae, Ostrinia nubilalis, Spodoptera frugiperda and S. littoralis) and the green-peach aphid Myzus persicae at doses as high as 4.8 µg/cm² and 1.5 mg/mL, respectively. At these protein concentrations, the recombinant 41.9-kDa protein caused no mortality or symptoms of impaired growth against any of the insects tested, suggesting that these species are outside the protein’s target range or that the protein may not, in fact, be toxic. While the use of the polymerase chain reaction has allowed a significant increase in the number of Bt insecticidal genes characterized to date, novel NGS technologies promise a much faster, cheaper and efficient screening of Bt pesticidal proteins. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

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21 pages, 586 KiB

Open AccessArticle

Changes in Gene Expression in the Larval Gut of Ostrinia nubilalis in Response to Bacillus thuringiensis Cry1Ab Protoxin Ingestion

by Jianxiu Yao, Lawrent L. Buschman, Nanyan Lu, Chitvan Khajuria and Kun Yan Zhu

Toxins 2014, 6(4), 1274-1294; https://doi.org/10.3390/toxins6041274 - 3 Apr 2014

Cited by 17 | Viewed by 9226

Abstract

We developed a microarray based on 2895 unique transcripts assembled from 15,000 cDNA sequences from the European corn borer (Ostrinia nubilalis) larval gut. This microarray was used to monitor gene expression in early third-instar larvae of Bacillus thuringiensis (Bt)-susceptible O. nubilalis [...] Read more.

We developed a microarray based on 2895 unique transcripts assembled from 15,000 cDNA sequences from the European corn borer (Ostrinia nubilalis) larval gut. This microarray was used to monitor gene expression in early third-instar larvae of Bacillus thuringiensis (Bt)-susceptible O. nubilalis after 6 h feeding on diet, with or without the Bt Cry1Ab protoxin. We identified 174 transcripts, for which the expression was changed more than two-fold in the gut of the larvae fed Cry1Ab protoxin (p < 0.05), representing 80 down-regulated and 94 up-regulated transcripts. Among 174 differentially expressed transcripts, 13 transcripts putatively encode proteins that are potentially involved in Bt toxicity, and these transcripts include eight serine proteases, three aminopeptidases, one alkaline phosphatase, and one cadherin. The expressions of trypsin-like protease and three aminopeptidase transcripts were variable, but two potential Bt-binding proteins, alkaline phosphatase and cadherin were consistently up-regulated in larvae fed Cry1Ab protoxin. The significantly up and down-regulated transcripts may be involved in Cry1Ab toxicity by activation, degradation, toxin binding, and other related cellular responses. This study is a preliminary survey of Cry1Ab protoxin-induced transcriptional responses in O. nubilalis gut and our results are expected to help with further studies on Bt toxin-insect interactions at the molecular level. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

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Review

Jump to: Research, Other

30 pages, 793 KiB

Open AccessReview

Bacillus thuringiensis Toxins: An Overview of Their Biocidal Activity

by Leopoldo Palma, Delia Muñoz, Colin Berry, Jesús Murillo and Primitivo Caballero

Toxins 2014, 6(12), 3296-3325; https://doi.org/10.3390/toxins6123296 - 11 Dec 2014

Cited by 571 | Viewed by 45669

Abstract

Bacillus thuringiensis (Bt) is a Gram positive, spore-forming bacterium that synthesizes parasporal crystalline inclusions containing Cry and Cyt proteins, some of which are toxic against a wide range of insect orders, nematodes and human-cancer cells. These toxins have been successfully used as bioinsecticides [...] Read more.

Bacillus thuringiensis (Bt) is a Gram positive, spore-forming bacterium that synthesizes parasporal crystalline inclusions containing Cry and Cyt proteins, some of which are toxic against a wide range of insect orders, nematodes and human-cancer cells. These toxins have been successfully used as bioinsecticides against caterpillars, beetles, and flies, including mosquitoes and blackflies. Bt also synthesizes insecticidal proteins during the vegetative growth phase, which are subsequently secreted into the growth medium. These proteins are commonly known as vegetative insecticidal proteins (Vips) and hold insecticidal activity against lepidopteran, coleopteran and some homopteran pests. A less well characterized secretory protein with no amino acid similarity to Vip proteins has shown insecticidal activity against coleopteran pests and is termed Sip (secreted insecticidal protein). Bin-like and ETX_MTX2-family proteins (Pfam PF03318), which share amino acid similarities with mosquitocidal binary (Bin) and Mtx2 toxins, respectively, from Lysinibacillus sphaericus, are also produced by some Bt strains. In addition, vast numbers of Bt isolates naturally present in the soil and the phylloplane also synthesize crystal proteins whose biological activity is still unknown. In this review, we provide an updated overview of the known active Bt toxins to date and discuss their activities. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

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23 pages, 790 KiB

Open AccessReview

Bt Toxin Modification for Enhanced Efficacy

by Benjamin R. Deist, Michael A. Rausch, Maria Teresa Fernandez-Luna, Michael J. Adang and Bryony C. Bonning

Toxins 2014, 6(10), 3005-3027; https://doi.org/10.3390/toxins6103005 - 22 Oct 2014

Cited by 63 | Viewed by 15618

Abstract

Insect-specific toxins derived from Bacillus thuringiensis (Bt) provide a valuable resource for pest suppression. Here we review the different strategies that have been employed to enhance toxicity against specific target species including those that have evolved resistance to Bt, or to modify the [...] Read more.

Insect-specific toxins derived from Bacillus thuringiensis (Bt) provide a valuable resource for pest suppression. Here we review the different strategies that have been employed to enhance toxicity against specific target species including those that have evolved resistance to Bt, or to modify the host range of Bt crystal (Cry) and cytolytic (Cyt) toxins. These strategies include toxin truncation, modification of protease cleavage sites, domain swapping, site-directed mutagenesis, peptide addition, and phage display screens for mutated toxins with enhanced activity. Toxin optimization provides a useful approach to extend the utility of these proteins for suppression of pests that exhibit low susceptibility to native Bt toxins, and to overcome field resistance. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

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39 pages, 3204 KiB

Open AccessReview

Structural Insights into Bacillus thuringiensis Cry, Cyt and Parasporin Toxins

by Chengchen Xu, Bi-Cheng Wang, Ziniu Yu and Ming Sun

Toxins 2014, 6(9), 2732-2770; https://doi.org/10.3390/toxins6092732 - 16 Sep 2014

Cited by 131 | Viewed by 20661

Abstract

Since the first X-ray structure of Cry3Aa was revealed in 1991, numerous structures of B. thuringiensis toxins have been determined and published. In recent years, functional studies on the mode of action and resistance mechanism have been proposed, which notably promoted the developments [...] Read more.

Since the first X-ray structure of Cry3Aa was revealed in 1991, numerous structures of B. thuringiensis toxins have been determined and published. In recent years, functional studies on the mode of action and resistance mechanism have been proposed, which notably promoted the developments of biological insecticides and insect-resistant transgenic crops. With the exploration of known pore-forming toxins (PFTs) structures, similarities between PFTs and B. thuringiensis toxins have provided great insights into receptor binding interactions and conformational changes from water-soluble to membrane pore-forming state of B. thuringiensis toxins. This review mainly focuses on the latest discoveries of the toxin working mechanism, with the emphasis on structural related progress. Based on the structural features, B. thuringiensis Cry, Cyt and parasporin toxins could be divided into three categories: three-domain type α-PFTs, Cyt toxin type β-PFTs and aerolysin type β-PFTs. Structures from each group are elucidated and discussed in relation to the latest data, respectively. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

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31 pages, 7741 KiB

Open AccessReview

Molecular Approaches to Improve the Insecticidal Activity of Bacillus thuringiensis Cry Toxins

by Wagner A. Lucena, Patrícia B. Pelegrini, Diogo Martins-de-Sa, Fernando C. A. Fonseca, Jose E. Gomes, Jr., Leonardo L. P. De Macedo, Maria Cristina M. Da Silva, Raquel S. Oliveira and Maria F. Grossi-de-Sa

Toxins 2014, 6(8), 2393-2423; https://doi.org/10.3390/toxins6082393 - 13 Aug 2014

Cited by 24 | Viewed by 10307

Abstract

Bacillus thuringiensis (Bt) is a gram-positive spore-forming soil bacterium that is distributed worldwide. Originally recognized as a pathogen of the silkworm, several strains were found on epizootic events in insect pests. In the 1960s, Bt began to be successfully used to control insect [...] Read more.

Bacillus thuringiensis (Bt) is a gram-positive spore-forming soil bacterium that is distributed worldwide. Originally recognized as a pathogen of the silkworm, several strains were found on epizootic events in insect pests. In the 1960s, Bt began to be successfully used to control insect pests in agriculture, particularly because of its specificity, which reflects directly on their lack of cytotoxicity to human health, non-target organisms and the environment. Since the introduction of transgenic plants expressing Bt genes in the mid-1980s, numerous methodologies have been used to search for and improve toxins derived from native Bt strains. These improvements directly influence the increase in productivity and the decreased use of chemical insecticides on Bt-crops. Recently, DNA shuffling and in silico evaluations are emerging as promising tools for the development and exploration of mutant Bt toxins with enhanced activity against target insect pests. In this report, we describe natural and in vitro evolution of Cry toxins, as well as their relevance in the mechanism of action for insect control. Moreover, the use of DNA shuffling to improve two Bt toxins will be discussed together with in silico analyses of the generated mutations to evaluate their potential effect on protein structure and cytotoxicity. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

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17 pages, 1142 KiB

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Quorum Sensing in Bacillus thuringiensis Is Required for Completion of a Full Infectious Cycle in the Insect

by Leyla Slamti, Stéphane Perchat, Eugénie Huillet and Didier Lereclus

Toxins 2014, 6(8), 2239-2255; https://doi.org/10.3390/toxins6082239 - 31 Jul 2014

Cited by 79 | Viewed by 14127

Abstract

Bacterial cell-cell communication or quorum sensing (QS) is a biological process commonly described as allowing bacteria belonging to a same pherotype to coordinate gene expression to cell density. In Gram-positive bacteria, cell-cell communication mainly relies on cytoplasmic sensors regulated by secreted and re-imported [...] Read more.

Bacterial cell-cell communication or quorum sensing (QS) is a biological process commonly described as allowing bacteria belonging to a same pherotype to coordinate gene expression to cell density. In Gram-positive bacteria, cell-cell communication mainly relies on cytoplasmic sensors regulated by secreted and re-imported signaling peptides. The Bacillus quorum sensors Rap, NprR, and PlcR were previously identified as the first members of a new protein family called RNPP. Except for the Rap proteins, these RNPP regulators are transcription factors that directly regulate gene expression. QS regulates important biological functions in bacteria of the Bacillus cereus group. PlcR was first characterized as the main regulator of virulence in B. thuringiensis and B. cereus. More recently, the PlcR-like regulator PlcRa was characterized for its role in cysteine metabolism and in resistance to oxidative stress. The NprR regulator controls the necrotrophic properties allowing the bacteria to survive in the infected host. The Rap proteins negatively affect sporulation via their interaction with a phosphorelay protein involved in the activation of Spo0A, the master regulator of this differentiation pathway. In this review we aim at providing a complete picture of the QS systems that are sequentially activated during the lifecycle of B. cereus and B. thuringiensis in an insect model of infection. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

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10 pages, 871 KiB

Open AccessReview

Thuringiensin: A Thermostable Secondary Metabolite from Bacillus thuringiensis with Insecticidal Activity against a Wide Range of Insects

by Xiaoyan Liu, Lifang Ruan, Donghai Peng, Lin Li, Ming Sun and Ziniu Yu

Toxins 2014, 6(8), 2229-2238; https://doi.org/10.3390/toxins6082229 - 25 Jul 2014

Cited by 42 | Viewed by 11975

Abstract

Thuringiensin (Thu), also known as β-exotoxin, is a thermostable secondary metabolite secreted by Bacillus thuringiensis. It has insecticidal activity against a wide range of insects, including species belonging to the orders Diptera, Coleoptera, Lepidoptera, Hymenoptera, Orthoptera, and Isoptera, and several nematode species. [...] Read more.

Thuringiensin (Thu), also known as β-exotoxin, is a thermostable secondary metabolite secreted by Bacillus thuringiensis. It has insecticidal activity against a wide range of insects, including species belonging to the orders Diptera, Coleoptera, Lepidoptera, Hymenoptera, Orthoptera, and Isoptera, and several nematode species. The chemical formula of Thu is C₂₂H₃₂O₁₉N₅P, and it is composed of adenosine, glucose, phosphoric acid, and gluconic diacid. In contrast to the more frequently studied insecticidal crystal protein, Thu is not a protein but a small molecule oligosaccharide. In this review, a detailed and updated description of the characteristics, structure, insecticidal mechanism, separation and purification technology, and genetic determinants of Thu is provided. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

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16 pages, 777 KiB

Open AccessReview

Regulation of cry Gene Expression in Bacillus thuringiensis

by Chao Deng, Qi Peng, Fuping Song and Didier Lereclus

Toxins 2014, 6(7), 2194-2209; https://doi.org/10.3390/toxins6072194 - 23 Jul 2014

Cited by 50 | Viewed by 14021

Abstract

Bacillus thuringiensis differs from the closely related Bacillus cereus group species by its ability to produce crystalline inclusions. The production of these crystals mainly results from the expression of the cry genes, from the stability of their transcripts and from the synthesis, accumulation [...] Read more.

Bacillus thuringiensis differs from the closely related Bacillus cereus group species by its ability to produce crystalline inclusions. The production of these crystals mainly results from the expression of the cry genes, from the stability of their transcripts and from the synthesis, accumulation and crystallization of large amounts of insecticidal Cry proteins. This process normally coincides with sporulation and is regulated by various factors operating at the transcriptional, post-transcriptional, metabolic and post-translational levels. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

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22 pages, 379 KiB

Open AccessReview

Bacillus thuringiensis subsp. israelensis and Its Dipteran-Specific Toxins

by Eitan Ben-Dov

Toxins 2014, 6(4), 1222-1243; https://doi.org/10.3390/toxins6041222 - 28 Mar 2014

Cited by 188 | Viewed by 14989

Abstract

Bacillus thuringiensis subsp. israelensis (Bti) is the first Bacillus thuringiensis to be found and used as an effective biological control agent against larvae of many mosquito and black fly species around the world. Its larvicidal activity resides in four major (of [...] Read more.

Bacillus thuringiensis subsp. israelensis (Bti) is the first Bacillus thuringiensis to be found and used as an effective biological control agent against larvae of many mosquito and black fly species around the world. Its larvicidal activity resides in four major (of 134, 128, 72 and 27 kDa) and at least two minor (of 78 and 29 kDa) polypeptides encoded respectively by cry4Aa, cry4Ba, cry11Aa, cyt1Aa, cry10Aa and cyt2Ba, all mapped on the 128 kb plasmid known as pBtoxis. These six δ-endotoxins form a complex parasporal crystalline body with remarkably high, specific and different toxicities to Aedes, Culex and Anopheles larvae. Cry toxins are composed of three domains (perforating domain I and receptor binding II and III) and create cation-selective channels, whereas Cyts are composed of one domain that acts as well as a detergent-like membrane perforator. Despite the low toxicities of Cyt1Aa and Cyt2Ba alone against exposed larvae, they are highly synergistic with the Cry toxins and hence their combinations prevent emergence of resistance in the targets. The lack of significant levels of resistance in field mosquito populations treated for decades with Bti-bioinsecticide suggests that this bacterium will be an effective biocontrol agent for years to come. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

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2 pages, 149 KiB

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Response to Leopoldo Palma. Comments on Ekino et al. Cloning and Characterization of a Unique Cytotoxic Protein Parasporin-5 Produced by Bacillus thuringiensis A1100 Strain. Toxins 2014, 6, 1882–1895

by Keisuke Ekino, Shiro Okumura, Tomoyuki Ishikawa, Sakae Kitada, Hiroyuki Saitoh, Tetsuyuki Akao, Takuji Oka, Yoshiyuki Nomura, Michio Ohba, Takashi Shin and Eiichi Mizuki

Toxins 2015, 7(12), 5096-5097; https://doi.org/10.3390/toxins7124866 - 27 Nov 2015

Viewed by 4770

Abstract

I appreciate the thoughtful comments from Dr. Leopoldo Palma [1] on our research about cytotoxic protein parasporin-5 produced by Bacillus thuringiensis (BT) A1100 strain [2]. [...] Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

2 pages, 149 KiB

Open AccessComment

Comments on Ekino et al. Cloning and Characterization of a Unique Cytotoxic Protein Parasporin-5 Produced by Bacillus thuringiensis A1100 Strain. Toxins 2014, 6, 1882–1895

by Leopoldo Palma

Toxins 2015, 7(12), 5094-5095; https://doi.org/10.3390/toxins7124865 - 27 Nov 2015

Cited by 1 | Viewed by 4549

Abstract

Ekino et al. [1] reported the cloning and characterization of a novel cytotoxic protein (Parasporin-5) produced by Bacillus thuringiensis strain A1100. [...] Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins)

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