Toxins

Editorial

Jump to: Research, Review, Other

3 pages, 226 KiB

Open AccessEditorial

Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action

by Yolanda Bel, Juan Ferré and Patricia Hernández-Martínez

Toxins 2020, 12(12), 785; https://doi.org/10.3390/toxins12120785 - 10 Dec 2020

Cited by 20 | Viewed by 4972

Abstract

Bacillus thuringiensis (Bt)-based products are the most successful microbial insecticides to date [...] Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

Research

Jump to: Editorial, Review, Other

15 pages, 5829 KiB

Open AccessArticle

Structural and Functional Insights into the C-terminal Fragment of Insecticidal Vip3A Toxin of Bacillus thuringiensis

by Kun Jiang, Yan Zhang, Zhe Chen, Dalei Wu, Jun Cai and Xiang Gao

Toxins 2020, 12(7), 438; https://doi.org/10.3390/toxins12070438 - 5 Jul 2020

Cited by 24 | Viewed by 4672

Abstract

The vegetative insecticidal proteins (Vips) secreted by Bacillus thuringiensis are regarded as the new generation of insecticidal toxins because they have different insecticidal properties compared with commonly applied insecticidal crystal proteins (Cry toxins). Vip3A toxin, representing the vast majority of Vips, has been [...] Read more.

The vegetative insecticidal proteins (Vips) secreted by Bacillus thuringiensis are regarded as the new generation of insecticidal toxins because they have different insecticidal properties compared with commonly applied insecticidal crystal proteins (Cry toxins). Vip3A toxin, representing the vast majority of Vips, has been used commercially in transgenic crops and bio-insecticides. However, the lack of both structural information on Vip3A and a clear understanding of its insecticidal mechanism at the molecular level limits its further development and broader application. Here we present the first crystal structure of the C-terminal fragment of Vip3A toxin (Vip3Aa11_200–789). Since all members of this insecticidal protein family are highly conserved, the structure of Vip3A provides unique insight into the general domain architecture and protein fold of the Vip3A family of insecticidal toxins. Our structural analysis reveals a four-domain organization, featuring a potential membrane insertion region, a receptor binding domain, and two potential glycan binding domains of Vip3A. In addition, cytotoxicity assays and insect bioassays show that the purified C-terminal fragment of Vip3Aa toxin alone have no insecticidal activity. Taken together, these findings provide insights into the mode of action of the Vip3A family of insecticidal toxins and will boost the development of Vip3A into more efficient bio-insecticides. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

► Show Figures

Figure 1

12 pages, 1260 KiB

Open AccessEditor’s ChoiceArticle

Reduced Membrane-Bound Alkaline Phosphatase Does Not Affect Binding of Vip3Aa in a Heliothis virescens Resistant Colony

by Daniel Pinos, Maissa Chakroun, Anabel Millán-Leiva, Juan Luis Jurat-Fuentes, Denis J. Wright, Patricia Hernández-Martínez and Juan Ferré

Toxins 2020, 12(6), 409; https://doi.org/10.3390/toxins12060409 - 19 Jun 2020

Cited by 15 | Viewed by 3892

Abstract

The Vip3Aa insecticidal protein from Bacillus thuringiensis (Bt) is produced by specific transgenic corn and cotton varieties for efficient control of target lepidopteran pests. The main threat to this technology is the evolution of resistance in targeted insect pests and understanding the mechanistic [...] Read more.

The Vip3Aa insecticidal protein from Bacillus thuringiensis (Bt) is produced by specific transgenic corn and cotton varieties for efficient control of target lepidopteran pests. The main threat to this technology is the evolution of resistance in targeted insect pests and understanding the mechanistic basis of resistance is crucial to deploy the most appropriate strategies for resistance management. In this work, we tested whether alteration of membrane receptors in the insect midgut might explain the >2000-fold Vip3Aa resistance phenotype in a laboratory-selected colony of Heliothis virescens (Vip-Sel). Binding of ¹²⁵I-labeled Vip3Aa to brush border membrane vesicles (BBMV) from 3rd instar larvae from Vip-Sel was not significantly different from binding in the reference susceptible colony. Interestingly, BBMV from Vip-Sel larvae showed dramatically reduced levels of membrane-bound alkaline phosphatase (mALP) activity, which was further confirmed by a strong downregulation of the membrane-bound alkaline phosphatase 1 (HvmALP1) gene. However, the involvement of HvmALP1 as a receptor for the Vip3Aa protein was not supported by results from ligand blotting and viability assays with insect cells expressing HvmALP1. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

► Show Figures

Figure 1

13 pages, 3817 KiB

Open AccessArticle

The Cadherin Protein Is Not Involved in Susceptibility to Bacillus thuringiensis Cry1Ab or Cry1Fa Toxins in Spodoptera frugiperda

by Jianfeng Zhang, Minghui Jin, Yanchao Yang, Leilei Liu, Yongbo Yang, Isabel Gómez, Alejandra Bravo, Mario Soberón, Yutao Xiao and Kaiyu Liu

Toxins 2020, 12(6), 375; https://doi.org/10.3390/toxins12060375 - 6 Jun 2020

Cited by 29 | Viewed by 4182

Abstract

It is well known that insect larval midgut cadherin protein serves as a receptor of Bacillus thuringiensis (Bt) crystal Cry1Ac or Cry1Ab toxins, since structural mutations and downregulation of cad gene expression are linked with resistance to Cry1Ac toxin in several lepidopteran insects. [...] Read more.

It is well known that insect larval midgut cadherin protein serves as a receptor of Bacillus thuringiensis (Bt) crystal Cry1Ac or Cry1Ab toxins, since structural mutations and downregulation of cad gene expression are linked with resistance to Cry1Ac toxin in several lepidopteran insects. However, the role of Spodoptera frugiperda cadherin protein (SfCad) in the mode of action of Bt toxins remains elusive. Here, we investigated whether SfCad is involved in susceptibility to Cry1Ab or Cry1Fa toxins. In vivo, knockout of the SfCad gene by CRISPR/Cas 9 did not increase tolerance to either of these toxins in S. frugiperda larvae. In vitro cytotoxicity assays demonstrated that cultured insect TnHi5 cells expressing GFP-tagged SfCad did not increase susceptibility to activated Cry1Ab or Cry1Fa toxins. In contrast, expression of another well recognized Cry1A receptor in this cell line, the ABCC2 transporter, increased the toxicity of both Cry1Ab and Cry1Fa toxins, suggesting that SfABCC2 functions as a receptor of these toxins. Finally, we showed that the toxin-binding region of SfCad did not bind to activated Cry1Ab, Cry1Ac, nor Cry1Fa. All these results support that SfCad is not involved in the mode of action of Cry1Ab or Cry1Fa toxins in S. frugiperda. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

► Show Figures

Figure 1

14 pages, 952 KiB

Open AccessArticle

Potential of Cry10Aa and Cyt2Ba, Two Minority δ-endotoxins Produced by Bacillus thuringiensis ser. israelensis, for the Control of Aedes aegypti Larvae

by Daniel Valtierra-de-Luis, Maite Villanueva, Liliana Lai, Trevor Williams and Primitivo Caballero

Toxins 2020, 12(6), 355; https://doi.org/10.3390/toxins12060355 - 29 May 2020

Cited by 28 | Viewed by 5325

Abstract

Bacillus thuringiensis ser. israelensis (Bti) has been widely used as microbial larvicide for the control of many species of mosquitoes and blackflies. The larvicidal activity of Bti resides in Cry and Cyt δ-endotoxins present in the parasporal crystal of this pathogen. The insecticidal [...] Read more.

Bacillus thuringiensis ser. israelensis (Bti) has been widely used as microbial larvicide for the control of many species of mosquitoes and blackflies. The larvicidal activity of Bti resides in Cry and Cyt δ-endotoxins present in the parasporal crystal of this pathogen. The insecticidal activity of the crystal is higher than the activities of the individual toxins, which is likely due to synergistic interactions among the crystal component proteins, particularly those involving Cyt1Aa. In the present study, Cry10Aa and Cyt2Ba were cloned from the commercial larvicide VectoBac-12AS^® and expressed in the acrystalliferous Bt strain BMB171 under the cyt1Aa strong promoter of the pSTAB vector. The LC₅₀ values for Aedes aegypti second instar larvae estimated at 24 hpi for these two recombinant proteins (Cry10Aa and Cyt2Ba) were 299.62 and 279.37 ng/mL, respectively. Remarkable synergistic mosquitocidal activity was observed between Cry10Aa and Cyt2Ba (synergistic potentiation of 68.6-fold) when spore + crystal preparations, comprising a mixture of both recombinant strains in equal relative concentrations, were ingested by A. aegypti larvae. This synergistic activity is among the most powerful described so far with Bt toxins and is comparable to that reported for Cyt1A when interacting with Cry4Aa, Cry4Ba or Cry11Aa. Synergistic mosquitocidal activity was also observed between the recombinant proteins Cyt2Ba and Cry4Aa, but in this case, the synergistic potentiation was 4.6-fold. In conclusion, although Cry10Aa and Cyt2Ba are rarely detectable or appear as minor components in the crystals of Bti strains, they represent toxicity factors with a high potential for the control of mosquito populations. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

► Show Figures

Graphical abstract

12 pages, 3411 KiB

Open AccessEditor’s ChoiceArticle

Oligomer Formation and Insecticidal Activity of Bacillus thuringiensis Vip3Aa Toxin

by Ensi Shao, Aishan Zhang, Yaqi Yan, Yaomin Wang, Xinyi Jia, Li Sha, Xiong Guan, Ping Wang and Zhipeng Huang

Toxins 2020, 12(4), 274; https://doi.org/10.3390/toxins12040274 - 23 Apr 2020

Cited by 11 | Viewed by 3460

Abstract

Bacillus thuringiensis (Bt) Vip3A proteins are important insecticidal proteins used for control of lepidopteran insects. However, the mode of action of Vip3A toxin is still unclear. In this study, the amino acid residue S164 in Vip3Aa was identified to be critical for the [...] Read more.

Bacillus thuringiensis (Bt) Vip3A proteins are important insecticidal proteins used for control of lepidopteran insects. However, the mode of action of Vip3A toxin is still unclear. In this study, the amino acid residue S164 in Vip3Aa was identified to be critical for the toxicity in Spodoptera litura. Results from substitution mutations of the S164 indicate that the insecticidal activity of Vip3Aa correlated with the formation of a >240 kDa complex of the toxin upon proteolytic activation. The >240 kDa complex was found to be composed of the 19 kDa and the 65 kDa fragments of Vip3Aa. Substitution of the S164 in Vip3Aa protein with Ala or Pro resulted in loss of the >240 kDa complex and loss of toxicity in Spodoptera litura. In contrast, substitution of S164 with Thr did not affect the >240 kDa complex formation, and the toxicity of the mutant was only reduced by 35%. Therefore, the results from this study indicated that formation of the >240 kDa complex correlates with the toxicity of Vip3Aa in insects and the residue S164 is important for the formation of the complex. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

► Show Figures

Figure 1

21 pages, 3351 KiB

Open AccessArticle

A Bacillus thuringiensis Chitin-Binding Protein is Involved in Insect Peritrophic Matrix Adhesion and Takes Part in the Infection Process

by Jiaxin Qin, Zongxing Tong, Yiling Zhan, Christophe Buisson, Fuping Song, Kanglai He, Christina Nielsen-LeRoux and Shuyuan Guo

Toxins 2020, 12(4), 252; https://doi.org/10.3390/toxins12040252 - 13 Apr 2020

Cited by 12 | Viewed by 4351

Abstract

Bacillus thuringiensis (Bt) is used for insect pest control, and its larvicidal activity is primarily attributed to Cry toxins. Other factors participate in infection, and limited information is available regarding factors acting on the peritrophic matrix (PM). This study aimed to investigate the [...] Read more.

Bacillus thuringiensis (Bt) is used for insect pest control, and its larvicidal activity is primarily attributed to Cry toxins. Other factors participate in infection, and limited information is available regarding factors acting on the peritrophic matrix (PM). This study aimed to investigate the role of a Bt chitin-binding protein (CBPA) that had been previously shown to be expressed at pH 9 in vitro and could therefore be expressed in the alkaline gut of lepidopteron larvae. A ∆cbpA mutant was generated that was 10-fold less virulent than wild-type Bt HD73 towards Ostrinia furnacalis neonate larvae, indicating its important role in infection. Purified recombinant Escherichia coli CBPA was shown to have a chitin affinity, thus indicating a possible interaction with the chitin-rich PM. A translational GFP–CBPA fusion elucidated the localization of CBPA on the bacterial surface, and the transcriptional activity of the promoter PcbpA was immediately induced and confirmed at pH 9. Next, in order to connect surface expression and possible in vivo gut activity, last instar Galleria mellonella (Gm) larvae (not susceptible to Bt HD-73) were used as a model to follow CBPA in gut expression, bacterial transit, and PM adhesion. CBPA-GFP was quickly expressed in the Gm gut lumen, and more Bt HD73 strain bacteria adhered to the PM than those of the ∆cbpA mutant strain. Therefore, CBPA may help to retain the bacteria, via the PM binding, close to the gut surface and thus takes part in the early steps of Bt gut interactions. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

► Show Figures

Figure 1

12 pages, 1465 KiB

Open AccessEditor’s ChoiceArticle

CRISPR-Mediated Knockout of the ABCC2 Gene in Ostrinia furnacalis Confers High-Level Resistance to the Bacillus thuringiensis Cry1Fa Toxin

by Xingliang Wang, Yanjun Xu, Jianlei Huang, Wenzhong Jin, Yihua Yang and Yidong Wu

Toxins 2020, 12(4), 246; https://doi.org/10.3390/toxins12040246 - 11 Apr 2020

Cited by 49 | Viewed by 4908

Abstract

The adoption of transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal crystalline (Cry) proteins has reduced insecticide application, increased yields, and contributed to food safety worldwide. However, the efficacy of transgenic Bt crops is put at risk by the adaptive resistance evolution of target [...] Read more.

The adoption of transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal crystalline (Cry) proteins has reduced insecticide application, increased yields, and contributed to food safety worldwide. However, the efficacy of transgenic Bt crops is put at risk by the adaptive resistance evolution of target pests. Previous studies indicate that resistance to Bacillus thuringiensis Cry1A and Cry1F toxins was genetically linked with mutations of ATP-binding cassette (ABC) transporter subfamily C gene ABCC2 in at least seven lepidopteran insects. Several strains selected in the laboratory of the Asian corn borer, Ostrinia furnacalis, a destructive pest of corn in Asian Western Pacific countries, developed high levels of resistance to Cry1A and Cry1F toxins. The causality between the O. furnacalis ABCC2 (OfABCC2) gene and resistance to Cry1A and Cry1F toxins remains unknown. Here, we successfully generated a homozygous strain (OfC2-KO) of O. furnacalis with an 8-bp deletion mutation of ABCC2 by the CRISPR/Cas9 approach. The 8-bp deletion mutation results in a frame shift in the open reading frame of transcripts, which produced a predicted protein truncated in the TM4-TM5 loop region. The knockout strain OfC2-KO showed much more than a 300-fold resistance to Cry1Fa, and low levels of resistance to Cry1Ab and Cry1Ac (<10-fold), but no significant effects on the toxicities of Cry1Aa and two chemical insecticides (abamectin and chlorantraniliprole), compared to the background NJ-S strain. Furthermore, we found that the Cry1Fa resistance was autosomal, recessive, and significantly linked with the 8-bp deletion mutation of OfABCC2 in the OfC2-KO strain. In conclusion, in vivo functional investigation demonstrates the causality of the OfABCC2 truncating mutation with high-level resistance to the Cry1Fa toxin in O. furnacalis. Our results suggest that the OfABCC2 protein might be a functional receptor for Cry1Fa and reinforces the association of this gene to the mode of action of the Cry1Fa toxin. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

► Show Figures

Figure 1

12 pages, 2942 KiB

Open AccessArticle

Protein-Lipid Interaction of Cytolytic Toxin Cyt2Aa2 on Model Lipid Bilayers of Erythrocyte Cell Membrane

by Sudarat Tharad, Boonhiang Promdonkoy and José L. Toca-Herrera

Toxins 2020, 12(4), 226; https://doi.org/10.3390/toxins12040226 - 3 Apr 2020

Cited by 4 | Viewed by 3443

Abstract

Cytolytic toxin (Cyt) is a toxin among Bacillus thuringiensis insecticidal proteins. Cyt toxin directly interacts with membrane lipids for cytolytic action. However, low hemolytic activity is desired to avoid non-specific effects in mammals. In this work, the interaction between Cyt2Aa2 toxin and model [...] Read more.

Cytolytic toxin (Cyt) is a toxin among Bacillus thuringiensis insecticidal proteins. Cyt toxin directly interacts with membrane lipids for cytolytic action. However, low hemolytic activity is desired to avoid non-specific effects in mammals. In this work, the interaction between Cyt2Aa2 toxin and model lipid bilayers mimicking the erythrocyte membrane was investigated for Cyt2Aa2 wild type (WT) and the T144A mutant, a variant with lower hemolytic activity. Quartz crystal microbalance with dissipation (QCM-D) results revealed a smaller lipid binding capacity for the T144A mutant than for the WT. In particular, the T144A mutant was unable to bind to the phosphatidylcholine lipid (POPC) bilayer. However, the addition of cholesterol (Chol) or sphingomyelin (SM) to the POPC bilayer promoted binding of the T144 mutant. Moreover, atomic force microscopy (AFM) images unveiled small aggregates of the T144A mutant on the 1:1 sphingomyelin/POPC bilayers. In contrast, the lipid binding trend for WT and T144A mutant was comparable for the 1:0.4 POPC/cholesterol and the 1:1:1 sphingomyelin/POPC/cholesterol bilayers. Furthermore, the binding of WT and T144A mutant onto erythrocyte cells was investigated. The experiments showed that the T144A mutant and the WT bind onto different areas of the erythrocyte membrane. Overall the results suggest that the T144 residue plays an important role for lipid binding. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

► Show Figures

Graphical abstract

15 pages, 1252 KiB

Open AccessCommunication

Study of the Bacillus thuringiensis Cry1Ia Protein Oligomerization Promoted by Midgut Brush Border Membrane Vesicles of Lepidopteran and Coleopteran Insects, or Cultured Insect Cells

by Ayda Khorramnejad, Mikel Domínguez-Arrizabalaga, Primitivo Caballero, Baltasar Escriche and Yolanda Bel

Toxins 2020, 12(2), 133; https://doi.org/10.3390/toxins12020133 - 21 Feb 2020

Cited by 8 | Viewed by 3774

Abstract

Bacillus thuringiensis (Bt) produces insecticidal proteins that are either secreted during the vegetative growth phase or accumulated in the crystal inclusions (Cry proteins) in the stationary phase. Cry1I proteins share the three domain (3D) structure typical of crystal proteins but are secreted to [...] Read more.

Bacillus thuringiensis (Bt) produces insecticidal proteins that are either secreted during the vegetative growth phase or accumulated in the crystal inclusions (Cry proteins) in the stationary phase. Cry1I proteins share the three domain (3D) structure typical of crystal proteins but are secreted to the media early in the stationary growth phase. In the generally accepted mode of action of 3D Cry proteins (sequential binding model), the formation of an oligomer (tetramer) has been described as a major step, necessary for pore formation and subsequent toxicity. To know if this could be extended to Cry1I proteins, the formation of Cry1Ia oligomers was studied by Western blot, after the incubation of trypsin activated Cry1Ia with insect brush border membrane vesicles (BBMV) or insect cultured cells, using Cry1Ab as control. Our results showed that Cry1Ia oligomers were observed only after incubation with susceptible coleopteran BBMV, but not following incubation with susceptible lepidopteran BBMV or non-susceptible Sf21 insect cells, while Cry1Ab oligomers were persistently detected after incubation with all insect tissues tested, regardless of its host susceptibility. The data suggested oligomerization may not necessarily be a requirement for the toxicity of Cry1I proteins. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

► Show Figures

Figure 1

15 pages, 4498 KiB

Open AccessEditor’s ChoiceArticle

Mitochondria and Lysosomes Participate in Vip3Aa-Induced Spodoptera frugiperda Sf9 Cell Apoptosis

by Xiaoyue Hou, Lu Han, Baoju An, Yanli Zhang, Zhanglei Cao, Yunda Zhan, Xia Cai, Bing Yan and Jun Cai

Toxins 2020, 12(2), 116; https://doi.org/10.3390/toxins12020116 - 13 Feb 2020

Cited by 30 | Viewed by 3473

Abstract

Vip3Aa, a soluble protein produced by certain Bacillus thuringiensis strains, is capable of inducing apoptosis in Sf9 cells. However, the apoptosis mechanism triggered by Vip3Aa is unclear. In this study, we found that Vip3Aa induces mitochondrial dysfunction, as evidenced by signs of collapse [...] Read more.

Vip3Aa, a soluble protein produced by certain Bacillus thuringiensis strains, is capable of inducing apoptosis in Sf9 cells. However, the apoptosis mechanism triggered by Vip3Aa is unclear. In this study, we found that Vip3Aa induces mitochondrial dysfunction, as evidenced by signs of collapse of mitochondrial membrane potential, accumulation of reactive oxygen species, release of cytochrome c, and caspase-9 and -3 activation. Meanwhile, our results indicated that Vip3Aa reduces the ability of lysosomes in Sf9 cells to retain acridine orange. Moreover, pretreatment with Z-Phe-Tyr-CHO (a cathepsin L inhibitor) or pepstatin (a cathepsin D inhibitor) increased Sf9 cell viability, reduced cytochrome c release, and decreased caspase-9 and -3 activity. In conclusion, our findings suggested that Vip3Aa promotes Sf9 cell apoptosis by mitochondrial dysfunction, and lysosomes also play a vital role in the action of Vip3Aa. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

► Show Figures

Figure 1

14 pages, 3226 KiB

Open AccessArticle

ATP-Binding Cassette Subfamily a Member 2 Is a Functional Receptor for Bacillus thuringiensis Cry2A Toxins in Bombyx mori, But Not for Cry1A, Cry1C, Cry1D, Cry1F, or Cry9A Toxins

by Xiaoyi Li, Kazuhisa Miyamoto, Yoko Takasu, Sanae Wada, Tetsuya Iizuka, Satomi Adegawa, Ryoichi Sato and Kenji Watanabe

Toxins 2020, 12(2), 104; https://doi.org/10.3390/toxins12020104 - 6 Feb 2020

Cited by 22 | Viewed by 3732

Abstract

Cry toxins are insecticidal proteins produced by Bacillus thuringiensis (Bt). They are used commercially to control insect pests since they are very active in specific insects and are harmless to the environment and human health. The gene encoding ATP-binding cassette subfamily A member [...] Read more.

Cry toxins are insecticidal proteins produced by Bacillus thuringiensis (Bt). They are used commercially to control insect pests since they are very active in specific insects and are harmless to the environment and human health. The gene encoding ATP-binding cassette subfamily A member 2 (ABCA2) was identified in an analysis of Cry2A toxin resistance genes. However, we do not have direct evidence for the role of ABCA2 for Cry2A toxins or why Cry2A toxin resistance does not cross to other Cry toxins. Therefore, we performed two experiments. First, we edited the ABCA2 sequence in Bombyx mori using transcription activator-like effector-nucleases (TALENs) and confirmed the susceptibility-determining ability in a diet overlay bioassay. Strains with C-terminal half-deleted BmABCA2 showed strong and specific resistance to Cry2A toxins; even strains carrying a deletion of 1 to 3 amino acids showed resistance. However, the C-terminal half-deleted strains did not show cross-resistance to other toxins. Second, we conducted a cell swelling assay and confirmed the specific ability of BmABCA2 to Cry2A toxins in HEK239T cells. Those demonstrated that BmABCA2 is a functional receptor for Cry2A toxins and that BmABCA2 deficiency-dependent Cry2A resistance does not confer cross-resistance to Cry1A, Cry1Ca, Cry1Da, Cry1Fa or Cry9Aa toxins. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

► Show Figures

Graphical abstract

15 pages, 3046 KiB

Open AccessEditor’s ChoiceArticle

Domain Shuffling between Vip3Aa and Vip3Ca: Chimera Stability and Insecticidal Activity against European, American, African, and Asian Pests

by Joaquín Gomis-Cebolla, Rafael Ferreira dos Santos, Yueqin Wang, Javier Caballero, Primitivo Caballero, Kanglai He, Juan Luis Jurat-Fuentes and Juan Ferré

Toxins 2020, 12(2), 99; https://doi.org/10.3390/toxins12020099 - 4 Feb 2020

Cited by 15 | Viewed by 3704

Abstract

The bacterium Bacillus thuringiensis produces insecticidal Vip3 proteins during the vegetative growth phase with activity against several lepidopteran pests. To date, three different Vip3 protein families have been identified based on sequence identity: Vip3A, Vip3B, and Vip3C. In this study, we report the [...] Read more.

The bacterium Bacillus thuringiensis produces insecticidal Vip3 proteins during the vegetative growth phase with activity against several lepidopteran pests. To date, three different Vip3 protein families have been identified based on sequence identity: Vip3A, Vip3B, and Vip3C. In this study, we report the construction of chimeras by exchanging domains between Vip3Aa and Vip3Ca, two proteins with marked specificity differences against lepidopteran pests. We found that some domain combinations made proteins insoluble or prone to degradation by trypsin as most abundant insect gut protease. The soluble and trypsin-stable chimeras, along with the parental proteins Vip3Aa and Vip3Ca, were tested against lepidopteran pests from different continents: Spodoptera exigua, Spodoptera littoralis, Spodoptera frugiperda, Helicoverpa armigera, Mamestra brassicae, Anticarsia gemmatalis, and Ostrinia furnacalis. The exchange of the Nt domain (188 N-terminal amino acids) had little effect on the stability and toxicity (equal or slightly lower) of the resulting chimeric protein against all insects except for S. frugiperda, for which the chimera with the Nt domain from Vip3Aa and the rest of the protein from Vip3Ca showed a significant increase in toxicity compared to the parental Vip3Ca. Chimeras with the C-terminal domain from Vip3Aa (from amino acid 510 of Vip3Aa to the Ct) with the central domain of Vip3Ca (amino acids 189–509 based on the Vip3Aa sequence) made proteins that could not be solubilized. Finally, the chimera including the Ct domain of Vip3Ca and the Nt and central domain from Vip3Aa was unstable. Importantly, an insect species tolerant to Vip3Aa but susceptible to Vip3Ca, such as Ostrinia furnacalis, was also susceptible to chimeras maintaining the Ct domain from Vip3Ca, in agreement with the hypothesis that the Ct region of the protein is the one conferring specificity to Vip3 proteins. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

► Show Figures

Graphical abstract

15 pages, 939 KiB

Open AccessArticle

Reduced Expression of a Novel Midgut Trypsin Gene Involved in Protoxin Activation Correlates with Cry1Ac Resistance in a Laboratory-Selected Strain of Plutella xylostella (L.)

by Lijun Gong, Shi Kang, Junlei Zhou, Dan Sun, Le Guo, Jianying Qin, Liuhong Zhu, Yang Bai, Fan Ye, Mazarin Akami, Qingjun Wu, Shaoli Wang, Baoyun Xu, Zhongxia Yang, Alejandra Bravo, Mario Soberón, Zhaojiang Guo, Lizhang Wen and Youjun Zhang

Toxins 2020, 12(2), 76; https://doi.org/10.3390/toxins12020076 - 23 Jan 2020

Cited by 18 | Viewed by 3695

Abstract

Bacillus thuringiensis (Bt) produce diverse insecticidal proteins to kill insect pests. Nevertheless, evolution of resistance to Bt toxins hampers the sustainable use of this technology. Previously, we identified down-regulation of a trypsin-like serine protease gene PxTryp_SPc1 in the midgut transcriptome and RNA-Seq data [...] Read more.

Bacillus thuringiensis (Bt) produce diverse insecticidal proteins to kill insect pests. Nevertheless, evolution of resistance to Bt toxins hampers the sustainable use of this technology. Previously, we identified down-regulation of a trypsin-like serine protease gene PxTryp_SPc1 in the midgut transcriptome and RNA-Seq data of a laboratory-selected Cry1Ac-resistant Plutella xylostella strain, SZ-R. We show here that reduced PxTryp_SPc1 expression significantly reduced caseinolytic and trypsin protease activities affecting Cry1Ac protoxin activation, thereby conferring higher resistance to Cry1Ac protoxin than activated toxin in SZ-R strain. Herein, the full-length cDNA sequence of PxTryp_SPc1 gene was cloned, and we found that it was mainly expressed in midgut tissue in all larval instars. Subsequently, we confirmed that the PxTryp_SPc1 gene was significantly decreased in SZ-R larval midgut and was further reduced when selected with high dose of Cry1Ac protoxin. Moreover, down-regulation of the PxTryp_SPc1 gene was genetically linked to resistance to Cry1Ac in the SZ-R strain. Finally, RNAi-mediated silencing of PxTryp_SPc1 gene expression decreased larval susceptibility to Cry1Ac protoxin in the susceptible DBM1Ac-S strain, supporting that low expression of PxTryp_SPc1 gene is involved in Cry1Ac resistance in P. xylostella. These findings contribute to understanding the role of midgut proteases in the mechanisms underlying insect resistance to Bt toxins. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

► Show Figures

Graphical abstract

Review

Jump to: Editorial, Research, Other

34 pages, 1985 KiB

Open AccessReview

Making 3D-Cry Toxin Mutants: Much More Than a Tool of Understanding Toxins Mechanism of Action

by Susana Vílchez

Toxins 2020, 12(9), 600; https://doi.org/10.3390/toxins12090600 - 16 Sep 2020

Cited by 18 | Viewed by 5257

Abstract

3D-Cry toxins, produced by the entomopathogenic bacterium Bacillus thuringiensis, have been extensively mutated in order to elucidate their elegant and complex mechanism of action necessary to kill susceptible insects. Together with the study of the resistant insects, 3D-Cry toxin mutants represent one [...] Read more.

3D-Cry toxins, produced by the entomopathogenic bacterium Bacillus thuringiensis, have been extensively mutated in order to elucidate their elegant and complex mechanism of action necessary to kill susceptible insects. Together with the study of the resistant insects, 3D-Cry toxin mutants represent one of the pillars to understanding how these toxins exert their activity on their host. The principle is simple, if an amino acid is involved and essential in the mechanism of action, when substituted, the activity of the toxin will be diminished. However, some of the constructed 3D-Cry toxin mutants have shown an enhanced activity against their target insects compared to the parental toxins, suggesting that it is possible to produce novel versions of the natural toxins with an improved performance in the laboratory. In this report, all mutants with an enhanced activity obtained by accident in mutagenesis studies, together with all the variants obtained by rational design or by directed mutagenesis, were compiled. A description of the improved mutants was made considering their historical context and the parallel development of the protein engineering techniques that have been used to obtain them. This report demonstrates that artificial 3D-Cry toxins made in laboratories are a real alternative to natural toxins. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

► Show Figures

Figure 1

24 pages, 1237 KiB

Open AccessEditor’s ChoiceReview

Current Insights on Vegetative Insecticidal Proteins (Vip) as Next Generation Pest Killers

by Tahira Syed, Muhammad Askari, Zhigang Meng, Yanyan Li, Muhammad Ali Abid, Yunxiao Wei, Sandui Guo, Chengzhen Liang and Rui Zhang

Toxins 2020, 12(8), 522; https://doi.org/10.3390/toxins12080522 - 14 Aug 2020

Cited by 43 | Viewed by 7338 | Correction

Abstract

Bacillus thuringiensis (Bt) is a gram negative soil bacterium. This bacterium secretes various proteins during different growth phases with an insecticidal potential against many economically important crop pests. One of the important families of Bt proteins is vegetative insecticidal proteins (Vip), which are [...] Read more.

Bacillus thuringiensis (Bt) is a gram negative soil bacterium. This bacterium secretes various proteins during different growth phases with an insecticidal potential against many economically important crop pests. One of the important families of Bt proteins is vegetative insecticidal proteins (Vip), which are secreted into the growth medium during vegetative growth. There are three subfamilies of Vip proteins. Vip1 and Vip2 heterodimer toxins have an insecticidal activity against many Coleopteran and Hemipteran pests. Vip3, the most extensively studied family of Vip toxins, is effective against Lepidopteron. Vip proteins do not share homology in sequence and binding sites with Cry proteins, but share similarities at some points in their mechanism of action. Vip3 proteins are expressed as pyramids alongside Cry proteins in crops like maize and cotton, so as to control resistant pests and delay the evolution of resistance. Biotechnological- and in silico-based analyses are promising for the generation of mutant Vip proteins with an enhanced insecticidal activity and broader spectrum of target insects. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

► Show Figures

Figure 1

17 pages, 1077 KiB

Open AccessReview

The Tripartite Interaction of Host Immunity–Bacillus thuringiensis Infection–Gut Microbiota

by Shuzhong Li, Surajit De Mandal, Xiaoxia Xu and Fengliang Jin

Toxins 2020, 12(8), 514; https://doi.org/10.3390/toxins12080514 - 12 Aug 2020

Cited by 32 | Viewed by 5552

Abstract

Bacillus thuringiensis (Bt) is an important cosmopolitan bacterial entomopathogen, which produces various protein toxins that have been expressed in transgenic crops. The evolved molecular interaction between the insect immune system and gut microbiota is changed during the Bt infection process. The host immune [...] Read more.

Bacillus thuringiensis (Bt) is an important cosmopolitan bacterial entomopathogen, which produces various protein toxins that have been expressed in transgenic crops. The evolved molecular interaction between the insect immune system and gut microbiota is changed during the Bt infection process. The host immune response, such as the expression of induced antimicrobial peptides (AMPs), the melanization response, and the production of reactive oxygen species (ROS), varies with different doses of Bt infection. Moreover, B. thuringiensis infection changes the abundance and structural composition of the intestinal bacteria community. The activated immune response, together with dysbiosis of the gut microbiota, also has an important effect on Bt pathogenicity and insect resistance to Bt. In this review, we attempt to clarify this tripartite interaction of host immunity, Bt infection, and gut microbiota, especially the important role of key immune regulators and symbiotic bacteria in the Bt killing activity. Increasing the effectiveness of biocontrol agents by interfering with insect resistance and controlling symbiotic bacteria can be important steps for the successful application of microbial biopesticides. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

► Show Figures

Figure 1

29 pages, 1998 KiB

Open AccessEditor’s ChoiceReview

Insecticidal Activity of Bacillus thuringiensis Proteins against Coleopteran Pests

by Mikel Domínguez-Arrizabalaga, Maite Villanueva, Baltasar Escriche, Carmen Ancín-Azpilicueta and Primitivo Caballero

Toxins 2020, 12(7), 430; https://doi.org/10.3390/toxins12070430 - 29 Jun 2020

Cited by 59 | Viewed by 8346

Abstract

Bacillus thuringiensis is the most successful microbial insecticide agent and its proteins have been studied for many years due to its toxicity against insects mainly belonging to the orders Lepidoptera, Diptera and Coleoptera, which are pests of agro-forestry and medical-veterinary interest. However, studies [...] Read more.

Bacillus thuringiensis is the most successful microbial insecticide agent and its proteins have been studied for many years due to its toxicity against insects mainly belonging to the orders Lepidoptera, Diptera and Coleoptera, which are pests of agro-forestry and medical-veterinary interest. However, studies on the interactions between this bacterium and the insect species classified in the order Coleoptera are more limited when compared to other insect orders. To date, 45 Cry proteins, 2 Cyt proteins, 11 Vip proteins, and 2 Sip proteins have been reported with activity against coleopteran species. A number of these proteins have been successfully used in some insecticidal formulations and in the construction of transgenic crops to provide protection against main beetle pests. In this review, we provide an update on the activity of Bt toxins against coleopteran insects, as well as specific information about the structure and mode of action of coleopteran Bt proteins. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

► Show Figures

Figure 1

22 pages, 1703 KiB

Open AccessEditor’s ChoiceReview

The Cytocidal Spectrum of Bacillus thuringiensis Toxins: From Insects to Human Cancer Cells

by Gretel Mendoza-Almanza, Edgar L. Esparza-Ibarra, Jorge L. Ayala-Luján, Marisa Mercado-Reyes, Susana Godina-González, Marisa Hernández-Barrales and Jorge Olmos-Soto

Toxins 2020, 12(5), 301; https://doi.org/10.3390/toxins12050301 - 6 May 2020

Cited by 33 | Viewed by 7772

Abstract

Bacillus thuringiensis (Bt) is a ubiquitous bacterium in soils, insect cadavers, phylloplane, water, and stored grain, that produces several proteins, each one toxic to different biological targets such as insects, nematodes, mites, protozoa, and mammalian cells. Most Bt toxins identify their particular target [...] Read more.

Bacillus thuringiensis (Bt) is a ubiquitous bacterium in soils, insect cadavers, phylloplane, water, and stored grain, that produces several proteins, each one toxic to different biological targets such as insects, nematodes, mites, protozoa, and mammalian cells. Most Bt toxins identify their particular target through the recognition of specific cell membrane receptors. Cry proteins are the best-known toxins from Bt and a great amount of research has been published. Cry are cytotoxic to insect larvae that affect important crops recognizing specific cell membrane receptors such as cadherin, aminopeptidase-N, and alkaline phosphatase. Furthermore, some Cry toxins such as Cry4A, Cry4B, and Cry11A act synergistically with Cyt toxins against dipteran larvae vectors of human disease. Research developed with Cry proteins revealed that these toxins also could kill human cancer cells through the interaction with specific receptors. Parasporins are a small group of patented toxins that may or may not have insecticidal activity. These proteins could kill a wide variety of mammalian cancer cells by recognizing specific membrane receptors, just like Cry toxins do. Surface layer proteins (SLP), unlike the other proteins produced by Bt, are also produced by most bacteria and archaebacteria. It was recently demonstrated that SLP produced by Bt could interact with membrane receptors of insect and human cancer cells to kill them. Cyt toxins have a structure that is mostly unrelated to Cry toxins; thereby, other mechanisms of action have been reported to them. These toxins affect mainly mosquitoes that are vectors of human diseases like Anopheles spp (malaria), Aedes spp (dengue, zika, and chikungunya), and Culex spp (Nile fever and Rift Valley fever), respectively. In addition to the Cry, Cyt, and parasporins toxins produced during spore formation as inclusion bodies, Bt strains also produce Vip (Vegetative insecticidal toxins) and Sip (Secreted insecticidal proteins) toxins with insecticidal activity during their vegetative growth phase. Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

► Show Figures

Figure 1

Other

Jump to: Editorial, Research, Review

1 pages, 206 KiB

Open AccessCorrection

Correction: Syed, T., et al. Current Insights on Vegetative Insecticidal Proteins (Vip) as Next Generation Pest Killers. Toxins 2020, 12, 522

by Tahira Syed, Muhammad Askari, Zhigang Meng, Yanyan Li, Muhammad Ali Abid, Yunxiao Wei, Sandui Guo, Chengzhen Liang and Rui Zhang

Toxins 2021, 13(3), 200; https://doi.org/10.3390/toxins13030200 - 11 Mar 2021

Cited by 2 | Viewed by 1721

Abstract

The authors wish to make the following corrections to their paper [...] Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

2 pages, 219 KiB

Open AccessObituary

A Tribute to a Bacillus thuringiensis Master: Professor David J. Ellar

by Susana Vílchez

Toxins 2020, 12(12), 764; https://doi.org/10.3390/toxins12120764 - 3 Dec 2020

Viewed by 1749

Abstract

This Special Issue, on Bacillus thuringiensis and its toxins, seems to be the right place to pay tribute to one of the most influential scientists in the field of research into this peculiar bacterium [...] Full article

(This article belongs to the Special Issue Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action)

Journal Menu

Journal Browser

Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action

Share This Special Issue

Special Issue Editors

Special Issue Information

Keywords

Benefits of Publishing in a Special Issue

Published Papers (21 papers)

Editorial

Research

Review

Other

Further Information

Guidelines

MDPI Initiatives

Follow MDPI