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

Abstract As a pore-forming toxin, oligomerization and pore-formation were both required for the mode of action of Cry toxins. Previous studies revealed that Domain I helices α4-α5 were involved in oligomerization of Cry2Ab, while the active residues in charge of Cry2Ab aggregation remained ambiguous. In present studies, we built 20 Cry2Ab alanine mutants site directed in helices α4-α5 and demonstrated that mutants N151A, T152A, F157A, L183A, L185A and I188A blocked the assembly of 250 kDa oligomers, suggesting that those residues were key residues for Cry2Ab oligomerization. As expected, those variants severely reduced the insecticidal activity against P. xylostella which was similar to our previous reports. Furthermore, we found that the pore-forming activities of non-oligomerization mutants sharply decreased compared to wild-type Cry2Ab. Taken together, our data comprehensively identified key residues for Cry2Ab for the first time and emphasized that oligomerization was closely related to insecticidal activity and pore-forming activity in Cry2Ab.

Download Full-text

Insect Hsp90 Chaperone Assists Bacillus thuringiensis Cry Toxicity by Enhancing Protoxin Binding to the Receptor and by Protecting Protoxin from Gut Protease Degradation

mBio ◽

10.1128/mbio.02775-19 ◽

2019 ◽

Vol 10 (6) ◽

Cited By ~ 1

Author(s):

Blanca I. García-Gómez ◽

Sayra N. Cano ◽

Erika E. Zagal ◽

Edgar Dantán-Gonzalez ◽

Alejandra Bravo ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Receptor Binding ◽

Insecticidal Activity ◽

Insect Pests ◽

Biotechnological Applications ◽

Cry Proteins ◽

Cry Toxins ◽

Content Type ◽

Hsp90 Chaperone ◽

Protease Degradation

ABSTRACT Bacillus thuringiensis Cry proteins are pore-forming insecticidal toxins with specificity against different crop pests and insect vectors of human diseases. Previous work suggested that the insect host Hsp90 chaperone could be involved in Cry toxin action. Here, we show that the interaction of Cry toxins with insect Hsp90 constitutes a positive loop to enhance the performance of these toxins. Plutella xylostella Hsp90 (PxHsp90) greatly enhanced Cry1Ab or Cry1Ac toxicity when fed together to P. xylostella larvae and also in the less susceptible Spodoptera frugiperda larvae. PxHsp90 bound Cry1Ab and Cry1Ac protoxins in an ATP- and chaperone activity-dependent interaction. The chaperone Hsp90 participates in the correct folding of proteins and may suppress mutations of some client proteins, and we show here that PxHsp90 recovered the toxicity of the Cry1AbG439D protoxin affected in receptor binding, in contrast to the Cry1AbR99E or Cry1AbE129K mutant, affected in oligomerization or membrane insertion, respectively, which showed a slight toxicity improvement. Specifically, PxHsp90 enhanced the binding of Cry1AbG439D protoxin to the cadherin receptor. Furthermore, PxHsp90 protected Cry1A protoxins from degradation by insect midgut proteases. Our data show that PxHsp90 assists Cry1A proteins by enhancing their binding to the receptor and by protecting Cry protoxin from gut protease degradation. Finally, we show that the insect cochaperone protein PxHsp70 also increases the toxicity of Cry1Ac in P. xylostella larvae, in contrast to a bacterial GroEL chaperone, which had a marginal effect, indicating that the use of insect chaperones along with Cry toxins could have important biotechnological applications for the improvement of Cry insecticidal activity, resulting in effective control of insect pests. IMPORTANCE Bacillus thuringiensis took advantage of important insect cellular proteins, such as chaperones, involved in maintaining protein homeostasis, to enhance its insecticidal activity. This constitutes a positive loop where the concentrations of Hsp90 and Hsp70 in the gut lumen are likely to increase as midgut cells burst due to Cry1A pore formation action. Hsp90 protects Cry1A protoxin from degradation and enhances receptor binding, resulting in increased toxicity. The effect of insect chaperones on Cry toxicity could have important biotechnological applications to enhance the toxicity of Cry proteins to insect pests, especially those that show low susceptibility to these toxins.

Download Full-text

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

Toxins ◽

10.3390/toxins12050301 ◽

2020 ◽

Vol 12 (5) ◽

pp. 301 ◽

Cited By ~ 4

Author(s):

Gretel Mendoza-Almanza ◽

Edgar L. Esparza-Ibarra ◽

Jorge L. Ayala-Luján ◽

Marisa Mercado-Reyes ◽

Susana Godina-González ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Cell Membrane ◽

Cancer Cells ◽

Insecticidal Activity ◽

Human Cancer ◽

Membrane Receptors ◽

Specific Cell ◽

Cry Proteins ◽

Cry Toxins ◽

Human Cancer Cells

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.

Download Full-text

Key residues of Bacillus thuringiensis Cry2Ab for oligomerization and pore-formation activity

AMB Express ◽

10.1186/s13568-021-01270-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Zhi-Zhen Pan ◽

Lian Xu ◽

Bo Liu ◽

Qing-Xi Chen ◽

Yu-Jing Zhu

Keyword(s):

Bacillus Thuringiensis ◽

Insecticidal Activity ◽

Mode Of Action ◽

Pore Formation ◽

Wild Type ◽

Cry Toxins ◽

Key Residues ◽

Domain I

AbstractAs a pore-forming toxin, activation, oligomerization and pore-formation were both required for the mode of action of Cry toxins. Previous results revealed that the helices α4–α5 of Domain I were involved in the oligomerization of Cry2Ab, however, the key residues for Cry2Ab aggregation remained ambiguous. In present studies, we built 20 Cry2Ab alanine mutants site-directed in the helices α4–α5 of Domain I and demonstrated that mutants N151A, T152A, F157A, L183A, L185A and I188A could reduce the assembly of the 250 kDa oligomers, suggesting that these mutation residues might be essential for Cry2Ab oligomerization. As expected, all of these variants showed lower insecticidal activity against P. xylostella. Furthermore, we found that the pore-forming activities of these mutants also decreased when compared to wild-type Cry2Ab. Taken together, our data identified key residues for Cry2Ab oligomerization and emphasized that oligomerization was closely related to the insecticidal activity and pore-forming activity of Cry2Ab.

Download Full-text

Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis

Peptides ◽

10.1016/j.peptides.2008.07.027 ◽

2009 ◽

Vol 30 (3) ◽

pp. 589-595 ◽

Cited By ~ 67

Author(s):

L. Pardo-López ◽

C. Muñoz-Garay ◽

H. Porta ◽

C. Rodríguez-Almazán ◽

M. Soberón ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Insecticidal Activity ◽

Cry Toxins

Download Full-text

Role of Tryptophan Residues in Toxicity of Cry1Ab Toxin from Bacillus thuringiensis

Applied and Environmental Microbiology ◽

10.1128/aem.72.1.901-907.2006 ◽

2006 ◽

Vol 72 (1) ◽

pp. 901-907 ◽

Cited By ~ 17

Author(s):

Cristopher Padilla ◽

Liliana Pardo-López ◽

Gustavo de la Riva ◽

Isabel Gómez ◽

Jorge Sánchez ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Insecticidal Activity ◽

Structural Changes ◽

Site Directed Mutagenesis ◽

Crystal Formation ◽

Cry Toxins ◽

Tryptophan Residues ◽

Cry1ab Toxin ◽

And Function

ABSTRACT Bacillus thuringiensis produces insecticidal proteins (Cry protoxins) during the sporulation phase as parasporal crystals. During intoxication, the Cry protoxins must change from insoluble crystals into membrane-inserted toxins which form ionic pores. The structural changes of Cry toxins during oligomerization and insertion into the membrane are still unknown. The Cry1Ab toxin has nine tryptophan residues; seven are located in domain I, the pore-forming domain, and two are located in domain II, which is involved in receptor recognition. Eight Trp residues are highly conserved within the whole family of three-domain Cry proteins, suggesting an essential role for these residues in the structural folding and function of the toxin. In this work, we analyzed the role of Trp residues in the structure and function of Cry1Ab toxin. We replaced the Trp residues with phenylalanine or cysteine using site-directed mutagenesis. Our results show that W65 and W316 are important for insecticidal activity of the toxin since their replacement by Phe reduced the toxicity against Manduca sexta. The presence of hydrophobic residue is important at positions 117, 219, 226, and 455 since replacement by Cys affected either the crystal formation or the insecticidal activity of the toxin in contrast to replacement by Phe in these positions. Additionally, some mutants in positions 219, 316, and 455 were also affected in binding to brush border membrane vesicles (BBMV). This is the first report that studies the role of Trp residues in the activity of Cry toxins.

Download Full-text