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Cadherin binding is not a limiting step for Bacillus thuringiensis subsp. israelensis Cry4Ba toxicity to Aedes
Claudia Rodríguez-Almazán1, Esmeralda Z Reyes, Fernando Zúñiga-Navarrete
1Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. postal 510-3, Cuernavaca 62250, Morelos, Mexico.
Abstract:
Bacillus thuringiensis subsp. israelensis produces three Cry toxins (Cry4Aa, Cry4Ba and Cry11Aa) that are active against Aedes aegypti larvae. The identification of the rate-limiting binding steps of Cry toxins that are used for insect control in the field, such as those of B. thuringiensis subsp. israelensis, should provide targets for improving insecticides against important insect pests. Previous studies showed that Cry11Aa binds to cadherin receptor fragment CR7-11 (cadherin repeats 7-11) with high affinity. Binding to cadherin has been proposed to facilitate Cry toxin oligomer formation. In the present study, we show that Cry4Ba binds to CR7-11 with 9-fold lower binding affinity compared with Cry11Aa. Oligomerization assays showed that Cry4Ba is capable of forming oligomers when proteolytically activated in vitro in the absence of the CR7-11 fragment in contrast with Cry11Aa that formed oligomers only in the presence of CR7-11. Pore-formation assays in planar lipid bilayers showed that Cry4Ba oligomers were proficient in opening ion channels. Finally, silencing the cadherin gene by dsRNA (double-stranded RNA) showed that silenced larvae were more tolerant to Cry11Aa in contrast with Cry4Ba, which showed similar toxic levels to those of control larvae. These findings show that cadherin binding is not a limiting step for Cry4Ba toxicity to A. aegypti larvae.
Insights
Cadherin binding is crucial for Cry11Aa toxicity in Aedes aegypti larvae. However, Cry4Ba toxicity is not limited by cadherin binding, suggesting alternative mechanisms for this important mosquito control agent.
Area of Science:
- Molecular biology
- Insect toxicology
- Biochemistry
Background:
- Bacillus thuringiensis subsp. israelensis produces Cry toxins targeting mosquito larvae.
- Cadherin binding is a proposed step in Cry toxin action.
- Cry11Aa binds strongly to cadherin fragment CR7-11.
Purpose of the Study:
- Investigate the role of cadherin binding in Cry4Ba toxicity.
- Compare Cry4Ba and Cry11Aa binding affinities and oligomerization.
- Determine if cadherin binding is rate-limiting for Cry4Ba.
Main Methods:
- Binding affinity assays using cadherin fragment CR7-11.
- In vitro proteolytic activation and oligomerization assays.
- Planar lipid bilayer pore-formation assays.
- Gene silencing of cadherin in Aedes aegypti larvae using dsRNA.
Main Results:
- Cry4Ba binds CR7-11 with 9-fold lower affinity than Cry11Aa.
- Cry4Ba oligomerizes independently of CR7-11, unlike Cry11Aa.
- Cry4Ba oligomers form functional ion channels.
- Cadherin silencing increases tolerance to Cry11Aa but not Cry4Ba.
Conclusions:
- Cadherin binding is essential for Cry11Aa toxicity but not rate-limiting for Cry4Ba.
- Cry4Ba toxicity mechanism differs from Cry11Aa, independent of high-affinity cadherin interaction.
