RESUMEN
Cry2Ab, a pore-forming toxin derived from Bacillus thuringiensis, is widely used as a bio-insecticide to control lepidopteran pests around the world. A previous study revealed that proteolytic activation of Cry2Ab by Plutella xylostella midgut juice was essential for its insecticidal activity against P. xylostella, although the exact molecular mechanism remained unknown. Here, we demonstrated for the first time that proteolysis of Cry2Ab uncovered an active region (the helices α4 and α5 in Domain I), which was required for the mode of action of Cry2Ab. Either the masking or the removal of helices α4 and α5 mediated the pesticidal activity of Cry2Ab. The exposure of helices α4 and α5 did not facilitate the binding of Cry2Ab to P. xylostella midgut receptors but did induce Cry2Ab monomer to aggregate and assemble a 250-kDa prepore oligomer. Site-directed mutagenesis assay was performed to generate Cry2Ab mutants site directed on the helices α4 and α5, and bioassays suggested that some Cry2Ab variants that could not form oligomers had significantly lowered their toxicities against P. xylostella. Taken together, our data highlight the importance of helices α4 and α5 in the mode of action of Cry2Ab and could lead to more detailed studies on the insecticidal activity of Cry2Ab.
Asunto(s)
Proteínas Bacterianas/farmacología , Endotoxinas/farmacología , Proteínas Hemolisinas/farmacología , Insecticidas/farmacología , Lepidópteros/efectos de los fármacos , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Análisis Mutacional de ADN , Endotoxinas/química , Endotoxinas/genética , Endotoxinas/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Insecticidas/química , Insecticidas/metabolismo , Peso Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/farmacología , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Proteolisis , Eliminación de SecuenciaRESUMEN
Bacillus thuringiensis insecticidal proteins (Bt toxins) have been widely used in crops for agricultural pest management and to reduce the use of chemical insecticides. Here, we have engineered Bt toxin Cry2Ab30 and bioconjugated it with 4"-O-succinyl avermectin (AVM) to synthesize Cry2Ab-AVM bioconjugate. It was found that Cry2Ab-AVM showed higher insecticidal activity against Plutella xylostella, up to 154.4 times compared to Cry2Ab30. The binding results showed that Cry2Ab-AVM binds to the cadherin-like binding protein fragments, the 10th and 11th cadherin repeat domains in the P. xylostella cadherin (PxCR10-11), with a much higher affinity (dissociation equilibrium constant KD = 3.44 nM) than Cry2Ab30 (KD = 28.7 nM). Molecular docking suggested that the macrolide lactone group of Cry2Ab-AVM ligand docking into the PxCR10-11 is a potential mechanism to enhance the binding affinity of Cry2Ab-AVM to PxCR10-11. These findings offer scope for the engineering of Bt toxins by bioconjugation for improved pest management.
Asunto(s)
Bacillus thuringiensis , Proteínas Bacterianas/farmacología , Endotoxinas/farmacología , Proteínas Hemolisinas/farmacología , Proteínas de Insectos/química , Insecticidas/farmacología , Ivermectina/análogos & derivados , Mariposas Nocturnas/efectos de los fármacos , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/química , Endotoxinas/química , Proteínas Hemolisinas/química , Insecticidas/síntesis química , Insecticidas/química , Ivermectina/química , Ivermectina/farmacología , Simulación del Acoplamiento Molecular , Unión ProteicaRESUMEN
Proteolysis of Vip3Aa by insect midgut proteases is essential for their toxicity against target insects. In the present study, proteolysis of Vip3Aa was evaluated by Spodoptera exigua midgut proteases (MJ). Trypsin was verified involved in the activation of Vip3Aa and three potential cleavage sites (Lys195, Lys197 and Lys198) were identified. Four Vip3Aa mutants (KKK195197198AAA, KK197198AA, KK195198AA and KK195197AA) were designed and constructed by replacing residues Lys195,197,198, Lys197,198, Lys195,198 and Lys195,197 with Ala, respectively. Proteolytic processing assays revealed that mutants KK197198AA, KK195198AA and KK195197AA could be processed into 66kDa activated toxins by trypsin or MJ while mutant KKK195197198AAA was not cleaved by trypsin and less susceptible to MJ. Bioassays demonstrated that mutants KK197198AA, KK195198AA and KK195197AA were toxic against S. exigua resembled that of wild-type Vip3Aa, however, the LC50 of mutant KKK195197198AAA against S. exigua was higher than wild-type. Those results suggested that proteolysis by MJ was associated with the insecticidal toxicity of Vip3Aa against S. exigua. It also revealed that trypsin played an important role in the formation of Vip3Aa activated toxin. Our studies characterized the proteolytic processing of Vip3Aa and provided new insight into the activation of this novel Bt toxin.