RESUMEN
The insecticidal Cry toxins are pore-forming toxins produced by the bacteria Bacillus thuringiensis that disrupt insect-midgut cells. Cells can trigger different survival mechanisms to counteract the effects of sub-lytic doses of pore forming toxins. Particularly, two signaling pathways have been demonstrated to play a role in the defense mechanism to other toxins in Caenorhabditis elegans and in mammalian cells. These are the unfolded protein response (UPR) and the sterol regulatory element binding proteins (SREBP) pathways, which are proposed to facilitate membrane repair responses. In this work we analyzed the role of these pathways in Aedes aegypti response to intoxication with Cry11Aa toxin. We show that UPR is activated upon toxin ingestion. The role of these two pathways was analyzed in vivo by using RNA interference. We silenced the expression of specific proteins in A. aegypti larvae. Gene silencing of Ire-1 and Xbp-1 proteins from UPR system, resulted in hypersensitive to Cry11Aa toxin action. In contrast, silencing of Cas-1, Scap and S2P from SREBP pathway had no affect on Cry11Aa toxicity in A. aegypti larvae. However, the role of SREBP pathway requires further studies to be conclusive. Our data indicate that the UPR pathway is involved in the insect defense against Cry toxins.
Asunto(s)
Aedes/metabolismo , Aedes/microbiología , Bacillus thuringiensis/patogenicidad , Proteínas Bacterianas/toxicidad , Endotoxinas/toxicidad , Proteínas Hemolisinas/toxicidad , Respuesta de Proteína Desplegada , Aedes/genética , Animales , Toxinas de Bacillus thuringiensis , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genes de Insecto , Proteínas de Insectos/antagonistas & inhibidores , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Larva/genética , Larva/metabolismo , Larva/microbiología , Filogenia , Interferencia de ARN , Factores de Transcripción del Factor Regulador X , Transducción de Señal , Proteínas de Unión a los Elementos Reguladores de Esteroles/antagonistas & inhibidores , Proteínas de Unión a los Elementos Reguladores de Esteroles/genética , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Estrés Fisiológico , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Bacillus thuringiensis subs. israelensis produces at least three Cry toxins (Cry4Aa, Cry4Ba, and Cry11Aa) that are active against Aedes aegypti larvae. Previous work characterized a GPI-anchored alkaline phosphatase (ALP1) as a Cry11Aa binding molecule from the gut of A. aegypti larvae. We show here that Cry4Ba binds ALP1, and that the binding and toxicity of Cry4Ba mutants located in loop 2 of domain II is correlated. Also, we analyzed the contribution of ALP1 toward the toxicity of Cry4Ba and Cry11Aa toxins by silencing the expression of this protein though RNAi. Efficient silencing of ALP1 was demonstrated by real-time quantitative PCR (qPCR) and Western blot. ALP1 silenced larvae showed tolerance to both Cry4Ba and Cry11Aa although the silenced larvae were more tolerant to Cry11Aa in comparison to Cry4Ba. Our results demonstrate that ALP1 is a functional receptor that plays an important role in the toxicity of the Cry4Ba and Cry11Aa proteins.