RESUMEN
Understanding how certain protein toxins from the normally insecticidal bacterium Bacillus thuringiensis (Bt) target human cell lines has implications for both the risk assessment of products containing these toxins and potentially for cancer therapy. This understanding requires knowledge of whether the human cell active toxins work by the same mechanism as their insecticidal counterparts or by alternative ones. The Bt Cry41Aa (also known as Parasporin3) toxin is structurally related to the toxins synthesised by commercially produced transgenic insect-resistant plants, with the notable exception of an additional C-terminal ß-trefoil ricin domain. To better understand its mechanism of action, we developed an efficient expression system for the toxin and created mutations in regions potentially involved in the toxic mechanism. Deletion of the ricin domain did not significantly affect the activity of the toxin against the human HepG2 cell line, suggesting that this region was not responsible for the mammalian specificity of Cry41Aa. Various biochemical assays suggested that unlike some other human cell active toxins from Bt Cry41Aa did not induce apoptosis, but that its mechanism of action was consistent with that of a pore-forming toxin. The toxin induced a rapid and significant decrease in metabolic activity. Adenosine triphosphate depletion, cell swelling and membrane damage were also observed. An exposed loop region believed to be involved in receptor binding of insecticidal Cry toxins was shown to be important for the activity of Cry41Aa against HepG2 cells.
Asunto(s)
Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/toxicidad , Endotoxinas/toxicidad , Hepatocitos/efectos de los fármacos , Modelos Moleculares , Proteínas Citotóxicas Formadoras de Poros/toxicidad , Adenosina Trifosfato/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Endotoxinas/química , Endotoxinas/genética , Endotoxinas/metabolismo , Células HeLa , Células Hep G2 , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Insecticidas/química , Insecticidas/metabolismo , Insecticidas/toxicidad , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidad , Homología Estructural de ProteínaRESUMEN
Previous studies reported "mode 1" Bacillus thuringiensis resistance in a colony of diamondback moths (NO-QA), and recently, this resistance has been mapped to an ABC transporter (ABCC2) locus. We report the lack of binding of Cry1Fa to insects derived from this colony and compare our data with those from other insects with ABCC2-associated resistance.