RESUMEN
Bacillus thuringiensis subsp. israelensis produces crystal inclusions composed of three-domain Cry proteins and cytolytic Cyt toxins, which are toxic to different mosquito larvae. A key component is the Cyt toxin, which synergizes the activity of the other Cry toxins, thereby resulting in high toxicity. The precise mechanism of action of Cyt toxins is still debated, and two models have been proposed: the pore formation model and the detergent effect. Here, we performed a systematic structural characterization of the Cyt toxin interaction with different membranes, including in Aedes aegypti larval brush border membrane vesicles, small unilamellar vesicle liposomes, and rabbit erythrocytes. We examined Cyt1Aa insertion into these membranes by analyzing fluorescence quenching in solution and in the membrane-bound state. For this purpose, we constructed several Cyt1Aa variants having substitutions with a single cysteine residue in different secondary structures, enabling Cys labeling with Alexa Fluor 488 for quenching analysis using I-soluble quencher in solution and in the membrane-bound state. We identified the Cyt1Aa residues exposed to the solvent upon membrane insertion, predicting a possible topology of the membrane-inserted toxin in the different membranes. Moreover, toxicity assays with these variants revealed that Cyt1Aa exerts its insecticidal activity and hemolysis through different mechanisms. We found that Cyt1Aa exhibits variable interactions with each membrane system, with deeper insertion into mosquito larva membranes, supporting the pore formation model, whereas in the case of erythrocytes and small unilamellar vesicles, Cyt1Aa's insertion was more superficial, supporting the notion that a detergent effect underlies its hemolytic activity.
Asunto(s)
Aedes/metabolismo , Toxinas de Bacillus thuringiensis/farmacología , Bacillus thuringiensis/química , Endotoxinas/farmacología , Membrana Eritrocítica/metabolismo , Proteínas Hemolisinas/farmacología , Animales , Larva , Liposomas , ConejosRESUMEN
Bacillus thuringiensis (Bt) Cyt1Aa toxin shows toxicity to mosquitoes, to certain coleopteran pests and also to red blood cells (RBC). However, its mode of action in the different target cells is not well defined. This protein is a single α-ß domain pore-forming toxin, where a ß sheet is wrapped by two α-helices layers. The Cyt1Aa α-helix hairpin in the N-terminal has been proposed to be involved in initial membrane binding and oligomerization, while the ß sheet inserts into the membrane to form a pore that lyze the cells. To determine the role of the N-terminal α-helix hairpin region of Cyt1Aa in its mode of action, we characterized different single point mutations located in helices α-1 and α-2. Eight cysteine substitutions in different residues were produced in Bt, and we found that three of them: Cyt1AaA65C, Cyt1AaL85C and Cyt1AaN89C, lost insecticidal toxicity against Aedes aegypti larvae but retained similar or increased hemolytic activity towards rabbit RBC. Analysis of toxin binding and oligomerization using Ae. aegypti midgut brush border membrane vesicles showed that the three Cyt1Aa mutants non-toxic to Ae. aegypti were affected in oligomerization. However, these mutants were still hemolytic. Our data shows that oligomerization of Cyt1Aa toxin is essential for its toxicity to Ae. aegypti but not for its toxicity against RBC indicating that the mode of action of Cyt1Aa is different in these distinct target membranes.
Asunto(s)
Aedes/efectos de los fármacos , Proteínas Bacterianas/química , Endotoxinas/química , Eritrocitos/efectos de los fármacos , Proteínas Hemolisinas/química , Insecticidas/química , Aedes/crecimiento & desarrollo , Animales , Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/toxicidad , Endotoxinas/toxicidad , Proteínas Hemolisinas/toxicidad , Insecticidas/toxicidad , Larva/efectos de los fármacos , Larva/crecimiento & desarrollo , Polimerizacion , ConejosRESUMEN
The western corn rootworm (WCR) Diabrotica virgifera virgifera causes substantial damage in corn. Genetically modified (GM) plants expressing some Bacillus thuringiensis (Bt) insecticidal Cry proteins efficiently controlled this pest. However, changes in WCR susceptibility to these Bt traits have evolved and identification of insecticidal proteins with different modes of action against WCR is necessary. We show here for the first time that Cyt1Aa from Bt exhibits toxicity against WCR besides to the dipteran Aedes aegypti larvae. Cyt1Aa is a pore-forming toxin that shows no cross-resistance with mosquitocidal Cry toxins. We characterized different mutations in helix α-A from Cyt1Aa. Two mutants (A61C and A59C) exhibited reduced or absent hemolytic activity but retained toxicity to A. aegypti larvae, suggesting that insecticidal and hemolytic activities of Cyt1Aa are independent activities. These mutants were still able to form oligomers in synthetic lipid vesicles and to synergize Cry11Aa toxicity. Remarkably, mutant A61C showed a five-fold increase insecticidal activity against mosquito and almost 11-fold higher activity against WCR. Cyt1Aa A61C mutant was as potent in killing WCR that were selected for resistance to mCry3A as it was against unselected WCR indicating that this toxin could be a useful resistance management option in the control of WCR.
Asunto(s)
Bacillus thuringiensis , Proteínas Bacterianas , Escarabajos/crecimiento & desarrollo , Endotoxinas , Proteínas Hemolisinas , Mutación Missense , Control Biológico de Vectores , Animales , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Proteínas Bacterianas/toxicidad , Endotoxinas/genética , Endotoxinas/toxicidad , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/toxicidad , Insecticidas/toxicidadRESUMEN
Cell lines have been use extensively for the study of the mode of action of different pore forming toxins produced by different bacterial species. Bacillus thuringiensis Cry toxins are not the exception and their mechanism of action has been analyzed in different cell lines. Here we review the data obtained with different cell lines, including those that are naturally susceptible to the three domain Cry toxins (3d-Cry) and other non-susceptible cell lines that have been transformed with 3d-Cry toxin binding molecules cloned from the susceptible insects. The effects on Cry toxin action after expressing different insect gut proteins, such as glycosyl-phosphatidyl-inositol (GPI) anchored proteins (like alkaline phosphatase (ALP) aminopeptidase (APN)), or trans-membrane proteins (like cadherin (CAD) or ATP-binding cassette subfamily C member 2 (ABCC2) transporter) in cell lines showed that, with few exceptions, expression of GPI-anchored proteins do not correlated with increased susceptibility to the toxin, while the expression of CAD or ABCC2 proteins correlated with induced susceptibility to Cry toxins in the transformed cells lines. Also, that the co-expression of CAD and ABCC2 transporter induced a synergistic effect in the toxicity of 3d-Cry toxins. Overall the data show that in susceptible cell lines, the 3d-Cry toxins induce pore formation that correlates with toxicity. However, the intracellular responses remain controversial since it was shown that the same 3d-Cry toxin in different cell lines activated different responses such as adenylate cyclase-PKA death response or apoptosis. Parasporins are Cry toxins that are toxic to cancer cell lines that have structural similarities with the insecticidal Cry toxins. They belong to the 3d-Cry toxin or to MTX-like Cry toxin families but also show important differences with the insecticidal Cry proteins. Some parasporins are pore-forming toxins, and some activate apoptosis. In this review we summarized the results of the different studies about the Cry toxins mode of action using cultured cell lines and discuss their relation with the studies performed in insect larvae.
Asunto(s)
Bacillus thuringiensis/química , Proteínas Bacterianas/farmacología , Endotoxinas/farmacología , Proteínas Hemolisinas/farmacología , Insectos/microbiología , Animales , Toxinas de Bacillus thuringiensis , Línea Celular , Línea Celular Tumoral , Insectos/crecimiento & desarrollo , Larva/crecimiento & desarrollo , Larva/microbiologíaRESUMEN
Bacillus thuringiensis Cry2Ab toxin has been used in combination with Cry1Ac for resistance management on the Bt-cotton that is widely planted worldwide. However, little is known regarding Cry2Ab mode of action. Particularly, there is a gap of knowledge on the identification of insect midgut proteins that bind Cry2Ab and mediate toxicity. In the case of Cry1Ab toxin, a transmembrane cadherin protein and glycosyl-phosphatidylinositol (GPI) anchored proteins like aminopeptidase-N1 (APN1) or alkaline-phosphatase (ALP) from Manduca sexta, have been shown to be important for oligomer formation and insertion into the membrane. Binding competition experiments showed that Cry2Ab toxin does not share binding sites with Cry1Ab toxin in M. sexta brush border membrane vesicles (BBMV). Also, that Cry2Ab shows reduced binding to the Cry1Ab binding molecules cadherin, APN1 or ALP. Finally, ligand blot experiments and protein sequence by LC-MS/MS identified APN2 isoform as a Cry2Ab binding protein. Cloning and expression of APN2 confirmed that APN2 is a Cry2Ab binding protein.