RESUMO
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.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Antígenos CD13/química , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insetos/química , Manduca/enzimologia , Receptores de Superfície Celular/química , Sequência de Aminoácidos , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Antígenos CD13/isolamento & purificação , Antígenos CD13/metabolismo , Endotoxinas/química , Proteínas Hemolisinas/química , Proteínas de Insetos/isolamento & purificação , Proteínas de Insetos/metabolismo , Resistência a Inseticidas , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Ligantes , Manduca/genética , Receptores de Superfície Celular/isolamento & purificação , Receptores de Superfície Celular/metabolismoRESUMO
Bacillus thuringiensis Cry1AbMod toxins are engineered versions of Cry1Ab that lack the amino-terminal end, including domain I helix α-1 and part of helix α-2. This deletion improves oligomerization of these toxins in solution in the absence of cadherin receptor and counters resistance to Cry1A toxins in different lepidopteran insects, suggesting that oligomerization plays a major role in their toxicity. However, Cry1AbMod toxins are toxic to Escherichia coli cells, since the cry1A promoter that drives its expression in B. thuringiensis has readthrough expression activity in E. coli, making difficult the construction of these CryMod toxins. In this work, we show that Cry1AbMod and Cry1AcMod toxins can be cloned efficiently under regulation of the cry3A promoter region to drive its expression in B. thuringiensis without expression in E. coli cells. However, p3A-Cry1Ab(c)Mod construction promotes the formation of Cry1AMod crystals in B. thuringiensis cells that were not soluble at pH 10.5 and showed no toxicity to Plutella xylostella larvae. Cysteine residues in the protoxin carboxyl-terminal end of Cry1A toxins have been shown to be involved in disulfide bond formation, which is important for crystallization. Six individual cysteine substitutions for serine residues were constructed in the carboxyl-terminal protoxin end of the p3A-Cry1AbMod construct and one in the carboxyl-terminal protoxin end of p3A-Cry1AcMod. Interestingly, p3A-Cry1AbMod C654S and C729S and p3A-Cry1AcMod C730S recover crystal solubility at pH 10.5 and toxicity to P. xylostella. These results show that combining the cry3A promoter expression system with single cysteine mutations is a useful system for efficient expression of Cry1AMod toxins in B. thuringiensis.
Assuntos
Bacillus thuringiensis/química , Proteínas de Bactérias/biossíntese , Cisteína/genética , Endotoxinas/biossíntese , Proteínas Hemolisinas/biossíntese , Regiões Promotoras Genéticas , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , DNA Bacteriano/genética , Endotoxinas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Hemolisinas/genética , Concentração de Íons de Hidrogênio , Larva , Lepidópteros , Mutagênese Sítio-Dirigida , Mutação , Controle Biológico de Vetores , Precursores de Proteínas/genética , Precursores de Proteínas/toxicidadeRESUMO
The venom of the scorpion Tityus costatus contains peptides toxic to humans but scarce information on their structure and function is available. Here, we report the separation of 50 different components by high performance liquid chromatography and the identification of approximately 90 distinct components by mass spectrometry analysis, with molecular weights varying from 413 to 45482 atomic mass units. Four peptides were fully sequenced: (i) a butantoxin-like peptide that blocks Shaker K+ channel; (ii) an insect toxin-like peptide; (iii) a scorpine-like peptide, and a short heptapeptide of unknown function. Fifteen peptides were directly sequenced at the N-terminal region, among which are components toxic to mice. A cDNA library was constructed and 13 clones were isolated and sequenced. Some of these peptides and genes are similar to other known scorpion toxins. Based on these results, stings by scorpions of the species Tityus costatus should be taken with caution by medical doctors.