Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domains II and III in specificity towards Spodoptera exigua larvae

Abstract

Several mutants of the Bacillus thuringiensis Cry1Ca toxin affected with regard to specific activity towards Spodoptera exigua were studied. Alanine was used to replace single residues in loops 2 and 3 of domain II (mutant pPB19) and to replace residues 541-544 in domain III (mutant pPB20). Additionally, a Cry1Ca mutant combining all mutations was constructed (mutant pPB21). Toxicity assays showed a marked decrease in toxicity against S. exigua for all mutants, while they retained their activity against Manduca sexta, confirming the importance of these residues in determining insect specificity. Parameters for binding to the specific receptors in BBMV (brush border membrane vesicles) of S. exigua were determined for all toxins. Compared with Cry1Ca, the affinity of mutant pPB19 was slightly affected (2-fold lower), whereas the affinity of the mutants with an altered domain III (pPB20 and pPB21) was approx. 8-fold lower. Activation of Cry1Ca protoxin by incubation with S. exigua or M. sexta BBMV revealed the transient formation of an oligomeric form of Cry1Ca. The presence of this oligomeric form was tested in the activation of the different Cry1Ca mutants, and we found that those mutated in domain II (pPB19 and pPB21) could not generate the oligomeric form when activated by S. exigua BBMV. In contrast, when oligomerization was tested using BBMV prepared from M. sexta, all of the Cry1Ca mutants showed the formation of a similar oligomeric form as did the wild-type toxin. Our results show how modification of insect specificity can be achieved by manipulation of different parts of the toxin structure involved in different steps of the mode of action of B. thuringiensis toxins.

The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta

“…Cry1Ca [18,30], Cry1Da, Cry1Ea, Cry1Fa [30]; coleopteran specific, Cry3Aa, Cry3Ba, Cry3Ca [37], and dipteran specific, Cry4Ba [26] and Cry11Aa (Pérez and Bravo, unpublished results). Protein samples containing Cry1Ab pre-pore oligomer were more toxic to M. sexta larvae than protein samples containing only monomeric Cry1Ab toxin [13].…”

“…After proteolytic activation of Cry1A protoxin by insect midgut proteases, the activated toxin binds to the Bt-R 1 receptor, and this interaction facilitates additional cleavage of the N-terminal end of the toxin, eliminating helix α-1, resulting in the formation of a pre-pore oligomeric structure [13]. The oligomeric structure has been observed in several Cry toxins [1,13,18,26,30,33,36,37,40]. The pre-pore binds to a second receptor, the GPI-anchored aminopeptidase, due to its higher affinity to this receptor [3], facilitating the insertion of the oligomer into membrane lipid rafts and forming pores [3,46].…”

Bacillus thuringiensis Cry1Ab Mutants Affecting Oligomer Formation Are Non-toxic to Manduca sexta Larvae

“…After proteolytic activation of Cry1A protoxin by insect midgut proteases, the activated toxin binds to a Bt-R receptor, and this interaction facilitates additional cleavage of the N-terminal end of the toxin, resulting in the formation of a pre-pore oligomer (10). This oligomeric structure has been observed in several Cry toxin members of the three-domain family (11)(12)(13)(14)(15)(16)(17). The prepore binds to a second receptor, a glycosylphosphatidylinositol-anchored aminopeptidase, due to its higher affinity to this receptor, facilitating insertion of the oligomeric toxin into membrane lipid rafts resulting in pore formation (3,18).…”

Interação de proteínas Cry1 e Vip3A de Bacillus thuringiensis para controle de lepidópteros-praga

“…A produção das proteínas Cry1 foi realizada de acordo com protocolo descrito por Herrero et al (2004). Para isso, os clones foram cultivados durante 60 horas, a 28°C, com agitação de 200 rpm, em meio TB ("terrific broth") constituído de: 12 g de triptona; 24 g de extrato de levedura; 2,31 g de KH 2 PO 4 ; 12,54 g de K 2 HPO 4 ; 4 mL de glicerol; q.s.p 1.000 mL; além de ampicilina (100 µg mL -1 ).…”

“…Para isso, os clones foram cultivados durante 60 horas, a 28°C, com agitação de 200 rpm, em meio TB ("terrific broth") constituído de: 12 g de triptona; 24 g de extrato de levedura; 2,31 g de KH 2 PO 4 ; 12,54 g de K 2 HPO 4 ; 4 mL de glicerol; q.s.p 1.000 mL; além de ampicilina (100 µg mL -1 ). Herrero et al (2004). Para a expressão das proteínas Vip3A, utilizou-se a metodologia descrita por Chakroun et al (2012), que altera a concentração de isopropil-D-tiogalactopiranosídeo (IPTG) para 1 mmol L -1 na indução da expressão.…”