Study of the Bacillus thuringiensis Cry1Ia Protein Oligomerization Promoted by Midgut Brush Border Membrane Vesicles of Lepidopteran and Coleopteran Insects, or Cultured Insect Cells

Abstract

Bacillus thuringiensis (Bt) produces insecticidal proteins that are either secreted during the vegetative growth phase or accumulated in the crystal inclusions (Cry proteins) in the stationary phase. Cry1I proteins share the three domain (3D) structure typical of crystal proteins but are secreted to the media early in the stationary growth phase. In the generally accepted mode of action of 3D Cry proteins (sequential binding model), the formation of an oligomer (tetramer) has been described as a major step, necessary for pore formation and subsequent toxicity. To know if this could be extended to Cry1I proteins, the formation of Cry1Ia oligomers was studied by Western blot, after the incubation of trypsin activated Cry1Ia with insect brush border membrane vesicles (BBMV) or insect cultured cells, using Cry1Ab as control. Our results showed that Cry1Ia oligomers were observed only after incubation with susceptible coleopteran BBMV, but not following incubation with susceptible lepidopteran BBMV or non-susceptible Sf21 insect cells, while Cry1Ab oligomers were persistently detected after incubation with all insect tissues tested, regardless of its host susceptibility. The data suggested oligomerization may not necessarily be a requirement for the toxicity of Cry1I proteins.

Which Is Stronger? A Continuing Battle Between Cry Toxins and Insects

“…As described, a rough but generally accepted insecticidal mechanism of Cry toxins is the sequential binding model or the classic mode of action ( Khorramnejad et al, 2020 ). The sequential binding model comprises a complicated multistep process ( Figure 2 ), emphasizing on the specific binding of Cry toxins to a variety of receptors ( Fu et al, 2018 ).…”

Insecticidal Activity of Bacillus thuringiensis Proteins against Coleopteran Pests

“…The oligomeric structure eventually leads to the lytic pore formation that disrupts the midgut insect cell by osmotic shock. However, oligomerization studies of Cry1Ab and Cry1Ia proteins incubated with lepidopteran and coleopteran BBMV, as well as culture insect cells, showed that Cry1Ia oligomerization may not be a requirement for toxicity [ 179 ]. Besides, the appearance of Cry1Ab oligomers when incubated with coleopteran BBMV could be due to an improper insertion of oligomers into the membrane or the inability to induce the post-pore events in the cells [ 179 ].…”

“…However, oligomerization studies of Cry1Ab and Cry1Ia proteins incubated with lepidopteran and coleopteran BBMV, as well as culture insect cells, showed that Cry1Ia oligomerization may not be a requirement for toxicity [ 179 ]. Besides, the appearance of Cry1Ab oligomers when incubated with coleopteran BBMV could be due to an improper insertion of oligomers into the membrane or the inability to induce the post-pore events in the cells [ 179 ]. Either way, susceptible insects can withstand minor damage, but greater damage destroys the epithelium of the midgut, leading to a disruption in feeding and subsequent starvation death.…”

<em>Bacillus thuringiensis</em> Toxins: Functional Characterization and Mechanism of Action

“…In addition, other steps in the mode of action (that comprises protein solubilization, activation, binding, oligomerization, and pore formation) have also been addressed. Examples of these steps include the involvement of a novel trypsin protein for toxin activation in Plutella xylostella, discovered after studying a Cry1Ac resistant strain [9], and the promotion of oligomerization of the activated Cry1Ia with insect brush border midgut vesicles, in vitro [10]. The toxicity-promoting effect of a Bt chitin-binding protein that binds to the insect peritrophic matrix has also been studied [11].…”