Oligomer Formation and Insecticidal Activity of Bacillus thuringiensis Vip3Aa Toxin

Abstract

Bacillus thuringiensis (Bt) Vip3A proteins are important insecticidal proteins used for control of lepidopteran insects. However, the mode of action of Vip3A toxin is still unclear. In this study, the amino acid residue S164 in Vip3Aa was identified to be critical for the toxicity in Spodoptera litura. Results from substitution mutations of the S164 indicate that the insecticidal activity of Vip3Aa correlated with the formation of a >240 kDa complex of the toxin upon proteolytic activation. The >240 kDa complex was found to be composed of the 19 kDa and the 65 kDa fragments of Vip3Aa. Substitution of the S164 in Vip3Aa protein with Ala or Pro resulted in loss of the >240 kDa complex and loss of toxicity in Spodoptera litura. In contrast, substitution of S164 with Thr did not affect the >240 kDa complex formation, and the toxicity of the mutant was only reduced by 35%. Therefore, the results from this study indicated that formation of the >240 kDa complex correlates with the toxicity of Vip3Aa in insects and the residue S164 is important for the formation of the complex. Key Contribution:Our results correlated the formation of a >240 kDa protein complex with the insecticidal activity of Vip3Aa toxin. The residue S164 in Vip3Aa protein was identified to be important for the formation of the >240 kDa protein complex.

Current Insights on Vegetative Insecticidal Proteins (Vip) as Next Generation Pest Killers

“…In another case, the Vip3Aa protein substitutions at site S164 with alanine or proline resulted in a loss of oligomer formation, and an ablation of the insecticidal potential against S. litura. Notably, substitution with threonine resulted in only a 35% reduction in toxicity [58].…”

“…Mutations at this site lead to a loss of insecticidal activity [58]. In addition, the two fragments are eluted together in gel permeation chromatography, emphasizing the fact that they may remain together after cleavage [55].…”

“…Moreover, another study proposed the formation of a >240 kDa complex and identified a novel site, S164, crucial for the formation and stability of this complex. Mutations at this site lead to a loss of insecticidal activity [ 58 ]. In addition, the two fragments are eluted together in gel permeation chromatography, emphasizing the fact that they may remain together after cleavage [ 55 ].…”

Structural and Functional Insights into the C-terminal Fragment of Insecticidal Vip3A Toxin of Bacillus thuringiensis

“…In addition, the~65 kDa C-terminal fragment of Vip3A used to be considered as the toxic core [8]. However, recent studies indicated that the~20 kDa N-terminal fragment and the~65 kDa C-terminal fragment of Vip3A still bind together after digestion, and the N-terminus is required for the stability and toxicity of Vip3A [20,21,41]. Moreover, several studies further demonstrated that Vip3A remains tetrameric after being activated by trypsin or midgut fluid [20,22].…”

Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action

“…In the Special Issue, five papers analyze different aspects of its biology. They cover aspects ranging from its crystal structure [ 14 ] and structural–functional domain analyses [ 15 ] to different aspects in the mode of action, such as a study of a possible receptor (the alkaline phosphatase) in a resistant strain [ 16 ], the role of oligomerization in toxicity [ 17 ], and the study of intracellular events promoted by Vip3A intoxication in Spodoptera frugiperda Sf9 cells [ 18 ].…”