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.

Keywords: Bacillus thuringiensis; Spodoptera litura; Vip3A; site-directed mutagenesis.

Figure 1

Analysis of Vip3Aa proteins after treatment by trypsin or midgut proteases of S. litura. Purified Vip3Aa-WT, Vip3Aa-S164T, Vip3Aa-S164A and Vip3Aa-S164P were in vitro digested by commercial trypsin or midgut proteases of S. litura. Processed proteins were mixed with 5 × SDS-PAGE sample buffer followed by heat denaturation and analyzed by the electrophoretic analysis in an SDS-PAGE gel.

Figure 2

Analysis of native Vip3Aa proteins after proteolytic processing. Protease treated Vip3Aa-WT, Vip3Aa-S164T, Vip3Aa-S164A and Vip3Aa-S164P by either commercial trypsin or midgut proteases of S. litura larvae were analyzed by the electrophoretic analysis without heat denaturation. (a) Vip3Aa proteins after tryptic processing were analyzed in a native gel; (b): Vip3Aa proteins after processing by midgut proteases were analyzed in a native gel; (c): Vip3Aa proteins after tryptic processing were mixed with 5 × SDS-PAGE sample buffer without β-mercaptoethanol and analyzed in an SDS-PAGE gel. Protein complex 1, protein complex 2 and protein complex 3 in panel (a) indicate gel bands sliced from each lane in the native gel.

Figure 3

Separation of peptides from protein complexes of tryptic Vip3Aa proteins. Major protein bands representing different protein complexes in Figure 2a were sliced and separated in an SDS-PAGE gel. (a) peptides separated from the protein complex 3 in Figure 2a; (b) peptides separated from the protein complexes 1 and 2 respectively in Figure 2a. The yellow, red, black and white arrows indicate the bands of 95 kDa, 19 kDa, 17 kDa and 15 kDa fragments respectively.

Figure 4

Schematic representation of the 15 kDa, 17 kDa and 19 kDa fragments from Vip3Aa protein. The first 198 amino acids at the N terminus of Vip3Aa were presented. The red box indicates amino acids corresponding to the N terminal 19 kDa fragment of Vip3Af. The yellow, blue and green boxes represent LC-MS/MS identified peptides from the 15 kDa, 19 kDa and 17 kDa fragments respectively.

Figure 5

Mortality of S. litura larvae fed with trypsin-processed Vip3Aa proteins. After tryptic processing, Vip3Aa-WT, Vip3Aa-S164T, Vip3Aa-S164A and Vip3Aa-S164P were respectively fed to neonates of S. litura for 96 h to test their insecticidal activity. Error bars indicate the standard error of mortality among five replications.