Domain Shuffling between Vip3Aa and Vip3Ca: Chimera Stability and Insecticidal Activity against European, American, African, and Asian Pests

Abstract

The bacterium Bacillus thuringiensis produces insecticidal Vip3 proteins during the vegetative growth phase with activity against several lepidopteran pests. To date, three different Vip3 protein families have been identified based on sequence identity: Vip3A, Vip3B, and Vip3C. In this study, we report the construction of chimeras by exchanging domains between Vip3Aa and Vip3Ca, two proteins with marked specificity differences against lepidopteran pests. We found that some domain combinations made proteins insoluble or prone to degradation by trypsin as most abundant insect gut protease. The soluble and trypsin-stable chimeras, along with the parental proteins Vip3Aa and Vip3Ca, were tested against lepidopteran pests from different continents: Spodopteraexigua, Spodopteralittoralis, Spodopterafrugiperda,Helicoverpaarmigera, Mamestrabrassicae, Anticarsiagemmatalis, and Ostriniafurnacalis. The exchange of the Nt domain (188 N-terminal amino acids) had little effect on the stability and toxicity (equal or slightly lower) of the resulting chimeric protein against all insects except for S.frugiperda, for which the chimera with the Nt domain from Vip3Aa and the rest of the protein from Vip3Ca showed a significant increase in toxicity compared to the parental Vip3Ca. Chimeras with the C-terminal domain from Vip3Aa (from amino acid 510 of Vip3Aa to the Ct) with the central domain of Vip3Ca (amino acids 189-509 based on the Vip3Aa sequence) made proteins that could not be solubilized. Finally, the chimera including the Ct domain of Vip3Ca and the Nt and central domain from Vip3Aa was unstable. Importantly, an insect species tolerant to Vip3Aa but susceptible to Vip3Ca, such as Ostriniafurnacalis, was also susceptible to chimeras maintaining the Ct domain from Vip3Ca, in agreement with the hypothesis that the Ct region of the protein is the one conferring specificity to Vip3 proteins.

Keywords: Anticarsia gemmatalis; Bacillus thuringiensis; Mamestra brassicae; Ostrinia furnacalis; Spodoptera spp., Helicoverpa armigera.

Figure 1

Protein sequence alignment of Vip3Aa45 and Vip3Ca2. Black background shading is used to highlight the conserved amino acid between proteins. The proposed structural domains (based on the Vip3Af proteolysis mutants) are indicated with colored lines above the sequences [19]. The purple box indicates the position of the cleavage site (PPS1), while the red vertical lines show the sites chosen to generate the chimeric proteins.

Figure 2

Summary of the combinations of the different Vip3 protein domains expressed in the heterologous Escherichia coli expression system. The “single” chimeric Vip3 proteins (Vip3_ch1, Vip3_ch2, Vip3_ch5, and Vip3_ch6) were obtained from the Vip3Aa and Vip3Ca as a template, while the “double” chimeric Vip3 proteins (Vip3_ch3 and Vip3_ch4) were amplified from the Vip3_ch5 and Vip3_ch6. The percentage of similarity of the different proteins vs. the parental proteins, Vip3Aa and Vip3Ca, was calculated with the NCBI Blast align tool.

Figure 3

Time course of trypsin activation of Vip3 parental and chimeric protoxins. The reaction was carried out using 1% trypsin (w:w) at 37 °C for increasing incubation periods. (A) Vip3Aa protein and Vip3_ch1, (B) Vip3_ch5. (C) Vip3Ca and Vip3_ch2; (D) Vip3_ch4. The arrowheads indicate the protein bands corresponding to the 62–67 kDa fragment, while the asterisks indicate the protein bands corresponding to the 19–22 kDa fragment. M1: Molecular Mass Marker.

Figure 4

SDS-PAGE analysis of the purified parental (Vip3Aa and Vip3Ca) and chimeric (Vip3_ch1, Vip3_ch2, Vip3_ch4, and Vip3_ch5) proteins. (A) Parental proteins and chimeric Vip3 proteins (5 µg) purified by isoelectric point precipitation (IPP). (B) Parental proteins and chimeric Vip3 proteins (5 µg) purified by ion metal affinity chromatography (IMAC) on a Hi-Trap chelating HP column (GE Healthcare). (C) Vip3_ch5 protein (2 µg) purified by IPP and IMAC on a Hi-Trap chelating HP column (GE Healthcare). The arrowheads indicate the protein band corresponding to the chimeric Vip3 proteins. M1: Molecular Mass Marker.