Proteolytic activation of Bacillus thuringiensis Vip3Aa protein by Spodoptera exigua midgut protease

Abstract

Proteolysis of Vip3Aa by insect midgut proteases is essential for their toxicity against target insects. In the present study, proteolysis of Vip3Aa was evaluated by Spodoptera exigua midgut proteases (MJ). Trypsin was verified involved in the activation of Vip3Aa and three potential cleavage sites (Lys¹⁹⁵, Lys¹⁹⁷ and Lys¹⁹⁸) were identified. Four Vip3Aa mutants (KKK195197198AAA, KK197198AA, KK195198AA and KK195197AA) were designed and constructed by replacing residues Lys^195,197,198, Lys^197,198, Lys^195,198 and Lys^195,197 with Ala, respectively. Proteolytic processing assays revealed that mutants KK197198AA, KK195198AA and KK195197AA could be processed into 66kDa activated toxins by trypsin or MJ while mutant KKK195197198AAA was not cleaved by trypsin and less susceptible to MJ. Bioassays demonstrated that mutants KK197198AA, KK195198AA and KK195197AA were toxic against S. exigua resembled that of wild-type Vip3Aa, however, the LC₅₀ of mutant KKK195197198AAA against S. exigua was higher than wild-type. Those results suggested that proteolysis by MJ was associated with the insecticidal toxicity of Vip3Aa against S. exigua. It also revealed that trypsin played an important role in the formation of Vip3Aa activated toxin. Our studies characterized the proteolytic processing of Vip3Aa and provided new insight into the activation of this novel Bt toxin.

Keywords: Bacillus thuringiensis; Cleavage site; Insecticidal toxicity; Proteolysis; Vip3Aa.