Proteolytic processing and in vivo binding of the Bacillus thuringiensis Vip3Ca insecticidal protein

Abstract

A screening of collections of Bacillus thuringiensis strains led to the discovery of novel vip3 genes encoding the new family of Vip proteins Vip3Ca (Palma et al., 2012, Appl. Environ. Microbiol. 78: 7163). The present study examines the insecticidal spectrum of the Vip3Ca protein and reports the first results on the mode of action of this protein. The proteolytic processing of Vip3Ca was studied by incubating the protoxin with midgut juice from a susceptible insect (Mamestra brassicae), a moderately susceptible insect (Agrotis ipsilon) and nonsusceptible insect (Ostrinia nubilalis). In all cases, the ca. 90 kDa protoxin was converted into a ca. 60 kDa toxin, suggesting that the activation is not critical in determining the susceptibility of an insect species. Binding of Vip3Ca to the epithelial membrane of M. brassicae midgut larvae was shown after ingestion of the protoxin and further detection with an anti-Vip3 protoxin polyclonal antibody. The binding experiment was carried out in parallel with Vip3Aa, a more potent toxin against this insect species, and with a nontoxic Vip3 protein. Histopathological inspection showed swelling of the epithelial cells with further disruption, which suggests that the mode of action of Vip3Ca is similar to that described for Vip3Aa.