A new strategy to improve the virulence of Bacillus thuringiensis supernatant based on enhancing Vip3A secretion

Vegetative insecticidal protein (Vip3), which has potent insecticidal activity against lepidopteran insect pests, is a non-classical secreted insecticidal protein produced by Bacillus thuringiensis during vegetative growth. Strains with high levels of Vip3A expression have been developed, but a large amount of Vip3A is retained inside the cell, failing to be secreted. In this study, we found that cell wall hydrolases (CWHs) acting on different sites of peptidoglycan cause differences in the level of Vip3A secretion. Vip3A secretion was significantly increased when MltE and BioL (two cell wall hydrolases acting on glycosidic bonds) were abundantly expressed. On the contrary, overexpression of EnvC (a cell wall hydrolase that acted on amide bonds) decreased Vip3A secretion. Furthermore, we demonstrated that overexpression of MltE significantly increased the secretion of other proteins in MV (Membrane vesicles) and increased MV production. The fermentation broth of MltE overexpression strain demonstrated a potent insecticidal effect merely 12 h after cultivation. Our results suggest that CWHs cooperate in controlling bacterial MV formation and disruption of cell wall homeostasis can cause massive MV release. These findings provide new ideas for enhancing Vip3A secretion in B. thuringiensis. They also have significant implications for improving the secretion of other MV proteins and studying MV biogenesis and release in Gram-positive bacteria.