Bacterial Extracellular Vesicles from Chromobacterium subtsugae and Bacillus thuringiensis as Cell-Free Bioinsecticidal Nanocarriers Against the Soybean Pest Euschistus heros

Bacterial extracellular vesicles (bEVs) are membrane-enclosed nanoparticles that carry bioactive cargo and mediate bacteria–environment interactions, but their use in pest biocontrol has been largely unexplored. This study provides proof-of-concept that bEVs isolated from two entomopathogenic bacteria—Chromobacterium subtsugae and Bacillus thuringiensis var. kurstaki—have insecticidal activity against Euschistus heros, a major soybean pest. The vesicles were characterized using tunable resistive pulse sensing, nano-flow cytometry, transmission electron microscopy, SDS-PAGE, MALDI-TOF mass spectrometry, and label-free quantitative proteomics.

C. subtsugae bEVs showed a proteome distinctly remodeled relative to the soluble protein fraction, enriched in outer-membrane, secretion-associated, proteolytic, and membrane-active proteins. MALDI-TOF detected a violacein-associated ion selectively in the bEV fraction, indicating that this hydrophobic bioactive metabolite is associated with the vesicles. In survival assays, C. subtsugae bEVs reduced E. heros nymph survival strongly (hazard ratio 4.0, p < 0.0001), while the soluble protein fraction had no significant effect (HR 1.2, p = 0.50). By contrast, B. thuringiensis bEVs and soluble fractions both showed moderate, similar activity (HR 2.1 for each), consistent with their overlapping proteomic profiles; Cry1Ab was found mainly in the soluble fraction rather than enriched in bEVs.

These findings support a multi-component cargo model in which C. subtsugae bEVs combine vesicle-associated violacein with enriched protein cargo to exert insecticidal effects. The study positions bacterial EVs as promising natural nanocarriers for next-generation, cell-free bioinsecticides, particularly against Cry-resistant hemipteran pests such as E. heros.