Extracellular proteomic profiling of Bacillus toyonensis biovar Thuringiensis Bto_UNVM-42 identifies candidate proteins potentially associated with its nematicidal activity

Previous studies demonstrated that cell-free supernatants of Bacillus toyonensis biovar Thuringiensis Bto_UNVM-42 exhibit nematicidal activity against Panagrellus redivivus, although the molecular basis of this phenotype remains unclear. While pesticidal proteins in Bacillus thuringiensis and related species are classically considered intracellular and associated with parasporal crystals, their occurrence in extracellular fractions remains poorly explored. Here, we characterized the extracellular protein repertoire of biologically active supernatants and evaluated their effects on Caenorhabditis elegans N2. LC-MS/MS analysis of LB-derived cell-free supernatants revealed the presence of pesticidal protein homologs related to Cry32-, Cyt1, and Mpp3-like protein families, together with degradative enzymes including collagenases, chitinases, proteases, phospholipases, and cytolysins. Signal peptide prediction suggested classical secretion pathways for several proteins, whereas others may reach extracellular fractions through alternative localization mechanisms. Comparative DIA-based proteomic analyses of LB and CCY supernatants revealed marked medium-dependent differences in the extracellular protein repertoire, including differential enrichment of Bt-like pesticidal homologs and degradative enzymes associated with distinct physiological states. Exposure assays showed that concentrated cell-free supernatants from both conditions caused severe loss of mobility and morphological alterations consistent with tissue disruption in C. elegans. Together, these findings provide proteomic evidence for the extracellular occurrence of pesticidal and degradative protein homologs in Bto_UNVM-42 and identify candidate extracellular factors potentially associated with the nematicidal activity of this strain. More broadly, the results support the view that soluble extracellular components may contribute to the biological activity of Bt-related bacteria beyond the classical spore-crystal paradigm.