Investigation of protease-mediated cuticle-degradation of nematodes by using an improved immunofluorescence-localization method

In order to facilitate the understanding of the actual process of enzyme-based degradation of nematodes, we visualized the localization of BLG4, a cuticle-degrading protease from the nematophagous bacterium Brevibacillus laterosporus G4, on nematode cuticle by using an improved immuno-labeled fluorescent method. Live nematodes, heat-killed nematodes and extracted nematode cuticles were exposed to the protease, and the localization of the protease and the resulting tissue degradation and destruction were observed microscopically. The bioassay findings showed that live nematodes were significantly more resistant to the protease than the dead nematodes and the extracted cuticles were. The observation of the immuno-labeling fluorescence for BLG4 revealed that the protease localized first in the tail region of the live target; and then spread over the entire target and ultimately destroyed it, including the cuticle. The results indicated the resistance of nematode cuticles to enzymatic attacks and the differences in protease susceptibilities at different regions on the nematode cuticles.