Overcoming insect immune response: The role of Pseudomonas aeruginosa alkaline protease in phenoloxidase inhibition

The role of Pseudomonas aeruginosa alkaline protease, one of the important virulence factors of these bacteria, in the prophenoloxidase (proPO) system activation in Galleria mellonella haemolymph was investigated. Immunization of G. mellonella larvae with alkaline protease led to an increase in phenoloxidase (PO) activity in haemolymph 2–8 h after the injection. However, 15 h after the challenge, almost no PO activity was detected, in contrast to insects immunized with heat‐killed P. aeruginosa, where an elevated PO activity in haemolymph persisted at all the time points of the experiment (2–24 h). To test the effect of alkaline protease on already activated PO in vitro, non‐immune G. mellonella haemolymph with proPO system pre‐activated in vitro by heat‐killed P. aeruginosa was used. Subsequently, direct incubation of the protease with non‐immune haemolymph was carried out to test the effect of the alkaline protease on the proPO system activation. It can be postulated that P. aeruginosa alkaline protease affected not only the active PO, but also significantly inhibited proPO cascade activation in the haemolymph of G. mellonella. Pseudomonas aeruginosa alkaline protease increases Galleria mellonella PO activity 2–8 h post‐challenge, however, protease drastically decreases PO activity 15 h after the injection. Alkaline protease decreases PO activity in G. mellonella haemolymph in vitro, but bacterial protease does not inhibit already activated insect PO. Alkaline protease inhibits earlier steps of G. mellonella proPO system.