N-Acetylcysteine Attacks Phenoloxidase to Inhibit Insect Melanization and Boost Fungal Virulence

The prophenoloxidase (PPO) melanization cascade is a cornerstone of insect humoral immunity against fungal pathogens, yet its direct fungicidal mechanism remains undefined, and strategies to safely manipulate this pathway for biological control are lacking. Here, we demonstrate that infection ofDrosophilaby the entomopathogenic fungusBeauveria bassianaelicits a surge in phenoloxidase (PO) activity directly triggered by the fungal cell wall component β-1,3-Glucan. This activated PO generates reactive oxygen species (ROS) that are essential for restricting fungal proliferation, thereby establishing a mechanistic link between cascade activation and fungicidal outcome. Critically, we uncover that N-acetylcysteine (NAC), a safe, low-cost pharmaceutical in global clinical use, acts as a potent direct inhibitor of PO enzymatic activity, neutralizing the melanization-driven ROS burst. NAC-mediated inhibition significantly acceleratesB. bassianavirulence and host mortalityin vivo. Our work defines a new molecular function for NAC as a melanization inhibitor and reveals a safe, translatable strategy to suppress host immune defenses, offering a promising approach to enhance the efficacy of fungal biopesticides for sustainable pest control worldwide.