Activation of Aedes aegypti prophenoloxidase-3 and its role in the immune response against entomopathogenic fungi

Serine protease cascade-mediated melanization is an important innate immune response in insects and crustaceans, which involves the proteolytic activation of prophenoloxidase (PPO). In this study, we investigated the role of Aedes aegypti PPO3 in antifungal immune defence. We expressed and purified recombinant PPO3 (rPPO3) in Escherichia coli and demonstrated that rPPO3 was activated by ethanol and, to a lesser extent, by cetylpyridinium chloride. In the presence of Cu²⁺ , rPPO3 exhibited enzyme activity. Immunoblot results revealed that the rPPO3 was cleaved by the haemolymph from immune-challenged mosquitoes or purified Ostrinia furnacalis serine protease 105 in vitro. The cleaved rPPO3 converted dopamine to toxic intermediates that killed fungal conidia of Beauveria bassiana in vitro. In mosquitoes challenged with Be. bassiana, cleavage of rPPO3 produced a 50 kDa phenoloxidase (PO) fragment. Further analysis revealed that the survival rate of mosquitoes with fungal infection increased significantly following injection of rPPO3 into the haemocoel. Taken together, our results suggest that proteolytic cleavage of the mosquito PPO3 plays an important role in the antifungal immune response. This has led to a better understanding of the mechanism of PPO activation in the mosquito and the role of melanization in the antifungal immune response.