Date Published: May 1, 2014
Publisher: Public Library of Science
Author(s): Olivier Binggeli, Claudine Neyen, Mickael Poidevin, Bruno Lemaitre, David S. Schneider.
The melanization reaction is a major immune response in Arthropods and involves the rapid synthesis of melanin at the site of infection and injury. A key enzyme in the melanization process is phenoloxidase (PO), which catalyzes the oxidation of phenols to quinones, which subsequently polymerize into melanin. The Drosophila genome encodes three POs, which are primarily produced as zymogens or prophenoloxidases (PPO). Two of them, PPO1 and PPO2, are produced by crystal cells. Here we have generated flies carrying deletions in PPO1 and PPO2. By analyzing these mutations alone and in combination, we clarify the functions of both PPOs in humoral melanization. Our study shows that PPO1 and PPO2 are responsible for all the PO activity in the hemolymph. While PPO1 is involved in the rapid early delivery of PO activity, PPO2 is accumulated in the crystals of crystal cells and provides a storage form that can be deployed in a later phase. Our study also reveals an important role for PPO1 and PPO2 in the survival to infection with Gram-positive bacteria and fungi, underlining the importance of melanization in insect host defense.
One of the most immediate immune responses in arthropods is the melanization reaction , . It involves the rapid synthesis of melanin at the site of infection or injury in order to contain a microbial pathogen as well as to facilitate wound healing. A key enzyme in melanin biosynthesis is phenoloxidase (PO), which catalyzes the oxidation of phenols to quinones, which subsequently polymerize into melanin. PO is usually synthesized as an inactive zymogen called proPO (PPO), which is cleaved to generate active PO as a result of proteolytic cascade activation. Several roles have been ascribed to the melanization reaction in insects , . PO activity contributes to wound healing by forming a scab at the epithelial site of injury. By-products of PO activity are reactive oxygen species (ROS), which are thought to contribute to the killing of microbes and pathogens. Finally, melanization participates in the encapsulation reaction against parasites. Deposition of melanin on the parasite forms a physical barrier, allowing the localized and confined production of toxic compounds while ensuring the protection of the host.
Although melanization is a well-established immune reaction of insects, the precise role of PO genes has never been addressed by loss-of-function mutations. In this study, we have generated deletions of PPO1 and PPO2. We observed that PPO mutants are viable and do not show any pigmentation defect. This is in agreement with the early assumption that another type of enzymes, laccases, but not POs, participate in cuticle tanning . An important finding is that PPO1Δ, PPO2Δ double mutants do not develop any hemolymphatic PO activity upon wounding or following microbial infection. This indicates that PPO1 and PPO2 are the two main sources of PO activity in the hemolymph of Drosophila. Antimicrobial peptide genes such as Diptericin and Drosomycin remain inducible in PPO1Δ, PPO2Δ double mutants. This is consistent with many studies, which showed that PO activity is not required for Toll and Imd pathway activation , , . Interestingly, Toll pathway activation tends to be stronger in PPO deficient flies, as revealed by a higher level of Drosomycin expression. Higher Toll activity was also observed upon injection of dead bacteria, indicating that this effect is not caused by an increased bacterial proliferation in melanization deficient flies. A possible explanation is that melanin alters the microbes rendering their PAMPs (most probably peptidoglycans) less accessible to pattern-recognition receptors that act upstream of the Toll pathway. Since the PPO and Toll cascades are downstream of the same Pattern-Recognition Receptors , another explanation is that loss of one downstream arm frees up effector proteases for the other arm, resulting in over-activation of Toll signaling in PPO mutants.