Prophenoloxidase Activation Is Required for Survival to Microbial Infections in Drosophila

Abstract

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.

Analysis of the Contribution of Hemocytes and Autophagy to Drosophila Antiviral Immunity

“…They play a key role in host defense through melanization and they participate in wound healing. Lamellocytes are large (15 to 40 m in diameter), flat adherent cells which encapsulate and neutralize objects too large to be phagocytosed by plasmatocytes (30)(31)(32). Of note, the question of the involvement of hemocytes in antiviral immunity has not been a focus of interest so far for Drosophila.…”

Methods to study Drosophila immunity

“…Enterococcus faecalis and Staphylococcus aureus have been used to probe the Toll pathway in survival experiments, and occasionally Aerococcus viridans has been used to stimulate a Toll-dependent immune response. S. aureus is especially interesting to probe the role of phagocytosis and melanization [22,23,131]. Among DAP-type Gram-positive bacteria, Listeria monocytogenes, Listeria innocua, Bacillus megaterium and Bacillus subtilis are currently being used (for example [24]).…”

“…The most historic is Black cells, a gain-of function mutation in PPO1 that affects both PPO1 and PPPO2 proteins ( [51], Neyen, unpublished observations). More recently, a double PPO1, PPO2 mutant was generated which shows complete absence of PO activity in the hemolymph [131].…”

“…As a complement to the PO assay, a Western Blot using antibodies against Drosophila PO1 and PO2 can be performed on larval or adult hemolymph samples [97,131]. By this method, both the naïve PPO (75 kDa) and the mature form of PO (70 kDa), resulting from PO activation upon proteolytic cleavage by the upstream serine protease Hayan, can be detected.…”

Suppression of Shrimp Melanization during White Spot Syndrome Virus Infection

“…The melanization cascade, activated by the prophenoloxidase (proPO) system, has been documented as an important immune mechanism in shrimp and other arthropods in their defense against pathogens (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Most studies have shown that the melanization reaction is initiated by microbial elicitors, which then activate the proteolytic cascade that terminates with the activation of the zymogen proPO into the active phenoloxidase (PO) enzyme.…”