Prophenoloxidase Activation Is Required for Survival to Microbial Infections in Drosophila

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.

Role and regulation of the melanization reaction in Drosophila immune response

Olivier Binggeli

In arthropods, the melanization reaction consists in 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 that subsequently polymerize into melanin. PO is synthesized under an inactive form called pro-PO (PPO) that is activated upon cleavage by a terminal protease of a serine protease cascade. The main objective of my PhD thesis is to understand the role and regulation of the melanization reaction in the Drosophila immune response. The Drosophila genome encodes three PPOs, two of which, PPO1 and PPO2, are produced by crystal cells, a larval-specific type of hemocyte. I generated flies carrying null mutations in PPO1 and PPO2 as well as PPO1, PPO2 double mutant flies. My results show that both PPO1 and PPO2 contribute to the PO activity in the hemolymph induced by septic injury. While PPO1 is involved in the early delivery of PO activity, PPO2 is stored in crystalline form in the crystal cells and can be deployed at a later phase of the immune response. My genetic analysis also reveals an important role of PPO1 and PPO2 in the resistance of flies to infection with Gram-positive bacteria and fungi. This study definitively establishes the importance of melanization in insect host defense. Serine proteases regulate many immune reactions, including blood clotting, the complement system in mammals, and melanization and Toll pathway activation in insects. A second objective of my thesis is to identify new serine proteases or serine protease inhibitors (serpins) participating in the Drosophila immune response. I performed an RNAi screen to identify genes encoding serine protease or serpins whose inactivation caused an immune phenotype. In parallel, I identified or generated mutations affecting some serine proteases and serpins and described their immune phenotype. This led to the identification of SPH9, a gene strongly induced by microbial infection, which encodes a serine protease homologue that negatively regulates the melanization reaction. Together, my results provide new insights on the melanization reaction in Drosophila.

EPFL2013

The Nimrod transmembrane receptor Eater is required for hemocyte attachment to the sessile compartment in Drosophila melanogaster

Olivier Binggeli, Andrew Jonathan Bretscher, Olivier Burri, Bruno Lemaitre

Eater is an EGF-like repeat transmembrane receptor of the Nimrod family and is expressed in Drosophila hemocytes. Eater was initially identified for its role in phagocytosis of both Gram-positive and Gram-negative bacteria. We have deleted eater and show that it appears to be required for efficient phagocytosis of Gram-positive but not Gram-negative bacteria. However, the most striking phenotype of eater deficient larvae is the near absence of sessile hemocytes, both plasmatocyte and crystal cell types. The eater deletion is the first loss of function mutation identified that causes absence of the sessile hemocyte state. Our study shows that Eater is required cell-autonomously in plasmatocytes for sessility. However, the presence of crystal cells in the sessile compartment requires Eater in plasmatocytes. We also show that eater deficient hemocytes exhibit a cell adhesion defect. Collectively, our data uncovers a new requirement of Eater in enabling hemocyte attachment at the sessile compartment and points to a possible role of Nimrod family members in hemocyte adhesion.

Company Of Biologists Ltd2015

Analysis of a specific modular serine protease acting in the Drosophila Toll pathway

Valentin Sottas

In Drosophila, the Toll pathway plays an important role in the immune defense against Gram-positive bacteria and fungi. Molecular determinants coming from those pathogens are directly detected by pattern recognition receptors (PPRs) circulating in the hemolymph. It has been proposed that several serine protease cascades are activated by PRRs, leading to the activation of a cleaved form of the cytokine-like molecule Spätzle, the ligand of the Toll receptor. The results obtained during my master project demonstrate an essential role for ModSP, a modular serine protease acting in the activation of the Toll pathway upon Gram-positive and fungal infections. We demonstrate that ModSP integrates signals coming from GNBP3 and PGRP-SA recognition molecules and that ModSP sends this signal to Grass, a serine protease already known to activate SPE and thereby Spätzle. Further biochemical experiments show the interaction between ModSP and GNBP1 demonstrating an apical role of this serine protease in the proteolytic cascades leading to Toll pathway activation. We also find that ModSP is expressed in specific vesicles released from the fat bodies into the hemolymph. Also, preliminary studies suggest that ModSP does not participate in the melanization reaction, a secondary but important insect immune mechanism. Biochemical analysis done by some collaborators indicates that ModSP does not cleave Grass and that ModSP exhibits a high level of auto-proteolysis when this molecule is expressed. Our data reveal a conserved role of modular serine protease in the regulation of immune proteolytic cascade in insects

2009

Role and regulation of the melanization reaction in Drosophila immune response

Olivier Binggeli

EPFL2013