Role and regulation of the melanization reaction in Drosophila immune response

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.

The melanization reaction in Drosophila: More than black or white

Jan Paul Dudzic

The melanization reaction is a rapid and important immune mechanism in arthropods. It results in the production of melanin at the site of injury and around invading microbes. The enzymes responsible for melanogenesis are phenoloxidases (PO), which catalyze the oxidation of phenols to quinones, which then polymerize into melanin. A by-product of melanogenesis are cytotoxic compounds that can pose a threat to the host organism itself due to their unspecific effects. Melanogenesis is therefore tightly regulated: POs are produced in an inactive form as prophenoloxidases (PPOs), which get activated by the sequential cleavage of an extracellular serine protease (SP) cascade. The aims of this PhD thesis were to better understand the melanization reaction in Drosophila melanogaster, at both the effector and the regulation levels. D. melanogaster has three PPO genes. We generated for the first time a mutant for the third PPO gene, PPO3, and analyzed its function. We demonstrated that PPO3 has an important role in the melanotic encapsulation reaction, a defense mechanism against parasitization. Additionally, we extended our knowledge about the other PPOs, PPO1 and PPO2. We confirmed their role in the defense against septic infections and ascribed a new role for PPO2 in the melanotic encapsulation. The use of single or combined mutations allowed us to show that each PPO mutant has a specific phenotype, and that knocking out two of three genes is required to abolish a particular function completely. Thus, Drosophila PPOs have partially overlapping functions to optimize melanization. Finally, we demonstrated that PPO3 is the result of a gene duplication of PPO2, restricted to a subgroup of Drosophila, and likely evolved as an additional defense mechanism in the cellular encapsulation process, probably due to the evolutionary pressure from parasitoid wasps. In the second part of this thesis, we re-addressed the roles and functions of three SPs involved in the melanization process. We developed a novel screening method for defects in melanization by infecting flies with a low-dose of S. aureus. We found that only one of the three SPs, Sp7, is involved in survival upon septic infections. Additionally, we demonstrated that the melanization reaction resulting in the clearance of systemic infections is regulated by extracellular components of the Toll pathway. While a connection between the SPs regulating the melanization and the Toll pathway was found in other insects, our study provides the first demonstration in D. melanogaster. We also present evidence of a disconnect between the melanin production at wound sites and the melanization reaction resulting in the clearance of infections, indicating a role of cytotoxic compounds in the killing of microbes. Finally, we ascribed a new role to Hayan, another SP implicated in melanization. Hayan is dispensable for the clearance of septic infections, but is important for wound melanization. We also demonstrated that Hayan plays an important role in the activation of the Toll pathway. Hayan, together with the SP Persephone, is necessary to activate Toll after infection. We propose that both Hayan and Persephone are the result of a recent gene duplication event. This can explain why they still have overlapping functions, but these genes also show signs of early sub-functionalization. Collectively, our work provides important insights on both melanization and the activation of the Toll pathway.

EPFL2018

Elucidating regulatory mechanisms shaping the transcriptome and proteome of the gut immune response in Drosophila melanogaster

Michael Vincent Frochaux

Work on Drosophila melanogaster paved the way to our current understanding of modern genetics. Since then, this model organism has contributed greatly to various fields such as neurobiology, development, and immunology. The discovery and analysis of the various pathways of the Drosophila immune response led the way to the in-depth characterization of the gut immune response. Furthermore, a genome-wide association study on a population of Drosophila identified resistance to infection by the entomopathogenic bacteria Pseudomonas entomophila (P.e.) as a complex phenotype, with highly resistant and susceptible lines, and highlighted genes and pathways mediating inter-individual differences. However, the molecular mechanisms mediating the resistance to enteric infection remain largely uncharacterized. This study uses the same Drosophila population to analyze what are the molecular mechanisms mediating the resistance to enteric infection. We analyzed the genetic determinants of gene expression variation among the most resistant and susceptible lines in response to infection using expression quantitative trait loci (eQTL) analysis. We also characterized the mode of action of these eQTLs into cis- and trans-acting. Furthermore, we identified the gene nutcracker (ntc) as the only differentially-expressed gene between resistance classes. We then demonstrate that loss of function ntc mutants are significantly more susceptible to infection and then identify a single nucleotide polymorphism (SNP) located in a transcription factor binding site (TFBS) which affects the binding affinity of the transcription factor Broad leading to allele-specific expression variation of the gene ntc. If the transcriptomic response to infection has been thoroughly characterized, it is not the case for the proteomic response. We thus sought to characterize for the first time the proteomic response of the Drosophila to enteric infection. We performed mass spectrometry and RNA sequencing on dissected guts from flies infected by two Gram-negative bacteria, Erwinia carotovora carotovora 15 (Ecc15) or Pseudomonas entomophila, 4h and 16h after infection. We found that a large portion of the measurable proteome (12%) varies after infection and that protein changes are strongly time- and infection-dependent. We showed the relatively poor correlation between gene expression and protein abundance in the Drosophila enteric immune response. Finally, we performed a screen of several proteins that were identified in our proteomics work but had previously not been found when assessing the gene expression response to infection using loss-of-function mutants and identifying 7 genes that modulate overall susceptibility to P.e. infection. In summary, this work analyze the genetic determinants of gene expression variation in the DGRP population upon infection and characterize them according to their mode of action. Furthermore, we describe how a non-coding variant lowers resistance to infection by modulating ntc gene expression through altered Broad repressor binding. Finally, it provides the first comprehensive characterization of the Drosophila gut proteome upon infection by Gram-negative bacteria which can be the basis of future proteomic analysis of the enteric immune response

EPFL2019

Prophenoloxidase Activation Is Required for Survival to Microbial Infections in Drosophila

Olivier Binggeli, Bruno Lemaitre

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.