The melanization reaction in Drosophila: More than black or white

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.

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

Iron sequestration by transferrin 1 mediates nutritional immunity in Drosophila melanogaster

Jean Philippe Boquete, Igor Iatsenko, Bruno Lemaitre, Alice Marra, Jasquelin Peña

Iron sequestration is a recognized innate immune mechanism against invading pathogens mediated by iron-binding proteins called transferrins. Despite many studies on antimicrobial activity of transferrins in vitro, their specific in vivo functions are poorly understood. Here we use Drosophila melanogaster as an in vivo model to investigate the role of transferrins in host defense. We find that systemic infections with a variety of pathogens trigger a hypoferremic response in flies, namely, iron withdrawal from the hemolymph and accumulation in the fat body. Notably, this hypoferremia to infection requires Drosophila nuclear factor kappa B (NF-kappa B) immune pathways, Toll and Imd, revealing that these pathways also mediate nutritional immunity in flies. Next, we show that the iron transporter Tsf1 is induced by infections downstream of the Toll and Imd pathways and is necessary for iron relocation from the hemolymph to the fat body. Consistent with elevated iron levels in the hemolymph, Tsf1 mutants exhibited increased susceptibility to Pseudomonas bacteria and Mucorales fungi, which could be rescued by chemical chelation of iron. Furthermore, using siderophore-deficient Pseudomonas aeruginosa, we discover that the siderophore pyoverdine is necessary for pathogenesis in wild-type flies, but it becomes dispensable in Tsf1 mutants due to excessive iron present in the hemolymph of these flies. As such, our study reveals that, similar to mammals, Drosophila uses iron limitation as an immune defense mechanism mediated by conserved iron-transporting proteins transferrins. Our in vivo work, together with accumulating in vitro studies, supports the immune role of insect transferrins against infections via an iron withholding strategy.

NATL ACAD SCIENCES2020

Infection Dynamics and Immune Response in a Newly Described Drosophila-Trypanosomatid Association

Anna Dostalova, Bruno Lemaitre

Trypanosomatid parasites are significant causes of human disease and are ubiquitous in insects. Despite the importance of Drosophila melanogaster as a model of infection and immunity and a long awareness that trypanosomatid infection is common in the genus, no trypanosomatid parasites naturally infecting Drosophila have been characterized. Here, we establish a new model of trypanosomatid infection in Drosophila-Jaenimonas drosophilae, gen. et sp. nov. As far as we are aware, this is the first Drosophila-parasitic trypanosomatid to be cultured and characterized. Through experimental infections, we find that Drosophila falleni, the natural host, is highly susceptible to infection, leading to a substantial decrease in host fecundity. J. drosophilae has a broad host range, readily infecting a number of Drosophila species, including D. melanogaster, with oral infection of D. melanogaster larvae resulting in the induction of numerous immune genes. When injected into adult hemolymph, J. drosophilae kills D. melanogaster, although interestingly, neither the Imd nor the Toll pathway is induced and Imd mutants do not show increased susceptibility to infection. In contrast, mutants deficient in drosocrystallin, a major component of the peritrophic matrix, are more severely infected during oral infection, suggesting that the peritrophic matrix plays an important role in mediating trypanosomatid infection in Drosophila. This work demonstrates that the J. drosophilae-Drosophila system can be a powerful model to uncover the effects of trypanosomatids in their insect hosts. IMPORTANCE Trypanosomatid parasites are ubiquitous in insects and are significant causes of disease when vectored to humans by blood-feeding insects. In recent decades, Drosophila has emerged as the predominant insect model of infection and immunity and is also known to be infected by trypanosomatids at high rates in the wild. Despite this, there has been almost no work on their trypanosomatid parasites, in part because Drosophila-specific trypanosomatids have been resistant to culturing. Here, we present the first isolation and detailed characterization of a trypanosomatid from Drosophila, finding that it represents a new genus and species, Jaenimonas drosophilae. Using this parasite, we conducted a series of experiments that revealed many of the unknown aspects of trypanosomatid infection in Drosophila, including host range, transmission biology, dynamics of infection, and host immune response. Taken together, this work establishes J. drosophilae as a powerful new opportunity to study trypanosomatid infections in insects.