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Eater and NimC1 are transmembrane receptors of the Drosophila Nimrod family, specifically expressed in haemocytes, the insect blood cells. Previous ex vivo and in vivo RNAi studies have pointed to their role in the phagocytosis of bacteria. Here, we have created a novel NimC1 null mutant to re‐evaluate the role of NimC1, alone or in combination with Eater, in the cellular immune response. We show that NimC1 functions as an adhesion molecule ex vivo, but in contrast to Eater it is not required for haemocyte sessility in vivo. Ex vivo phagocytosis assays and electron microscopy experiments confirmed that Eater is the main phagocytic receptor for Gram‐positive, but not Gram‐negative bacteria, and contributes to microbe tethering to haemocytes. Surprisingly, NimC1 deletion did not impair phagocytosis of bacteria, nor their adhesion to the haemocytes. However, phagocytosis of both types of bacteria was almost abolished in NimC1 1 ;eater 1 haemocytes. This indicates that both receptors contribute synergistically to the phagocytosis of bacteria, but that Eater can bypass the requirement for NimC1. Finally, we uncovered that NimC1, but not Eater, is essential for uptake of latex beads and zymosan particles. We conclude that Eater and NimC1 are the two main receptors for phagocytosis of bacteria in Drosophila, and that each receptor likely plays distinct roles in microbial uptake., Eater and NimC1 are transmembrane receptors belonging to the Drosophila Nimrod family. These receptors are expressed in insect blood cells (haemocytes) and have been implicated in bacterial phagocytosis. In this study, Bruno Lemaitre and colleagues created a novel NimC1 null mutant and used this to characterize the role of NimC1 in cellular immune responses, alone and in combination with Eater. The authors reveal that Eater and NimC1 contribute synergistically to the initial step of phagocytosis, notably adhesion to bacteria, and that the receptors play distinct roles in this process.

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 κB (NF-κB) 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.

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.

The fruit fly Drosophila melanogaster combats microbial infection by producing a battery of effector peptides that are secreted into the haemolymph. Technical difficulties prevented the investigation of these short effector genes until the recent advent of the CRISPR/CAS era. As a consequence, many putative immune effectors remain to be formally described, and exactly how each of these effectors contribute to survival is not well characterized. Here we describe a novel Drosophila antifungal peptide gene that we name Baramicin A. We show that BaraA encodes a precursor protein cleaved into multiple peptides via furin cleavage sites. BaraA is strongly immune-induced in the fat body downstream of the Toll pathway, but also exhibits expression in other tissues. Importantly, we show that flies lacking BaraA are viable but susceptible to the entomopathogenic fungus Beauveria bassiana. Consistent with BaraA being directly antimicrobial, overexpression of BaraA promotes resistance to fungi and the IM10-like peptides produced by BaraA synergistically inhibit growth of fungi in vitro when combined with a membrane-disrupting antifungal. Surprisingly, BaraA mutant males but not females display an erect wing phenotype upon infection. Here, we characterize a new antifungal immune effector downstream of Toll signalling, and show it is a key contributor to the Drosophila antimicrobial response., Author summary The ways that animals combat infection involve complex molecular pathways that are triggered upon microbial challenge. While a great deal is known about which pathways are key to a successful defence response, far less is known about exactly what elements of that response are critical to combat a given infection. Using the fruit fly–a genetic workhorse of Biology–we recently showed that a class of host-encoded antibiotics called “antimicrobial peptides” are essential for defence against bacterial infection, but do not contribute as strongly to defence against fungi. However a number of fly immune peptides remain uncharacterized, possibly explaining this gap in our understanding of the fly antifungal defence. Here we describe a novel antifungal peptide gene of fruit flies, and show that it is a major contributor to the fly antifungal defence response. We also found that this gene seems to regulate a behaviour that flies perform after infection, paralleling exciting recent findings that these genes are involved in neurological processes. Collectively, we clarify a key part of the fly antifungal defence, and contribute an important piece to help explain the logical organization of the immune defence against microbial infection.

Background Members of the thioester-containing protein (TEP) family contribute to host defence in both insects and mammals. However, their role in the immune response of Drosophila is elusive. In this study, we address the role of TEPs in Drosophila immunity by generating a mutant fly line, referred to as TEPq Δ, lacking the four immune-inducible TEPs, TEP1, 2, 3 and 4. Results Survival analyses with TEPq Δ flies reveal the importance of these proteins in defence against entomopathogenic fungi, Gram-positive bacteria and parasitoid wasps. Our results confirm that TEPs are required for efficient phagocytosis of bacteria, notably for the two Gram-positive species tested, Staphylococcus aureus and Enterococcus faecalis. Furthermore, we show that TEPq Δ flies have reduced Toll pathway activation upon microbial infection, resulting in lower expression of antimicrobial peptide genes. Epistatic analyses suggest that TEPs function upstream or independently of the serine protease ModSP at an initial stage of Toll pathway activation. Conclusions Collectively, our study brings new insights into the role of TEPs in insect immunity. It reveals that TEPs participate in both humoral and cellular arms of immune response in Drosophila. In particular, it shows the importance of TEPs in defence against Gram-positive bacteria and entomopathogenic fungi, notably by promoting Toll pathway activation. Electronic supplementary material The online version of this article (doi:10.1186/s12915-017-0408-0) contains supplementary material, which is available to authorized users.

Sugars are important energy sources, but high sugar intake poses a metabolic challenge and leads to diseases. Drosophila melanogaster is a generalist fruit breeder that encounters high levels of dietary sugars in its natural habitat. Consequently, Drosophila displays adaptive responses to dietary sugars, including highly conserved and unique metabolic adaptations not described in mammals. Carbohydrate homeostasis is maintained by a network comprising intracellular energy sensors, transcriptional regulators, and hormonal and neuronal mechanisms that together coordinate animal behavior, gut function, and metabolic flux. Here we give an overview of the physiological responses associated with sugar intake and discuss some of the emerging themes and applications of the Drosophila model in understanding sugar sensing and carbohydrate metabolism.

In animals, growth is regulated by the complex interplay between paracrine and endocrine signals. When food is scarce, tissues compete for nutrients, leading to critical resource allocation and prioritization. Little is known about how the immune system maturation is coordinated with the growth of other tissues. Here, we describe a signaling mechanism that regulates the number of hemocytes (blood cells) according to the nutritional state of the Drosophila larva. Specifically, we found that a secreted protein, NimB5, is produced in the fat body upon nutrient scarcity downstream of metabolic sensors and ecdysone signaling. NimB5 is then secreted and binds to hemocytes to down-regulate their proliferation and adhesion. Blocking this signaling loop results in conditional lethality when larvae are raised on a poor diet, due to excessive hemocyte numbers and insufficient energy storage. Similar regulatory mechanisms shaping the immune system in response to nutrient availability are likely to be widespread in animals.

There is a developing interest in how immune genes may function in other physiological roles, and how traditionally non-immune peptides may, in fact, be active in immune contexts. In the absence of infection, the induction of the immune response is costly, and there are well-characterized trade-offs between immune defense and fitness. The agents behind these fitness costs are less understood. Here we implicate antimicrobial peptides (AMPs) as particularly costly effectors of immunity using an evolutionary framework. We describe the independent loss of AMPs in multiple lineages of Diptera (true flies), tying these observations back to life history. We then focus on the intriguing case of the glycine-rich AMP, Diptericin, and find several instances of loss, pseudogenization, and segregating null alleles. We suggest that Diptericin may be a particularly toxic component of the Dipteran immune response lost in flies either with reduced pathogen pressure or other environmental factors. As Diptericins have recently been described to have neurological roles, these findings parallel a developing interest in AMPs as potentially harmful neuropeptides, and AMPs in other roles beyond immunity.

Summary Animals require complex metabolic and physiological adaptations to maintain the function of vital organs in response to environmental stresses and infection. Here, we found that infection or injury in Drosophila induced the excretion of hemolymphatic lipids by Malpighian tubules, the insect kidney. This lipid purge was mediated by a stress-induced lipid-binding protein, Materazzi, which was enriched in Malpighian tubules. Flies lacking materazzi had higher hemolymph concentrations of reactive oxygen species (ROS) and increased lipid peroxidation. These flies also displayed Malpighian tubule dysfunction and were susceptible to infections and environmental stress. Feeding flies with antioxidants rescued the materazzi phenotype, indicating that the main role of Materazzi is to protect the organism from damage caused by stress-induced ROS. Our findings suggest that purging hemolymphatic lipids presents a physiological adaptation to protect host tissues from excessive ROS during immune and stress responses, a process that is likely to apply to other organisms.

Melanization contributes to arthropod-specific innate immunity through deposition of melanin at wound sites or around parasites, with concomitant release of microbicidal reactive oxygen species. Melanization requires sequential activation of proteolytic enzymes in the hemolymph, including the final enzyme pro-phenoloxidase. Black cells (BC) is a mutation causing spontaneous melanization of Drosophila crystal cells, a hemocyte cell type producing phenoloxidases. BC individuals exhibit circulating black spots but fail to melanize upon injury. Although BC is widely used as a loss-of-function mutant of phenoloxidases, the mutation causing BC remained unknown. Here, we identified a single point mutation in the prophenoloxidase I (PPO1) gene of Bc flies causing an Alanine to Valine change in the C-terminal domain of PPO1, predicted to affect the conformation of the N-terminal pro-domain cleavage site at a distance and causing uncontrolled catalytic activity. Genomic insertion of a PPO1(A480V)transgene phenocopies Black cells, proving that A480V is indeed the causal mutation of the historical Bc phenotype. (C) 2014 Elsevier Ltd. All rights reserved.

The digestive tract plays a central role in the digestion and absorption of nutrients. Far from being a passive tube, it provides the first line of defense against pathogens and maintains energy homeostasis by exchanging neuronal and endocrine signals with other organs. Historically neglected, the gut of the fruit fly Drosophila melanogaster has recently come to the forefront of Drosophila research. Areas as diverse as stem cell biology, neurobiology, metabolism, and immunity are benefitting from the ability to study the genetics of development, growth regulation, and physiology in the same organ. In this review, we summarize our knowledge of the Drosophila digestive tract, with an emphasis on the adult midgut and its functional underpinnings.

International audience; Activation of the innate immune response in Metazoans is initiated through the recognition of microbes by host pattern-recognition receptors. In Drosophila, diaminopimelic acid (DAP)-containing peptidoglycan from Gram-negative bacteria is detected by the transmembrane receptor PGRP-LC and by the intracellular receptor PGRP-LE. Here, we show that PGRP-SD acted upstream of PGRP-LC as an extracellular receptor to enhance peptidoglycan-mediated activation of Imd signaling. Consistent with this, PGRP-SD mutants exhibited impaired activation of the Imd pathway and increased susceptibility to DAP-type bacteria. PGRP-SD enhanced the localization of peptidoglycans to the cell surface and hence promoted signaling. Moreover, PGRP-SD antagonized the action of PGRP-LB, an extracellular negative regulator, to fine-tune the intensity of the immune response. These data reveal that Drosophila PGRP-SD functions as an extracellular receptor similar to mammalian CD14 and demonstrate that, comparable to lipopolysaccharide sensing in mammals, Drosophila relies on both intra- and extracellular receptors for the detection of bacteria.

The JAK/STAT pathway is a key signaling pathway in the regulation of development and immunity in metazoans. In contrast to the multiple combinatorial JAK/STAT pathways in mammals, only one canonical JAK/STAT pathway exists in Drosophila. It is activated by three secreted proteins of the Unpaired family (Upd): Upd1, Upd2 and Upd3. Although many studies have established a link between JAK/STAT activation and tissue damage, the mode of activation and the precise function of this pathway in the Drosophila systemic immune response remain unclear. In this study, we used mutations in upd2 and upd3 to investigate the role of the JAK/STAT pathway in the systemic immune response. Our study shows that haemocytes express the three upd genes and that injury markedly induces the expression of upd3 by the JNK pathway in haemocytes, which in turn activates the JAK/STAT pathway in the fat body and the gut. Surprisingly, release of Upd3 from haemocytes upon injury can remotely stimulate stem cell proliferation and the expression of Drosomycin-like genes in the intestine. Our results also suggest that a certain level of intestinal epithelium renewal is required for optimal survival to septic injury. While haemocyte-derived Upd promotes intestinal stem cell activation and survival upon septic injury, haemocytes are dispensable for epithelium renewal upon oral bacterial infection. Our study also indicates that intestinal epithelium renewal is sensitive to insults from both the lumen and the haemocoel. It also reveals that release of Upds by haemocytes coordinates the wound-healing program in multiple tissues, including the gut, an organ whose integrity is critical to fly survival., Author Summary Innate immunity acts as the primary line of defense to overcome invading organisms. This response starts through sensing of microbe-specific molecules such as lipopolysaccharide and peptidoglycan by host receptors. Metazoans can also recognize signals that are associated with tissue damage and wounding, to then activate specific pathways involved in tissue repair or inflammation. Previous studies have suggested a link between JAK/STAT activation and tissue repair in Drosophila, but the mode of activation and the precise function of this pathway remain unclear. In this article, we analyze the role of the JAK/STAT pathway in the resistance to wounding in Drosophila. We show that this pathway contributes to fly resistance to wounding and bacterial infection. Mechanistically, injury induces the production of secreted molecules called Unpaireds by blood cells, which then activate the JAK/STAT pathway in the fat body (an organ equivalent to the vertebrate liver) and in the gut. One of the most surprising findings of our study is that Unpaireds released from blood cells can remotely stimulate intestinal stem cell proliferation. Thus, we uncover an unexpected interaction between circulating blood cells and intestinal stem cell proliferation that contributes to fly survival after injury.

The Drosophila antimicrobial response is one of the best characterized systems of pattern recognition receptor-mediated defense in metazoans. Drosophila senses Gram-negative bacteria via two peptidoglycan recognition proteins (PGRPs), membrane-bound PGRP-LC and secreted/cytosolic PGRP-LE, which relay diaminopimelic acid (DAP)-type peptidoglycan sensing to the Imd signaling pathway. In the case of PGRP-LC, differential splicing of PGRP domain-encoding exons to a common intracellular domain-encoding exon generates three receptor isoforms, which differ in their peptidoglycan binding specificities. In this study, we used Phi31-mediated recombineering to generate fly lines expressing specific isoforms of PGRP-LC and assessed the tissue-specific roles of PGRP-LC isoforms and PGRP-LE in the antibacterial response. Our in vivo studies demonstrate the key role of PGRP-LCx in sensing DAP-type peptidoglycan-containing Gram-negative bacteria or Gram-positive bacilli during systemic infection. We also highlight the contribution of PGRP-LCa/x heterodimers to the systemic immune response to Gram-negative bacteria through sensing of tracheal cytotoxin (TCT), whereas PGRP-LCy may have a minor role in antagonizing the immune response. Our results reveal that both PGRP-LC and PGRP-LE contribute to the intestinal immune response, with a predominant role of cytosolic PGRP-LE in the midgut, the central section of endodermal origin where PGRP-LE is enriched. Our in vivo model also definitively establishes TCT as the long-distance elicitor of systemic immune responses to intestinal bacteria observed in a loss-of-tolerance model. In conclusion, our study delineates how a combination of extracellular sensing by PGRP-LC isoforms and intracellular sensing through PGRP-LE provides sophisticated mechanisms to detect and differentiate between infections by different DAP-type bacteria in Drosophila.

SummaryPeptidoglycan recognition proteins (PGRPs) are key regulators of insect immune responses. In addition to recognition PGRPs, which activate the Toll and Imd pathways, the Drosophila genome encodes six catalytic PGRPs with the capacity to scavenge peptidoglycan. We have performed a systematic analysis of catalytic PGRP function using deletions, separately and in combination. Our findings support the role of PGRP-LB as a negative regulator of the Imd pathway and brought to light a synergy of PGRP-SCs with PGRP-LB in the systemic response. Flies lacking all six catalytic PGRPs were still viable but exhibited deleterious immune responses to innocuous gut infections. Together with recent studies on mammalian PGRPs, our study uncovers a conserved role for PGRPs in gut homeostasis. Analysis of the immune phenotype of flies lacking all catalytic PGRPs and the Imd regulator Pirk reveals that the Imd-mediated immune response is highly constrained by the existence of multiple negative feedbacks.

Prenatal exposure to mercury causes neurodevelopmental disorders and neurological pathologies in infants, such as microcephaly and mental retardation. Despite critical importance, the molecular interactions leading to mercury toxicity are yet to be elucidated. We first used a cell-free assay to investigate mercury effects on purified γ-secretase activity. Next, we treated adult Drosophila melanogaster with mercury and collected control and mercury-treated embryos, which were subjected to mild hypotonic protein extraction, or immunostained to reveal nervous system morphology. Embryos left to develop into adults were examined for wing phenotypes. Relative to control metals, we found that mercury strongly inhibits in vitro γ-secretase processing of both amyloid-β precursor protein (APP) and Notch. Mercury inhibited APP and Notch cleavage in a dose-dependent manner, with IC(50) values of 50-125 nM, and is therefore comparable in potency to benchmark γ-secretase inhibitors. Immunoblot analysis of embryonic protein extracts showed that mercury inhibits Notch cleavage by γ-secretase in vivo. This is accompanied by severe neurodevelopmental abnormalities in embryos and adult wing-notching phenotypes. Our findings provide first evidence that mercury is a direct and potent γ-secretase inhibitor and suggest that inhibition of γ-secretase and disruption of the Notch developmental pathway potentially contribute to mercury-induced toxicity in the nervous system.

The peritrophic matrix (PM) forms a layer composed of chitin and glycoproteins that lines the insect intestinal lumen. This physical barrier plays a role analogous to that of mucous secretions of the vertebrate digestive tract and is thought to protect the midgut epithelium from abrasive food particles and microbes. Almost nothing is known about PM functions in Drosophila , and its function as an immune barrier has never been addressed by a genetic approach. Here we show that the Drosocrystallin (Dcy) protein, a putative component of the eye lens of Drosophila , contributes to adult PM formation. A loss-of-function mutation in the dcy gene results in a reduction of PM width and an increase of its permeability. Upon bacterial ingestion a higher level of expression of antibacterial peptides was observed in dcy mutants, pointing to an influence of this matrix on bacteria sensing by the Imd immune pathway. Moreover, dcy -deficient flies show an increased susceptibility to oral infections with the entomopathogenic bacteria Pseudomonas entomophila and Serratia marcescens . Dcy mutant flies also succumb faster than wild type upon ingestion of a P. entomophila toxic extract. We show that this lethality is due in part to an increased deleterious action of Monalysin, a pore-forming toxin produced by P. entomophila . Collectively, our analysis of the dcy immune phenotype indicates that the PM plays an important role in Drosophila host defense against enteric pathogens, preventing the damaging action of pore-forming toxins on intestinal cells.

Summary Intestinal infection triggers potent immune responses to combat pathogens and concomitantly drives epithelial renewal to maintain barrier integrity. Current models propose that epithelial renewal is primarily driven by damage caused by reactive oxygen species (ROS). Here we found that in Drosophila, the Imd-NF-κB pathway controlled enterocyte (EC) shedding upon infection, via a mechanism independent of ROS-associated apoptosis. Mechanistically, the Imd pathway synergized with JNK signaling to induce epithelial cell shedding specifically in the context of bacterial infection, requiring also the reduced expression of the transcription factor GATAe. Furthermore, cell-specific NF-κB responses enabled simultaneous production of antimicrobial peptides (AMPs) and epithelial shedding in different EC populations. Thus, the Imd-NF-κB pathway is central to the intestinal antibacterial response by mediating both AMP production and the maintenance of barrier integrity. Considering the similarities between Drosophila Imd signaling and mammalian TNFR pathway, our findings suggest the existence of an evolutionarily conserved genetic program in immunity-induced epithelial shedding.

The Drosophila Toll receptor does not interact directly with microbial determinants, but is instead activated by a cleaved form of the cytokine-like molecule Spätzle. During the immune response, Spätzle is processed by complex cascades of serine proteases, which are activated by secreted pattern-recognition receptors. Here, we demonstrate the essential role of ModSP, a modular serine protease, in the activation of the Toll pathway by Gram-positive bacteria and fungi. Our analysis shows that ModSP integrates signals originating from the circulating recognition molecules GNBP3 and PGRP-SA and connects them to the Grass-SPE-Spätzle extracellular pathway upstream of the Toll receptor. It also reveals the conserved role of modular serine proteases in the activation of insect immune reactions.

SummaryAlthough Drosophila systemic immunity is extensively studied, little is known about the fly's intestine-specific responses to bacterial infection. Global gene expression analysis of Drosophila intestinal tissue to oral infection with the Gram-negative bacterium Erwinia carotovora revealed that immune responses in the gut are regulated by the Imd and JAK-STAT pathways, but not the Toll pathway. Ingestion of bacteria had a dramatic impact on the physiology of the gut that included modulation of stress response and increased stem cell proliferation and epithelial renewal. Our data suggest that gut homeostasis is maintained through a balance between cell damage due to the collateral effects of bacteria killing and epithelial repair by stem cell division. The Drosophila gut provides a powerful model to study the integration of stress and immunity with pathways associated with stem cell control, and this study should prove to be a useful resource for such further studies.

In insects the enzyme phenoloxidase (PO) catalyzes melanin deposition at the wound site and around parasitoid eggs. Its proenzyme prophenoloxidase (proPO) is proteolytically cleaved to active phenoloxidase by a cascade consisting of serine proteases and inhibited by serpins. The Drosophila genome encodes 29 serpins, of which only two, Serpin-27A (Spn27A) and Necrotic, have been analyzed in detail. Using a genetic approach, we demonstrate that the so far uncharacterized Serpin-28D (Spn28D, CG7219) regulates the proPO cascade in both hemolymph and tracheal compartments. spn28D is the serpin gene most strongly induced upon injury. Inactivation of spn28D causes pupal lethality and a deregulated developmental PO activation leading to extensive melanization of tissues in contact with air and pigmentation defects of the adult cuticle. Our data also show that Spn28D regulates hemolymph PO activity in both larvae and adults at a different level than Spn27A. Our data support a model in which Spn28D confines PO availability by controlling its initial release, while Spn27A is rather limiting the melanization reaction to the wound site. This study further highlights the complexity of the proPO cascade that can be differentially regulated in different tissues during development.

SummaryMetazoans tolerate commensal-gut microbiota by suppressing immune activation while maintaining the ability to launch rapid and balanced immune reactions to pathogenic bacteria. Little is known about the mechanisms underlying the establishment of this threshold. We report that a recently identified Drosophila immune regulator, which we call PGRP-LC-interacting inhibitor of Imd signaling (PIMS), is required to suppress the Imd innate immune signaling pathway in response to commensal bacteria. pims expression is Imd (immune deficiency) dependent, and its basal expression relies on the presence of commensal flora. In the absence of PIMS, resident bacteria trigger constitutive expression of antimicrobial peptide genes (AMPs). Moreover, pims mutants hyperactivate AMPs upon infection with Gram-negative bacteria. PIMS interacts with the peptidoglycan recognition protein (PGRP-LC), causing its depletion from the plasma membrane and shutdown of Imd signaling. Therefore, PIMS is required to establish immune tolerance to commensal bacteria and to maintain a balanced Imd response following exposure to bacterial infections.

The Drosophila immune system discriminates between different classes of infectious microbes and responds with pathogen-specific defense reactions via the selective activation of the Toll and the immune deficiency (Imd) signaling pathways. The Toll pathway mediates most defenses against Gram-positive bacteria and fungi, whereas the Imd pathway is required to resist Gram-negative bacterial infection. Microbial recognition is achieved through peptidoglycan recognition proteins (PGRPs); Gram-positive bacteria activate the Toll pathway through a circulating PGRP (PGRP-SA), and Gram-negative bacteria activate the Imd pathway via PGRP-LC, a putative transmembrane receptor, and PGRP-LE. Gram-negative binding proteins (GNBPs) were originally identified in Bombyx mori for their capacity to bind various microbial compounds. Three GNBPs and two related proteins are encoded in the Drosophila genome, but their function is not known. Using inducible expression of GNBP1 double-stranded RNA, we now demonstrate that GNBP1 is required for Toll activation in response to Gram-positive bacterial infection; GNBP1 double-stranded RNA expression renders flies susceptible to Gram-positive bacterial infection and reduces the induction of the antifungal peptide encoding gene Drosomycin after infection by Gram-positive bacteria but not after fungal infection. This phenotype induced by GNBP1 inactivation is identical to a loss-of-function mutation in PGRP-SA, and our genetic studies suggest that GNBP1 acts upstream of the Toll ligand Spätzle. Altogether, our results demonstrate that the detection of Gram-positive bacteria in Drosophila requires two putative pattern recognition receptors, PGRP-SA and GNBP1.

Human immunodeficiency virus 1 (HIV-1) expresses several accessory proteins that manipulate various host-cell processes to achieve optimum replicative efficiency. One of them, viral protein U (Vpu), has been shown to interfere with the cellular degradation machinery through interaction with SCFβ-TrCP complexes. To learn more about Vpu function in vivo, we used the genetically tractable fruit fly, Drosophila melanogaster. Our results show that the directed expression of Vpu, but not the non-phosphorylated form, Vpu2/6, in fat-body cells affects Drosophila antimicrobial responses. In flies, the Toll and Imd pathways regulate antimicrobial-peptide gene expression. We show that Vpu specifically affects Toll pathway activation by inhibiting Cactus degradation. Given the conservation of the Toll/nuclear factor-κB (NF-κB) signalling pathways between flies and mammals, our results suggest a function for Vpu in the inhibition of host NF-κB-mediated innate immune defences and provide a powerful genetic approach for studying Vpu inhibition of NF-κB signalling in vivo.

The Drosophila immune system discriminates between different classes of infectious microbes and responds with pathogen-specific defense reactions through selective activation of the Toll and the immune deficiency (Imd) signaling pathways. The Toll pathway mediates most defenses against Gram-positive bacteria and fungi, whereas the Imd pathway is required to resist infection by Gram-negative bacteria. The bacterial components recognized by these pathways remain to be defined. Here we report that Gram-negative diaminopimelic acid-type peptidoglycan is the most potent inducer of the Imd pathway and that the Toll pathway is predominantly activated by Gram-positive lysine-type peptidoglycan. Thus, the ability of Drosophila to discriminate between Gram-positive and Gram-negative bacteria relies on the recognition of specific forms of peptidoglycan.

Background: The inducible production of antimicrobial peptides is a major immune response in Drosophila . The genes encoding these peptides are activated by NF-κB transcription factors that are controlled by two independent signaling cascades: the Toll pathway that regulates the NF-κB homologs, Dorsal and DIF; and the IMD pathway that regulates the compound NF-κB-like protein, Relish. Although numerous components of each pathway that are required to induce antimicrobial gene expression have been identified, less is known about the mechanisms that either repress antimicrobial genes in the absence of infection or that downregulate these genes after infection. Results: In a screen for factors that negatively regulate the IMD pathway, we isolated two partial loss-of-function mutations in the SkpA gene that constitutively induce the antibacterial peptide gene, Diptericin , a target of the IMD pathway. These mutations do not affect the systemic expression of the antifungal peptide gene, Drosomycin , a target of the Toll pathway. SkpA encodes a homolog of the yeast and human Skp1 proteins. Skp1 proteins function as subunits of SCF-E3 ubiquitin ligases that target substrates to the 26S proteasome, and mutations affecting either the Drosophila SCF components, Slimb and dCullin1, or the proteasome also induce Diptericin expression. In cultured cells, inhibition of SkpA and Slimb via RNAi increases levels of both the full-length Relish protein and the processed Rel-homology domain. Conclusions: In contrast to other NF-κB activation pathways, the Drosophila IMD pathway is repressed by the ubiquitin-proteasome system. A possible target of this proteolytic activity is the Relish transcription factor, suggesting a mechanism for NF-κB downregulation in Drosophila .

The speed of stem cell differentiation has to be properly coupled with self-renewal, both under basal conditions for tissue maintenance and during regeneration for tissue repair. Using the Drosophila midgut model, we analyze at the cellular and molecular levels the differentiation program required for robust regeneration. We observe that the intestinal stem cell (ISC) and its differentiating daughter, the enteroblast (EB), form extended cell-cell contacts in regenerating intestines. The contact between progenitors is stabilized by cell adhesion molecules, and can be dynamically remodeled to elicit optimal juxtacrine Notch signaling to determine the speed of progenitor differentiation. Notably, increasing the adhesion property of progenitors by expressing Connectin is sufficient to induce rapid progenitor differentiation. We further demonstrate that JAK/STAT signaling, Sox21a and GATAe form a functional relay to orchestrate EB differentiation. Thus, our study provides new insights into the complex and sequential events that are required for rapid differentiation following stem cell division during tissue replenishment., Author summary Adult tissue/organ function is maintained by stem cells. Key question in stem cell biology is how the pool of stem cells can be robustly expanded yet timely contracted through differentiation according to the need of a tissue. Over the last years, the mechanisms underlying stem cell activation have been extensively studied, while the genetic control of progenitor differentiation, especially during regeneration, is still poorly understood. Using the fruit fly Drosophila midgut as model, we investigate the cellular changes and the genetic program required for efficient progenitor differentiation during intestinal regeneration. We first detect the presence of extended cell-cell contact between a stem cell and its differentiating daughter in regenerating intestine, compared to homeostatic conditions. The extended cell-cell contact is consolidated by cell adhesion molecules and enhances Notch signaling in the differentiating progenitors leading to their fast differentiation into enterocytes. We further uncover a genetic program, involving the JAK/STAT and Dpp signaling, the Sox21a and GATAe transcription factors, which acts in the differentiating progenitors to instruct their terminal differentiation. Thus, our study presents an integrated view of stem cell differentiation during tissue regeneration and the findings here are likely to apply to mammals.

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., Author Summary The melanization reaction is a major immune response in Arthropods and involves the rapid synthesis of a black pigment, melanin, at the site of infection and injury. Melanization requires the activation of proPhenoloxidase, an enzyme that catalyzes the oxidation of phenols to quinones, which polymerize to melanin. The Drosophila genome contains three genes encoding prophenoloxidases (PPO). In this paper, we have generated flies carrying deletions in the PPO1 and PPO2 genes. By analyzing these mutations alone and in combination, we clarify the functions of both prophenoloxidases in humoral melanization. We report that PPO2 composes most of the crystals found in crystal cells, a specific hemocyte cell type. Although PPO1 and PPO2 both contribute to phenoloxidase activity in the insect blood, these PPOs are not fully redundant. Our study shows that PPO1 is involved in the rapid delivery of phenoloxidase activity when required, while PPO2 provides a storage form that can be deployed in a second phase. Some controversy exists in the Drosophila field about the importance of melanization in the Drosophila host defense. Our study demonstrates the important role of PPO1 and PPO2 in the survival to infection with both Gram-positive bacteria and fungi, underlining the importance of melanization in insect immunity.

In mammals, TAK1, a MAPKKK kinase, is implicated in multiple signaling processes, including the regulation of NF-κB activity via the IL1-R/TLR pathways. TAK1 function has largely been studied in cultured cells, and its in vivo function is not fully understood. We have isolated null mutations in the Drosophila dTAK1 gene that encodes dTAK1, a homolog of TAK1. dTAK1 mutant flies are viable and fertile, but they do not produce antibacterial peptides and are highly susceptible to Gram-negative bacterial infection. This phenotype is similar to the phenotypes generated by mutations in components of the Drosophila Imd pathway. Our genetic studies also indicate that dTAK1 functions downstream of the Imd protein and upstream of the IKK complex in the Imd pathway that controls the Rel/NF-κB like transactivator Relish. In addition, our epistatic analysis places the caspase, Dredd, downstream of the IKK complex, which supports the idea that Relish is processed and activated by a caspase activity. Our genetic demonstration of dTAK1's role in the regulation ofDrosophila antimicrobial peptide gene expression suggests an evolutionary conserved role for TAK1 in the activation of Rel/NF-κB-mediated host defense reactions.

PeptidoGlycan Recognition Proteins (PGRPs) are key regulators of the insect innate antibacterial response. Even if they have been intensively studied, some of them have yet unknown functions. Here, we present a functional analysis of PGRP-LA, an as yet uncharacterized Drosophila PGRP. The PGRP-LA gene is located in cluster with PGRP-LC and PGRP-LF, which encode a receptor and a negative regulator of the Imd pathway, respectively. Structure predictions indicate that PGRP-LA would not bind to peptidoglycan, pointing to a regulatory role of this PGRP. PGRP-LA expression was enriched in barrier epithelia, but low in the fat body. Use of a newly generated PGRP-LA deficient mutant indicates that PGRP-LA is not required for the production of antimicrobial peptides by the fat body in response to a systemic infection. Focusing on the respiratory tract, where PGRP-LA is strongly expressed, we conducted a genome-wide microarray analysis of the tracheal immune response of wild-type, Relish, and PGRP-LA mutant larvae. Comparing our data to previous microarray studies, we report that a majority of genes regulated in the trachea upon infection differ from those induced in the gut or the fat body. Importantly, antimicrobial peptide gene expression was reduced in the tracheae of larvae and in the adult gut of PGRP-LA-deficient Drosophila upon oral bacterial infection. Together, our results suggest that PGRP-LA positively regulates the Imd pathway in barrier epithelia. © 2013 Gendrin et al. publishersversion published

The cytokine-induced activation cascade of NF-kappaB in mammals and the activation of the morphogen dorsal in Drosophila embryos show striking structural and functional similarities (Toll/IL-1, Cactus/I-kappaB, and dorsal/NF-kappaB). Here we demonstrate that these parallels extend to the immune response of Drosophila. In particular, the intracellular components of the dorsoventral signaling pathway (except for dorsal) and the extracellular Toll ligand, spatzle, control expression of the antifungal peptide gene drosomycin in adults. We also show that mutations in the Toll signaling pathway dramatically reduce survival after fungal infection. Antibacterial genes are induced either by a distinct pathway involving the immune deficiency gene (imd) or by combined activation of both imd and dorsoventral pathways.

Summary The Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway is involved in the regulation of intestinal stem cell (ISC) activity to ensure a continuous renewal of the adult Drosophila midgut. Three ligands, Unpaired 1, Unpaired 2 and Unpaired 3 (Upd1, Upd2 and Upd3, respectively) are known to activate the JAK/STAT pathway in Drosophila. Using newly generated upd mutants and cell-specific RNAi, we showed that Upd1 is required throughout the fly life to maintain basal turnover of the midgut epithelium by controlling ISC maintenance in an autocrine manner. A role of Upd2 and Upd3 in basal conditions is discernible only in old gut, where they contribute to increased ISC abnormal division. Finally, upon an acute stress such as oral bacterial infection, we showed that Upd3 is released from enterocytes and has an additive effect with Upd2 to promote rapid epithelial regeneration. Taken together, our results show that Upd ligands are required to maintain the midgut homeostasis under both normal and pathological states.

The cytokine-induced activation cascade of NF-kappaB in mammals and the activation of the morphogen dorsal in Drosophila embryos show striking structural and functional similarities (Toll/IL-1, Cactus/I-kappaB, and dorsal/NF-kappaB). Here we demonstrate that these parallels extend to the immune response of Drosophila. In particular, the intracellular components of the dorsoventral signaling pathway (except for dorsal) and the extracellular Toll ligand, spätzle regulatory gene cassette, control expression of the antifungal peptide gene drosomycin in adults. We also show that mutations in the Toll signaling pathway dramatically reduce survival after fungal infection. Antibacterial genes are induced either by a distinct pathway involving the immune deficiency gene (imd) or by combined activation of both imd and dorsoventral pathways.

The systemic immune response of Drosophila is known to be induced both by septic injury and by oral infection with certain bacteria, and is characterized by the secretion of antimicrobial peptides (AMPs) into the haemolymph. To investigate other possible routes of bacterial infection, we deposited Erwinia carotovora (Ecc15) on various sites of the cuticle and monitored the immune response via expression of the AMP gene Diptericin. A strong response was observed to deposition on the genital plate of males (up to 20% of a septic injury response), but not females. We show that the principal response to genital infection is systemic, but that some AMPs, particularly Defensin, are induced locally in the genital tract. At late time points we detected bacteria in the haemolymph of immune deficient RelishE20 flies, indicating that the genital plate can be a route of entry for pathogens, and that the immune response protects flies against the progression of genital infection. The protective role of the immune response is further illustrated by our observation that RelishE20 flies exhibit significant lethality in response to genital Ecc15 infections. We next show that a systemic immune response can be induced by deposition of the bacterial elicitor peptidoglycan (PGN), or its terminal monomer tracheal cytotoxin (TCT), on the genital plate. This immune response is downregulated by PGRP-LB and Pirk, known regulators of the Imd pathway, and can be suppressed by the overexpression of PGRP-LB in the haemolymph compartment. Finally, we provide strong evidence that TCT can activate a systemic response by crossing epithelia, by showing that radiolabelled TCT deposited on the genital plate can subsequently be detected in the haemolymph. Genital infection is thus an intriguing new model for studying the systemic immune response to local epithelial infections and a potential route of entry for naturally occurring pathogens of Drosophila., Author Summary Innate immunity is the first line of antimicrobial defence for vertebrates and the only immune response present in invertebrates such as the fruitfly Drosophila, which provides a powerful model system to study innate immunity. Interestingly, local infections of epithelia like the gut and, in our study, the genital tract, result not only in a local immune response, but in an immune response of the whole body. The latter seems to protect Drosophila against the potential spread of local infections. We have investigated the immune response to bacteria placed on the genitalia, at the entrance to both the genital tract and hindgut. This could be a natural entry route of pathogens, possibly linked to sexually transmitted infections. We observe a strong immune response to Gram-negative bacteria, mediated by the immune responsive Imd signalling pathway. This response depends on peptidoglycan, a crucial component of the bacterial cell wall, as pure peptidoglycan placed on the genitalia is sufficient to trigger a whole body immune response. Finally, we present strong evidence that peptidoglycan fragments within the genital tract or hindgut can cross these epithelia, enter the body cavity and thus induce a system wide immune response to a local infection.

Invertebrates lack an adaptive immune system and rely on innate immunity to resist pathogens. The response of Drosophila melanogaster to bacterial and fungal infections involves two signaling pathways, Toll and Imd, both of which activate members of the nuclear factor (NF)-kappaB family of transcription factors, leading to antimicrobial peptide (AMP) gene expression. In this chapter, we present the current methods used in our laboratory to monitor the activity of both signaling pathways.

The founding member of the inhibitor of apoptosis protein (IAP) family was originally identified as a cell death inhibitor. However, recent evidence suggests that IAPs are multifunctional signaling devices that influence diverse biological processes. To investigate the in vivo function of Drosophila melanogaster IAP2, we have generated diap2 null alleles. diap2 mutant animals develop normally and are fully viable, suggesting that diap2 is dispensable for proper development. However, these animals were acutely sensitive to infection by gram-negative bacteria. In Drosophila, infection by gram-negative bacteria triggers the innate immune response by activating the immune deficiency (imd) signaling cascade, a NF-kappaB-dependent pathway that shares striking similarities with the pathway of mammalian tumor necrosis factor receptor 1 (TNFR1). diap2 mutant flies failed to activate NF-kappaB-mediated expression of antibacterial peptide genes and, consequently, rapidly succumbed to bacterial infection. Our genetic epistasis analysis places diap2 downstream of or in parallel to imd, Dredd, Tak1, and Relish. Therefore, DIAP2 functions in the host immune response to gram-negative bacteria. In contrast, we find that the Drosophila TNFR-associated factor (Traf) family member Traf2 is dispensable in resistance to gram-negative bacterial infection. Taken together, our genetic data identify DIAP2 as an essential component of the Imd signaling cascade, protecting the organism from infiltrating microbes.

SummaryUnlike mammalian Toll-like Receptors, the Drosophila Toll receptor does not interact directly with microbial determinants but is rather activated upon binding a cleaved form of the cytokine-like molecule Spatzle (Spz). During the immune response, Spz is thought to be processed by secreted serine proteases (SPs) present in the hemolymph that are activated by the recognition of gram-positive bacteria or fungi [1]. In the present study, we have used an in vivo RNAi strategy to inactivate 75 distinct Drosophila SP genes. We then screened this collection for SPs regulating the activation of the Toll pathway by gram-positive bacteria. Here, we report the identification of five novel SPs that function in an extracellular pathway linking the recognition proteins GNBP1 and PGRP-SA to Spz. Interestingly, four of these genes are also required for Toll activation by fungi, while one is specifically associated with signaling in response to gram-positive bacterial infections. These results demonstrate the existence of a common cascade of SPs upstream of Spz, integrating signals sent by various secreted recognition molecules via more specialized SPs.

SummaryThe Drosophila host defense against gram-negative bacteria is mediated by the Imd pathway upon sensing of peptidoglycan by the peptidoglycan recognition protein (PGRP)-LC. Here we report a functional analysis of PGRP-LB, a catalytic member of the PGRP family. We show that PGRP-LB is a secreted protein regulated by the Imd pathway. Biochemical studies demonstrate that PGRP-LB is an amidase that specifically degrades gram-negative bacteria peptidoglycan. In agreement with its amidase activity, PGRP-LB downregulates the Imd pathway. Hence, activation of PGRP-LB by the Imd pathway provides a negative feedback regulation to tightly adjust immune activation to infection. Our study also reveals that PGRP-LB controls the immune reactivity of flies to the presence of ingested bacteria in the gut. Our work highlights the key role of PGRPs that encode both sensors and scavengers of peptidoglycan, which modulate the level of the host immune response to the presence of infectious microorganisms.

Pathogens have developed multiple strategies that allow them to exploit host resources and resist the immune response. To study how Drosophila flies deal with infectious diseases in a natural context, we investigated the interactions between Drosophila and a newly identified entomopathogen, Pseudomonas entomophila. Flies orally infected with P. entomophila rapidly succumb despite the induction of both local and systemic immune responses, indicating that this bacterium has developed specific strategies to escape the fly immune response. Using a combined genetic approach on both host and pathogen, we showed that P. entomophila virulence is multi-factorial with a clear differentiation between factors that trigger the immune response and those that promote pathogenicity. We demonstrate that AprA, an abundant secreted metalloprotease produced by P. entomophila, is an important virulence factor. Inactivation of aprA attenuated both the capacity to persist in the host and pathogenicity. Interestingly, aprA mutants were able to survive to wild-type levels in immune-deficient Relish flies, indicating that the protease plays an important role in protection against the Drosophila immune response. Our study also reveals that the major contribution to the fly defense against P. entomophila is provided by the local, rather than the systemic immune response. More precisely, our data points to an important role for the antimicrobial peptide Diptericin against orally infectious Gram-negative bacteria, emphasizing the critical role of local antimicrobial peptide expression against food-borne pathogens., Synopsis Normal feeding and digestion involves the ingestion of many microorganisms. Many are innocuous, some are commensal, and others may be pathogenic. Eukaryotes have thus evolved complex mechanisms to detect, control, and if necessary, eliminate intestinal microbes. Insects are no exception, and the fruit fly, Drosophila, employs a physical barrier within the intestinal lumen and the peritrophic matrix, and an innate immune response which exhibits similarities to the mammalian counterpart. Pseudomonas entomophila was identified as a novel entomopathogenic bacterium that can infect and colonize the gut of Drosophila. In this paper, Liehl et al. describe one specific secreted virulence factor of P. entomophila, the zinc metalloprotease, AprA, which they demonstrate to be required for defense against the host gut epithelial immune response. AprA defends P. entomophila against the Drosophila antimicrobial peptides, produced by the gut innate immune response. P. entomophila aprA mutants are attenuated for virulence in wild-type Drosophila but are equally infective as wild-type bacteria in immune-deficient mutant flies that do not express these antimicrobial peptides. Although secreted proteases have previously been described as a potentially important defense against host immune proteins, this is one of the rare examples of an in vivo demonstration of such a specific role against insect antimicrobial peptides.

SummaryThe Toll receptor was originally identified as an indispensable molecule for Drosophila embryonic development and subsequently as an essential component of innate immunity from insects to humans. Although in Drosophila the Easter protease processes the pro-Spätzle protein to generate the Toll ligand during development, the identification of the protease responsible for pro-Spätzle processing during the immune response has remained elusive for a decade. Here, we report a protease, called Spätzle-processing enzyme (SPE), required for Toll-dependent antimicrobial response. Flies with reduced SPE expression show no noticeable pro-Spätzle processing and become highly susceptible to microbial infection. Furthermore, activated SPE can rescue ventral and lateral development in embryos lacking Easter, showing the functional homology between SPE and Easter. These results imply that a single ligand/receptor-mediated signaling event can be utilized for different biological processes, such as immunity and development, by recruiting similar ligand-processing proteases with distinct activation modes.

Clotting is critical in limiting hemolymph loss and initiating wound healing in insects as in vertebrates. It is also an important immune defense, quickly forming a secondary barrier to infection, immobilizing bacteria and thereby promoting their killing. However, hemolymph clotting is one of the least understood immune responses in insects. Here, we characterize fondue (fon; CG15825), an immune-responsive gene of Drosophila melanogaster that encodes an abundant hemolymph protein containing multiple repeat blocks. After knockdown of fon by RNAi, bead aggregation activity of larval hemolymph is strongly reduced, and wound closure is affected. fon is thus the second Drosophila gene after hemolectin (hml), for which a knockdown causes a clotting phenotype. In contrast to hml-RNAi larvae, clot fibers are still observed in samples from fon-RNAi larvae. However, clot fibers from fon-RNAi larvae are more ductile and longer than in wt hemolymph samples, indicating that Fondue might be involved in cross-linking of fiber proteins. In addition, fon-RNAi larvae exhibit melanotic tumors and constitutive expression of the antifungal peptide gene Drosomycin (Drs), while fon-RNAi pupae display an aberrant pupal phenotype. Altogether, our studies indicate that Fondue is a major hemolymph protein required for efficient clotting in Drosophila.

The Drosophila peptidoglycan recognition protein SA (PGRP-SA) is critically involved in sensing bacterial infection and activating the Toll signaling pathway, which induces the expression of specific antimicrobial peptide genes. We have determined the crystal structure of PGRP-SA to 2.2-Å resolution and analyzed its peptidoglycan (PG) recognition and signaling activities. We found an extended surface groove in the structure of PGRP-SA, lined with residues that are highly diverse among different PGRPs. Mutational analysis identified it as a PG docking groove required for Toll signaling and showed that residue Ser158 is essential for both PG binding and Toll activation. Contrary to the general belief that PGRP-SA has lost enzyme function and serves primarily for PG sensing, we found that it possesses an intrinsic L,D-carboxypeptidase activity for diaminopimelic acid-type tetrapeptide PG fragments but not lysine-type PG fragments, and that Ser158 and His42 may participate in the hydrolytic activity. As L,D-configured peptide bonds exist only in prokaryotes, this work reveals a rare enzymatic activity in a eukaryotic protein known for sensing bacteria and provides a possible explanation of how PGRP-SA mediates Toll activation specifically in response to lysine-type PG., The Drosophila protein PGRP-SA is found to have an intrinsic activity to cleave L,D-configured peptide bonds, which exist only in prokaryotes. This work reveals a rare enzymatic activity in a eukaryotic protein known for sensing bacteria

A few years ago, it would have been difficult to argue that elucidating the mechanisms of disease resistance in the fruit fly, Drosophila melanogaster, would provide new insights into mammalian immunity. Yet the finding that the Drosophila protein Toll mediates immune responses to fungal infection had a pioneering role in the identification of Toll-like receptors as essential regulators of mammalian host defence, and it fundamentally altered our understanding of innate immunity. In this Landmark article, I describe the thought processes and the experimental steps that defined Toll as a key regulator of Drosophila immune responses.

Gut immunocompetence involves immune, stress and regenerative processes. To investigate the determinants underlying inter-individual variation in gut immunocompetence, we perform enteric infection of 140 Drosophila lines with the entomopathogenic bacterium Pseudomonas entomophila and observe extensive variation in survival. Using genome-wide association analysis, we identify several novel immune modulators. Transcriptional profiling further shows that the intestinal molecular state differs between resistant and susceptible lines, already before infection, with one transcriptional module involving genes linked to reactive oxygen species (ROS) metabolism contributing to this difference. This genetic and molecular variation is physiologically manifested in lower ROS activity, lower susceptibility to ROS-inducing agent, faster pathogen clearance and higher stem cell activity in resistant versus susceptible lines. This study provides novel insights into the determinants underlying population-level variability in gut immunocompetence, revealing how relatively minor, but systematic genetic and transcriptional variation can mediate overt physiological differences that determine enteric infection susceptibility., Animals rely on a multitude of resistance and tolerance mechanisms to resist harmful gut microbes. Here, the authors explore the genetic, molecular and physiological basis underlying the remarkable phenotypic variation in resistance to enteric bacterial infection in Drosophila melanogaster.

In Drosophila, the immune deficiency (Imd) pathway controls antibacterial peptide gene expression in the fat body in response to Gram-negative bacterial infection [1, 2]. The ultimate target of the Imd pathway is Relish, a transactivator related to mammalian P105 and P100 NF-κB precursors [3]. Relish is processed in order to translocate to the nucleus, and this cleavage is dependent on both Dredd, an apical caspase related to caspase-8 of mammals, and the fly Iκ-B kinase complex (dmIKK) [4–9]. dTAK1, a MAPKKK, functions upstream of the dmIKK complex and downstream of Imd, a protein with a death domain similar to that of mammalian receptor interacting protein (RIP) [10, 11]. Finally, the peptidoglycan recognition protein-LC (PGRP-LC) acts upstream of Imd and probably functions as a receptor for the Imd pathway [12–14]. Using inducible expression of dFADD double-stranded RNA, we demonstrate that dFADD is a novel component of the Imd pathway: dFADD double-stranded RNA expression reduces the induction of antibacterial peptide-encoding genes after infection and renders the fly susceptible to Gram-negative bacterial infection. Epistatic studies indicate that dFADD acts between Imd and Dredd. Our results reinforce the parallels between the Imd and the TNF-R1 pathways.

Recent studies of Drosophila immune responses have defined the immune deficiency (IMD) signaling pathway that mediates defense against Gram-negative bacterial infection. Like the Toll pathway, the IMD pathway regulates antimicrobial peptide gene expression via a Rel/nuclear factor (NF)-kappaB-like transcription factor. However, the two pathways do not appear to share any intermediate components. Maintaining distinct immune response pathways might be one mechanism by which flies mount adapted immune responses.

International audience; The production of antimicrobial peptides is an important aspect of host defense in multicellular organisms. In Drosophila, seven antimicrobial peptides with different spectra of activities are synthesized by the fat body during the immune response and secreted into the hemolymph. Using GFP reporter transgenes, we show here that all seven Drosophila antimicrobial peptides can be induced in surface epithelia in a tissue-specific manner. The imd gene plays a critical role in the activation of this local response to infection. In particular, drosomycin expression, which is regulated by the Toll pathway during the systemic response, is regulated by imd in the respiratory tract, thus demonstrating the existence of distinct regulatory mechanisms for local and systemic induction of antimicrobial peptide genes in Drosophila.

From deciphering the principles of heredity to identifying the genes that control development, the fruit fly Drosophila melanogaster is being used to deconstruct an increasing number of biological processes. Genetic studies of Drosophila responses to microbial infection have identified regulators of innate immunity that are functionally conserved in mammals. These recent findings highlight the ancient origins of animal immune responses and demonstrate the potential of Drosophila for dissecting host-pathogen interactions. The sequencing of the Drosophila genome both enhances genetic approaches and provides new clues for the identification of key components of innate immunity. This article summarizes how information gained from genomic analysis contributes to our understanding of how animals cope with infectious disease.

Although Drosophila possesses potent immune responses, little is known about the microbial pathogens that infect Drosophila . We have identified members of the bacterial genus Erwinia that induce the systemic expression of genes encoding antimicrobial peptides in Drosophila larvae after ingestion. These Erwinia strains are phytopathogens and use flies as vectors; our data suggest that these strains have also evolved mechanisms for exploiting their insect vectors as hosts. Erwinia infections induce an antimicrobial response in Drosophila larvae with a preferential expression of antibacterial versus antifungal peptide-encoding genes. Antibacterial peptide gene expression after Erwinia infection is reduced in two Drosophila mutants that have reduced numbers of hemocytes, suggesting that blood cells play a role in regulating Drosophila antimicrobial responses and also illustrating that this Drosophila – Erwinia interaction provides a powerful model for dissecting host–pathogen relationships.