The Splicing Factor RNA-Binding Fox Protein 1 Mediates the Cellular Immune Response in Drosophila melanogaster.

The uptake and destruction of bacteria by phagocytic cells is an essential defense mechanism in metazoans. To identify novel genes involved in the phagocytosis of Staphylococcus aureus, a major human pathogen, we assessed the phagocytic capacity of adult blood cells (hemocytes) of the fruit fly, Drosophila melanogaster, by testing several lines of the Drosophila Genetic Reference Panel. Natural genetic variation in the gene RNA-binding Fox protein 1 (Rbfox1) correlated with low phagocytic capacity in hemocytes, pointing to Rbfox1 as a candidate regulator of phagocytosis. Loss of Rbfox1 resulted in increased expression of the Ig superfamily member Down syndrome adhesion molecule 4 (Dscam4). Silencing of Dscam4 in Rbfox1-depleted blood cells rescued the fly's cellular immune response to S. aureus, indicating that downregulation of Dscam4 by Rbfox1 is critical for S. aureus phagocytosis in Drosophila To our knowledge, this study is the first to demonstrate a link between Rbfox1, Dscam4, and host defense against S. aureus.

Atg6 is required for multiple vesicle trafficking pathways and hematopoiesis in Drosophila.

Atg6 (beclin 1 in mammals) is a core component of the Vps34 complex that is required for autophagy. Beclin 1 (Becn1) functions as a tumor suppressor, and Becn1(+/-) tumors in mice possess elevated cell stress and p62 levels, altered NF-κB signaling and genome instability. The tumor suppressor function of Becn1 has been attributed to its role in autophagy, and the potential functions of Atg6/Becn1 in other vesicle trafficking pathways for tumor development have not been considered. Here, we generate Atg6 mutant Drosophila and demonstrate that Atg6 is essential for autophagy, endocytosis and protein secretion. By contrast, the core autophagy gene Atg1 is required for autophagy and protein secretion, but it is not required for endocytosis. Unlike null mutants of other core autophagy genes, all Atg6 mutant animals possess blood cell masses. Atg6 mutants have enlarged lymph glands (the hematopoietic organ in Drosophila), possess elevated blood cell numbers, and the formation of melanotic blood cell masses in these mutants is not suppressed by mutations in either p62 or NFκB genes. Thus, like mammals, altered Atg6 function in flies causes hematopoietic abnormalities and lethality, and our data indicate that this is due to defects in multiple membrane trafficking processes.

Drosophila Rab14 mediates phagocytosis in the immune response to Staphylococcus aureus.

Drosophila haemocytes are essential for the animal to resist Staphylococcus aureus infections. Phagocytosis is a central component of the haemocyte-mediated immune response. It involves regulated interaction between the phagocytic and the endocytic compartments. RabGTPases are pivotal for the membrane trafficking and fusion events, and thus are often targets of intracellular pathogens that subvert phagocytosis. An in vivo screen identified Rab2 and Rab14 as candidates for proteins regulating phagosome maturation. Since Rab14 is often targeted by intracellular pathogens, an understanding of its function during phagocytosis and the overall immune response can give insight into the pathogenesis of intracellular microbes. We generated a Drosophila Rab14 mutant and characterized the resulting immune defects in animals and specifically in haemocytes in response to an S. aureus infection. Haemocyte based immunofluorescence studies indicate that Rab14 is recruited to the phagosome and like Rab7, a well-characterized regulator of the phagocytic pathway, is essential for progression of phagosome maturation. Rab14 mutant haemocytes show impaired recruitment of Rab7 and of a lysosomal marker onto S. aureus phagosomes. The defect in phagocytosis is associated with higher bacterial load and increased susceptibility to S. aureus in the animal.

Guts and glory: balancing microbes and the immune response.

Maintaining balance with intestinal flora is an important activity of the immune system in higher metazoans. In this issue of Developmental Cell, Ha et al. demonstrate a central role of a redox balance in microbial interactions in the fruit fly gut.

Lessons from the fly: pattern recognition in Drosophila melanogaster.

Drosophila have a variety of innate immune strategies for defending itself from infection, including humoral and cell mediated responses to invading microorganisms. At the front lines of these responses, are a diverse group of pattern recognition receptors that recognize pathogen associated molecular patterns. These patterns include bacterial lipopolysaccharides, peptidoglycans, and fungal beta-1,3 glucans. Some of the receptors catalytically modify the pathogenic determinant, but all are responsible for directly facilitating a signaling event that results in an immune response. Some of these events require multiple pattern recognition receptors acting sequentially to activate a pathway. In some cases, a signaling pathway may be activated by a variety of different pathogens, through parallel receptors detecting different pathogenic determinants. In this chapter, we review what is known about pattern recognition receptors in Drosophila, and how those lessons may be applied towards a broader understanding of immunity.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay.

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Microarray analyses reveal distinct roles for Rel proteins in the Drosophila immune response.

The NF-kappaB group of transcription factors play an important role in mediating immune responses in organisms as diverse as insects and mammals. The fruit fly Drosophila melanogaster express three closely related NF-kappaB-like transcription factors: Dorsal, Dif, and Relish. To study their roles in vivo, we used microarrays to determine the effect of null mutations in individual Rel transcription factors on larval immune gene expression. Of the 188 genes that were significantly up-regulated in wild-type larvae upon bacterial challenge, overlapping but distinct groups of genes were affected in the Rel mutants. We also ectopically expressed Dorsal or Dif and used cDNA microarrays to determine the genes that were up-regulated in the presence of these transcription factors. This expression was sufficient to drive expression of some immune genes, suggesting redundancy in the regulation of these genes. Combining this data, we also identified novel genes that may be specific targets of Dif.

The NF-κB Factor Relish Regulates Atg1 Expression and Controls Autophagy.

Macroautophagy and cell death both contribute to innate immunity, but little is known about how these processes integrate. Drosophila larval salivary glands require autophagy for developmentally programmed cell death, and innate immune signaling factors increase in these dying cells. Here, we show that the nuclear factor κB (NF-κB) factor Relish, a component of the immune deficiency (Imd) pathway, is required for salivary gland degradation. Surprisingly, of the classic Imd pathway components, only Relish and the PGRP receptors were involved in salivary gland degradation. Significantly, Relish controls salivary gland degradation by regulating autophagy but not caspases. In addition, expression of either Relish or PGRP-LC causes premature autophagy induction and subsequent gland degradation. Relish controls autophagy by regulating the expression of Atg1, a core component and activator of the autophagy pathway. Together these findings demonstrate that a NF-κB pathway regulates autophagy during developmentally programmed cell death.

Gutsy Microbes Fly High in the Antiviral War.

The importance of microbiomes in health and disease is now well appreciated. New work from Sansone and colleagues adds to this understanding by showing that gut microbes are key for the local induction of an ERK-dependent antiviral response in flies.

A glutamate-dependent redox system in blood cells is integral for phagocytosis in Drosophila melanogaster.

Glutamate transport is highly regulated as glutamate directly acts as a neurotransmitter and indirectly regulates the synthesis of antioxidants. Although glutamate deregulation has been repeatedly linked to serious human diseases such as HIV infection and Alzheimer's, glutamate's role in the immune system is still poorly understood. We find that a putative glutamate transporter in Drosophila melanogaster, polyphemus (polyph), plays an integral part in the fly's immune response. Flies with a disrupted polyph gene exhibit decreased phagocytosis of microbial-derived bioparticles. When infected with S. aureus, polyph flies show an increase in both susceptibility and bacterial growth. Additionally, the expression of two known glutamate transporters, genderblind and excitatory amino acid transporter 1, in blood cells affects the flies' ability to phagocytose and survive after an infection. Consistent with previous data showing a regulatory role for glutamate transport in the synthesis of the major antioxidant glutathione, polyph flies produce more reactive oxygen species (ROS) as compared to wild-type flies when exposed to S. aureus. In conclusion, we demonstrate that a polyph-dependent redox system in blood cells is necessary to maintain the cells' immune-related functions. Furthermore, our model provides insight into how deregulation of glutamate transport may play a role in disease.

Pattern recognition receptors in the fly: lessons we can learn from the Drosophila melanogaster immune system.

Drosophila have a variety of innate immune strategies for defending itself from infection, including humoral and cell mediated responses to invading microorganisms. At the front lines of these responses, are a diverse group of pattern recognition receptors that recognize pathogen associated molecular patterns. These patterns include bacterial lipopolysaccharides, peptidoglycans, and fungal beta-1,3 glucans. Some of the receptors catalytically modify the pathogenic determinant, but all are responsible for directly facilitating a signaling event that results in an immune response. Some of these events require multiple pattern recognition receptors acting sequentially to activate a pathway. In some cases, a signaling pathway may be activated by a variety of different pathogens, through parallel receptors detecting different pathogenic determinants. In this chapter, we review what is known about pattern recognition receptors in Drosophila, and how those lessons may be applied towards a broader understanding of immunity.

no poles encodes a predicted E3 ubiquitin ligase required for early embryonic development of Drosophila.

In a screen for cell-cycle regulators, we identified a Drosophila maternal effect-lethal mutant that we named ;no poles' (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the rapid S-M cycles of syncytial embryogenesis. We identified CG5140, which encodes a candidate RING domain-containing E3 ubiquitin ligase, as the nopo gene. A conserved residue in the RING domain is altered in our EMS-mutagenized allele of nopo, suggesting that E3 ligase activity is crucial for NOPO function. We show that mutation of a DNA checkpoint kinase, CHK2, suppresses the spindle and developmental defects of nopo-derived embryos, revealing that activation of a DNA checkpoint operational in early embryos contributes significantly to the nopo phenotype. CHK2-mediated mitotic arrest has been previously shown to occur in response to mitotic entry with DNA damage or incompletely replicated DNA. Syncytial embryos lacking NOPO exhibit a shorter interphase during cycle 11, suggesting that they may enter mitosis prior to the completion of DNA replication. We show that Bendless (BEN), an E2 ubiquitin-conjugating enzyme, interacts with NOPO in a yeast two-hybrid assay; furthermore, ben-derived embryos arrest with a nopo-like phenotype during syncytial divisions. These data support our model that an E2-E3 ubiquitination complex consisting of BEN-UEV1A (E2 heterodimer) and NOPO (E3 ligase) is required for the preservation of genomic integrity during early embryogenesis.

Fungal peptide Destruxin A plays a specific role in suppressing the innate immune response in Drosophila melanogaster.

Destruxins are a class of insecticidal, anti-viral, and phytotoxic cyclic depsipeptides that are also studied for their toxicity to cancer cells. They are produced by various fungi, and a direct relationship has been established between Destruxin production and the virulence of the entomopathogen Metarhizium anisopliae. Aside from opening calcium channels, their in vivo mode of action during pathogenesis remains largely uncharacterized. To better understand the effects of a Destruxin, we looked at changes in gene expression following injection of Destruxin A into the fruit fly Drosophila melanogaster. Microarray results revealed reduced expression of various antimicrobial peptides that play a major role in the humoral immune response of the fly. Flies co-injected with a non-lethal dose of Destruxin A and the normally innocuous Gram-negative bacteria Escherichia coli, showed increased mortality and an accompanying increase in bacterial titers. Mortality due to sepsis was rescued through ectopic activation of components in the IMD pathway, one of two signal transduction pathways that are responsible for antimicrobial peptide induction. These results demonstrate a novel role for Destruxin A in specific suppression of the humoral immune response in insects.

ird1 is a Vps15 homologue important for antibacterial immune responses in Drosophila.

The immune response-deficient 1 (ird1) gene was identified in a forward genetic screen as a novel regulator for the activation of Imd NFkappaB immune signalling pathway in Drosophila. ird1 animals are also more susceptible to Escherichia coli and Micrococcus luteus bacterial infection. ird1 encodes the Drosophila homologue of the Vps15/p150 serine/threonine kinase that regulates a class III phosphoinositide 3-kinase and is necessary for phagosome maturation and starvation-induced autophagy in yeast and mammalian cells. To gain insight into the role of ird1 in the immune response, we examine how amino acid starvation affects the immune signalling pathways in Drosophila. Starvation, in the absence of infection, leads to expression of antimicrobial peptide (AMP) genes and this response is dependent on ird1 and the Imd immune signalling pathway. Starvation, in addition to bacterial infection, suppresses the AMP response in wild-type animals and reduces the ability to survive M. luteus infection. Our results suggest that starvation and innate immune signalling may be intimately linked processes.

The peptidoglycan recognition protein PGRP-SC1a is essential for Toll signaling and phagocytosis of Staphylococcus aureus in Drosophila.

From a forward genetic screen for phagocytosis mutants in Drosophila melanogaster, we identified a mutation that affects peptidoglycan recognition protein (PGRP) SC1a and impairs the ability to phagocytose the bacteria Staphylococcus aureus, but not Escherichia coli and Bacillus subtilis. Because of the differences in peptidoglycan peptide linkages in these bacteria, our data suggest that PGRP-SC1a is necessary for recognition of the Lys-type peptidoglycan typical of most Gram(+) bacteria. PGRP-SC1a mutants also fail to activate the Toll/NF-kappaB signaling pathway and are compromised for survival after S. aureus infection. This mutant phenotype is the first found for an N-acetylmuramoyl-l-alanine amidase PGRP that cleaves peptidoglycan at the lactylamide bond between the glycan backbone and the crosslinking stem peptides. By generating transgenic rescue flies that express either wild-type or a noncatalytic cysteine-serine mutant PGRP-SC1a, we find that PGRP-SC1a amidase activity is not necessary for Toll signaling, but is essential for uptake of S. aureus into the host phagocytes and for survival after S. aureus infection. Furthermore, we find that the PGRP-SC1a amidase activity can be substituted by exogenous addition of free peptidoglycan, suggesting that the presence of peptidoglycan cleavage products is more important than the generation of cleaved peptidoglycan on the bacterial surface for PGRP-SC1a mediated phagocytosis.

RNAi is an antiviral immune response against a dsRNA virus in Drosophila melanogaster.

Drosophila melanogaster has a robust and efficient innate immune system, which reacts to infections ranging from bacteria to fungi and, as discovered recently, viruses as well. The known Drosophila immune responses rely on humoral and cellular activities, similar to those found in the innate immune system of other animals. Recently, RNAi or 'RNA silencing' has arisen as a possible means by which Drosophila can react to a specific pathogens, transposons and retroviral elements, in a fashion similar to that of a traditional mammalian adaptive immune system instead of in a more generalized and genome encoded innate immune-based response. RNAi is a highly conserved regulation and defence mechanism, which suppresses gene expression via targeted RNA degradation directed by either exogenous dsRNA (cleaved into siRNAs) or endogenous miRNAs. In plants, RNAi has been found to act as an antiviral immune response system. Here we show that RNAi is an antiviral response used by Drosophila to combat infection by Drosophila X Virus, a birnavirus, as well. Additionally, we identify multiple core RNAi pathway genes, including piwi, vasa intronic gene (vig), aubergine (aub), armitage (armi), Rm62, r2d2 and Argonaute2 (AGO2) as having vital roles in this response in whole organisms. Our findings establish Drosophila as an ideal model for the study of antiviral RNAi responses in animals.

The Toll pathway is important for an antiviral response in Drosophila.

The innate immune response of Drosophila melanogaster is governed by a complex set of signaling pathways that trigger antimicrobial peptide (AMP) production, phagocytosis, melanization, and encapsulation. Although immune responses against both bacteria and fungi have been demonstrated in Drosophila, identification of an antiviral response has yet to be found. To investigate what responses Drosophila mounts against a viral infection, we have developed an in vivo Drosophila X virus (DXV)-based screening system that identifies altered sensitivity to viral infection by using DXV's anoxia-induced death pathology. Using this system to screen flies with mutations in genes with known or suggested immune activity, we identified the Toll pathway as a vital part of the Drosophila antiviral response. Inactivation of this pathway instigated a rapid onset of anoxia induced death in infected flies and increases in viral titers compared to those in WT flies. Although constitutive activation of the pathway resulted in similar rapid onset of anoxia sensitivity, it also resulted in decreased viral titer. Additionally, AMP genes were induced in response to viral infection similar to levels observed during Escherichia coli infection. However, enhanced expression of single AMPs did not alter resistance to viral infection or viral titer levels, suggesting that the main antiviral response is cellular rather than humoral. Our results show that the Toll pathway is required for efficient inhibition of DXV replication in Drosophila. Additionally, our results demonstrate the validity of using a genetic approach to identify genes and pathways used in viral innate immune responses in Drosophila.