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.Drosophila X virus ͉ innate immunity ͉ virus ͉ Dif

SummaryDrosophila 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.

CDP-diacylglycerol synthase (CDS) is an enzyme required for the regeneration of the signalling molecule phosphatidylinositol-4,5-bisphosphate (PtdlnsP2) from phosphatidic acid. A photo-receptor cell-specific isoform of CDS from Drosophila is a key regulator of phototransduction, a G-protein-coupled signalling cascade mediated by phospholipase C. cds mutants cannot sustain a light-activated current as a result of depletion of PtdlnsP2. Overexpression of CDS increases the amplitude of the light response, demonstrating that availability of PtdlnsP2 is a determinant in the gain of this pathway. cds mutants undergo light-dependent retinal degeneration which can be suppressed by a mutation in phospholipase C. Thus, enzymes involved in PtdlnsP2 metabolism regulate phosphoinositide-mediated signalling cascades in vivo.

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.

The ird5 gene was identified in a genetic screen for Drosophila immune response mutants. Mutations in ird5 prevent induction of six antibacterial peptide genes in response to infection but do not affect the induction of an antifungal peptide gene. Consistent with this finding, Escherichia coli survive 100 times better in ird5 adults than in wild-type animals. The ird5 gene encodes a Drosophila homolog of mammalian IB kinases (IKKs). The ird5 phenotype and sequence suggest that the gene is specifically required for the activation of Relish, a Drosophila NF-B family member. In both mammals and Drosophila, microbial infection activates Toll-like receptor (TLR) signaling pathways as a part of the innate host defense response (for review, see Anderson 2000). TLR-mediated signaling pathways are essential for appropriate responses to bacterial infection. In addition, mouse Tlr4 mediates septic shock associated with infection by gram-negative bacteria (Vogel 1992;Poltorak et al. 1998).The available data indicate that different microbial cell wall components activate different Toll-like receptor signaling pathways, which regulate distinct sets of target genes. In mammals, TLR4 is the prime mediator of responses to bacterial lipopolysaccharide, while TLR2 mediates responses to bacterial peptidoglycans (Poltorak et al. 1998;Takeuchi et al. 1999; for review, see Beutler 2000). The best-studied aspect of the Drosophila innate immune response is the rapid transcriptional induction of antimicrobial peptide genes in response to infection (Hultmark 1993;Hoffmann 1995). Infection by different classes of microorganisms leads to the preferential induction of particular subsets of antimicrobial peptides (Lemaitre et al. 1997), indicating that different microbial components activate different signaling pathways.At least two Toll-related signaling pathways are required for the activation of the Drosophila antimicrobial peptide genes. The Toll pathway itself, which was first identified because of its essential role in Drosophila embryonic patterning (Anderson et al. 1985), is essential for the induction of an antifungal peptide gene, drosomycin, although the antibacterial peptide genes are still induced in Toll pathway mutants (Lemaitre et al. 1996). Another Drosophila member of the Toll family, 18-wheeler, is required for the normal induction of attacin, an antibacterial peptide gene, but mutations in 18-wheeler do not prevent the induction of other antibacterial peptides (Williams et al. 1997). The imd gene is important for the induction of Diptericin and other antibacterial peptides (Lemaitre et al. 1995a;Corbo and Levine 1996) and, therefore, appears to be a component of a third signaling pathway activated by infection, but its biochemical function is not known.Each of the three Drosophila signaling pathways activated by infection leads to activation of NF-B/Rel dimers, just as the mammalian TLRs activate NF-B. All three Drosophila Rel proteins, Dorsal, Dif, and Relish, are expressed in the fat body cells that produce the antimicr...

The Drosophila immune response uses many of the same components as the mammalian innate immune response, including signalling pathways that activate transcription factors of the Rel/NK-kappaB family. In response to infection, two Rel proteins, Dif and Dorsal, translocate from the cytoplasm to the nuclei of larval fat-body cells. The Toll signalling pathway, which controls dorsal-ventral patterning during Drosophila embryogenesis, regulates the nuclear import of Dorsal in the immune response, but here we show that the Toll pathway is not required for nuclear import of Dif. Cytoplasmic retention of both Dorsal and Dif depends on Cactus protein; nuclear import of Dorsal and Dif is accompanied by degradation of Cactus. Therefore the two signalling pathways that target Cactus for degradation must discriminate between Cactus-Dorsal and Cactus-Dif complexes. We identified new genes that are required for normal induction of transcription of an antibacterial peptide during the immune response. Mutations in three of these genes prevent nuclear import of Dif in response to infection, and define new components of signalling pathways involving Rel. Mutations in three other genes cause constitutive nuclear localization of Dif; these mutations may block Rel protein activity by a novel mechanism.

The PCR was used to alter transcriptionkl and translational signals surrounding the Flavobacterium okeanokoites restriction endonuclease (fokiR) gene, so as to achieve high expression in Eseherichia coli. By changing the ribosome-binding site sequence preceding the fokiR gene tO match the consensus E. coli signal and by placing a podive retroregulator stefla-loop sequence downstream of the gene, Fok I yield was increased to 5-8% of total cellular protein. Fok I was purified to homogeneity with phosphocellulose, DEAESephadex, and gel chromatography, yielding 50 mg of pure Fok I endonuclease per liter of culture medium. The recognition and cleavage domains of Fok I were analyzed by trypsin digestion. Fok I in the absence of a DNA substrate cleaves into a 58-kDa carboxyl-terminal and 8-kDa amino-terminal fngment. The 58-kDa fragment does not bind the DNA substrate. Fok I in the presence of a DNA substrate cleaves into a 41-kDa amino-terminal fragment and a 25-kDa carboxyl-terminal fragment. On further digestion, the 41-kDa fragment degrades into 30-kDa amino-terminal and 11-kDa carboxyl-terminal fragments. The cleaved fragments both bind DNA substrates, as does the 41-kDa fragment. Gel-mobility-shift assays indicate that all the protein contacts necessary for the sequence-speciftl recognition of DNA substrates are encoded within the 41-kDa fragment. Thus, the 41-kDa amino-terminal fragment constitutes the Fok I recognition domain. The 25-kDa fragment, purified by using a DEAE-Sephadex column, cleaves nonspecifically both methylated (pACYCfokIM) and nonmethylated (pTZ19R) DNA substrates in the presence of MgC12. Thus, the 25-kDa carboxyl-terminal fragment constitutes the Fok I cleavage domain.We have undertaken a detailed study of the Fok I restriction modification system from Flavobacterium okeanokoites. Fok

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 Toll͞NF-B 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.antimicrobial peptides ͉ N-acetylmuramoyl-L-alanine amidase ͉ pattern recognition receptor