Drosophila immunity: two paths to NF-kappaB

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

DIAP2 functions as a mechanism-based regulator of drICE that contributes to the caspase activity threshold in living cells.

In addition to their well-known function in apoptosis, caspases are also important in several nonapoptotic processes. How caspase activity is restrained and shut down under such nonapoptotic conditions remains unknown. Here, we show that Drosophila melanogaster inhibitor of apoptosis protein 2 (DIAP2) controls the level of caspase activity in living cells. Animals that lack DIAP2 have higher levels of drICE activity. Although diap2-deficient cells remain viable, they are sensitized to apoptosis following treatment with sublethal doses of x-ray irradiation. We find that DIAP2 regulates the effector caspase drICE through a mechanism that resembles the one of the caspase inhibitor p35. As for p35, cleavage of DIAP2 is required for caspase inhibition. Our data suggest that DIAP2 forms a covalent adduct with the catalytic machinery of drICE. In addition, DIAP2 also requires a functional RING finger domain to block cell death and target drICE for ubiquitylation. Because DIAP2 efficiently interacts with drICE, our data suggest that DIAP2 controls drICE in its apoptotic and nonapoptotic roles.P.S. Ribeiro is supported by a PhD fellowship (SFRH/BD/15219/2004) from the Fundação para a Ciência e Tecnologia and F. Leulier is supported by a Human Frontier Science Program fellowship (LT-0177/2004). E. Kuranaga and M. Miura are supported in part by grants from the Japanese Ministry of Education, Science, Sports, Culture and Technology (19109003, 17025010, 19040006, 19041026, and 19044013) and the Takeda Science Foundation

A Quantitative RNAi Screen for JNK Modifiers Identifies Pvr as a Novel Regulator of Drosophila Immune Signaling

Drosophila melanogaster responds to gram-negative bacterial challenges through the IMD pathway, a signal transduction cassette that is driven by the coordinated activities of JNK, NF-κB and caspase modules. While many modifiers of NF-κB activity were identified in cell culture and in vivo assays, the regulatory apparatus that determines JNK inputs into the IMD pathway is relatively unexplored. In this manuscript, we present the first quantitative screen of the entire genome of Drosophila for novel regulators of JNK activity in the IMD pathway. We identified a large number of gene products that negatively or positively impact on JNK activation in the IMD pathway. In particular, we identified the Pvr receptor tyrosine kinase as a potent inhibitor of JNK activation. In a series of in vivo and cell culture assays, we demonstrated that activation of the IMD pathway drives JNK-dependent expression of the Pvr ligands, Pvf2 and Pvf3, which in turn act through the Pvr/ERK MAP kinase pathway to attenuate the JNK and NF-κB arms of the IMD pathway. Our data illuminate a poorly understood arm of a critical and evolutionarily conserved innate immune response. Furthermore, given the pleiotropic involvement of JNK in eukaryotic cell biology, we believe that many of the novel regulators identified in this screen are of interest beyond immune signaling

Evolutionary origin of peptidoglycan recognition proteins in vertebrate innate immune system

<p>Abstract</p> <p>Background</p> <p>Innate immunity is the ancient defense system of multicellular organisms against microbial infection. The basis of this first line of defense resides in the recognition of unique motifs conserved in microorganisms, and absent in the host. Peptidoglycans, structural components of bacterial cell walls, are recognized by Peptidoglycan Recognition Proteins (PGRPs). PGRPs are present in both vertebrates and invertebrates. Although some evidence for similarities and differences in function and structure between them has been found, their evolutionary history and phylogenetic relationship have remained unclear. Such studies have been severely hampered by the great extent of sequence divergence among vertebrate and invertebrate PGRPs. Here we investigate the birth and death processes of PGRPs to elucidate their origin and diversity.</p> <p>Results</p> <p>We found that (i) four rounds of gene duplication and a single domain duplication have generated the major variety of present vertebrate PGRPs, while in invertebrates more than ten times the number of duplications are required to explain the repertoire of present PGRPs, and (ii) the death of genes in vertebrates appears to be almost null whereas in invertebrates it is frequent.</p> <p>Conclusion</p> <p>These results suggest that the emergence of new <it>PGRP </it>genes may have an impact on the availability of the repertoire and its function against pathogens. These striking differences in PGRP evolution of vertebrates and invertebrates should reflect the differences in the role of their innate immunity. Insights on the origin of <it>PGRP </it>genes will pave the way to understand the evolution of the interaction between host and pathogens and to lead to the development of new treatments for immune diseases that involve proteins related to the recognition of self and non-self.</p

Lethality and Developmental Delay in Drosophila melanogaster Larvae after Ingestion of Selected Pseudomonas fluorescens Strains

The fruit fly, Drosophila melanogaster, is a well-established model organism for probing the molecular and cellular basis of physiological and immune system responses of adults or late stage larvae to bacterial challenge. However, very little is known about the consequences of bacterial infections that occur in earlier stages of development. We have infected mid-second instar larvae with strains of Pseudomonas fluorescens to determine how infection alters the ability of larvae to survive and complete development.We mimicked natural routes of infection using a non-invasive feeding procedure to study the toxicity of the three sequenced P. fluorescens strains (Pf0-1, SBW25, and Pf-5) to Drosophila melanogaster. Larvae fed with the three strains of P. fluorescens showed distinct differences in developmental trajectory and survival. Treatment with SBW25 caused a subset of insects to die concomitant with a systemic melanization reaction at larval, pupal or adult stages. Larvae fed with Pf-5 died in a dose-dependent manner with adult survivors showing eye and wing morphological defects. In addition, larvae in the Pf-5 treatment groups showed a dose-dependent delay in the onset of metamorphosis relative to control-, Pf0-1-, and SBW25-treated larvae. A functional gacA gene is required for the toxic properties of wild-type Pf-5 bacteria.These experiments are the first to demonstrate that ingestion of P. fluorescens bacteria by D. melanogaster larvae causes both lethal and non-lethal phenotypes, including delay in the onset of metamorphosis and morphological defects in surviving adult flies, which can be decoupled

The Imd Pathway Is Involved in Antiviral Immune Responses in Drosophila

Cricket Paralysis virus (CrPV) is a member of the Dicistroviridae family of RNA viruses, which infect a broad range of insect hosts, including the fruit fly Drosophila melanogaster. Drosophila has emerged as an effective system for studying innate immunity because of its powerful genetic techniques and the high degree of gene and pathway conservation. Intra-abdominal injection of CrPV into adult flies causes a lethal infection that provides a robust assay for the identification of mutants with altered sensitivity to viral infection. To gain insight into the interactions between viruses and the innate immune system, we injected wild type flies with CrPV and observed that antimicrobial peptides (AMPs) were not induced and hemocytes were depleted in the course of infection. To investigate the contribution of conserved immune signaling pathways to antiviral innate immune responses, CrPV was injected into isogenic mutants of the Immune Deficiency (Imd) pathway, which resembles the mammalian Tumor Necrosis Factor Receptor (TNFR) pathway. Loss-of-function mutations in several Imd pathway genes displayed increased sensitivity to CrPV infection and higher CrPV loads. Our data show that antiviral innate immune responses in flies infected with CrPV depend upon hemocytes and signaling through the Imd pathway

NF-κB/Rel-Mediated Regulation of the Neural Fate in Drosophila

Two distinct roles are described for Dorsal, Dif and Relish, the three NF-κB/Rel proteins of Drosophila, in the development of the peripheral nervous system. First, these factors regulate transcription of scute during the singling out of sensory organ precursors from clusters of cells expressing the proneural genes achaete and scute. This effect is possibly mediated through binding sites for NF-κB/Rel proteins in a regulatory module of the scute gene required for maintenance of scute expression in precursors as well as repression in cells surrounding precursors. Second, genetic evidence suggests that the receptor Toll-8, Relish, Dif and Dorsal, and the caspase Dredd pathway are active over the entire imaginal disc epithelium, but Toll-8 expression is excluded from sensory organ precursors. Relish promotes rapid turnover of transcripts of the target genes scute and asense through an indirect, post-transcriptional mechanism. We propose that this buffering of gene expression levels serves to keep the neuro-epithelium constantly poised for neurogenesis

Anopheles Imd Pathway Factors and Effectors in Infection Intensity-Dependent Anti-Plasmodium Action

The Anopheles gambiae immune response against Plasmodium falciparum, an etiological agent of human malaria, has been identified as a source of potential anti-Plasmodium genes and mechanisms to be exploited in efforts to control the malaria transmission cycle. One such mechanism is the Imd pathway, a conserved immune signaling pathway that has potent anti-P. falciparum activity. Silencing the expression of caspar, a negative regulator of the Imd pathway, or over-expressing rel2, an Imd pathway-controlled NFkappaB transcription factor, confers a resistant phenotype on A. gambiae mosquitoes that involves an array of immune effector genes. However, unexplored features of this powerful mechanism that may be essential for the implementation of a malaria control strategy still remain. Using RNA interference to singly or dually silence caspar and other components of the Imd pathway, we have identified genes participating in the anti-Plasmodium signaling module regulated by Caspar, each of which represents a potential target to achieve over-activation of the pathway. We also determined that the Imd pathway is most potent against the parasite's ookinete stage, yet also has reasonable activity against early oocysts and lesser activity against late oocysts. We further demonstrated that caspar silencing alone is sufficient to induce a robust anti-P. falciparum response even in the relative absence of resident gut microbiota. Finally, we established the relevance of the Imd pathway components and regulated effectors TEP1, APL1, and LRIM1 in parasite infection intensity-dependent defense, thereby shedding light on the relevance of laboratory versus natural infection intensity models. Our results highlight the physiological considerations that are integral to a thoughtful implementation of Imd pathway manipulation in A. gambiae as part of an effort to limit the malaria transmission cycle, and they reveal a variety of previously unrecognized nuances in the Imd-directed immune response against P. falciparum

cIAP-1 Controls Innate Immunity to C. pneumoniae Pulmonary Infection

The resistance of epithelial cells infected with Chlamydophila pneumoniae for apoptosis has been attributed to the induced expression and increased stability of anti-apoptotic proteins called inhibitor of apoptosis proteins (IAPs). The significance of cellular inhibitor of apoptosis protein-1 (cIAP-1) in C. pneumoniae pulmonary infection and innate immune response was investigated in cIAP-1 knockout (KO) mice using a novel non-invasive intra-tracheal infection method. In contrast to wildtype, cIAP-1 knockout mice failed to clear the infection from their lungs. Wildtype mice responded to infection with a strong inflammatory response in the lung. In contrast, the recruitment of macrophages was reduced in cIAP-1 KO mice compared to wildtype mice. The concentration of Interferon gamma (IFN-γ) was increased whereas that of Tumor Necrosis Factor (TNF-α) was reduced in the lungs of infected cIAP-1 KO mice compared to infected wildtype mice. Ex vivo experiments on mouse peritoneal macrophages and splenocytes revealed that cIAP-1 is required for innate immune responses of these cells. Our findings thus suggest a new immunoregulatory role of cIAP-1 in the course of bacterial infection

Expression and Putative Function of Innate Immunity Genes under in situ Conditions in the Symbiotic Hydrothermal Vent Tubeworm Ridgeia piscesae

The relationships between hydrothermal vent tubeworms and sulfide-oxidizing bacteria have served as model associations for understanding chemoautotrophy and endosymbiosis. Numerous studies have focused on the physiological and biochemical adaptations that enable these symbioses to sustain some of the highest recorded carbon fixation rates ever measured. However, far fewer studies have explored the molecular mechanisms underlying the regulation of host and symbiont interactions, specifically those mediated by the innate immune system of the host. To that end, we conducted a series of studies where we maintained the tubeworm, Ridgeia piscesae, in high-pressure aquaria and examined global and quantitative changes in gene expression via high-throughput transcriptomics and quantitative real-time PCR (qPCR). We analyzed over 32,000 full-length expressed sequence tags as well as 26 Mb of transcript sequences from the trophosome (the organ that houses the endosymbiotic bacteria) and the plume (the gas exchange organ in contact with the free-living microbial community). R. piscesae maintained under conditions that promote chemoautotrophy expressed a number of putative cell signaling and innate immunity genes, including pattern recognition receptors (PRRs), often associated with recognizing microbe-associated molecular patterns (MAMPs). Eighteen genes involved with innate immunity, cell signaling, cell stress and metabolite exchange were further analyzed using qPCR. PRRs, including five peptidoglycan recognition proteins and a Toll-like receptor, were expressed significantly higher in the trophosome compared to the plume. Although PRRs are often associated with mediating host responses to infection by pathogens, the differences in expression between the plume and trophosome also implicate similar mechanisms of microbial recognition in interactions between the host and symbiont. We posit that regulation of this association involves a molecular “dialogue” between the partners that includes interactions between the host’s innate immune system and the symbiont