Ylva Engström

Dif, a dorsal-related gene that mediates an immune response in Drosophila

There are striking parallels between the regulation of gene expression along the dorsoventral (DV) axis of Drosophila embryos and lymphoid-restricted expression in the mammalian immune system. Both depend on regulatory factors containing rel domains (dorsal and NF-kappa B) that are controlled at the level of nuclear transport. A novel Rel-containing gene in Drosophila, Dif (dorsal-related immunity factor), provides a potential link between these seemingly disparate processes. Although Dif maps close to dorsal, it does not appear to participate in DV patterning, but instead mediates an immune response in Drosophila larvae. Dif is normally localized in the cytoplasm of the larval fat body, but quickly accumulates in the nucleus upon bacterial infection or injury. Evidence is presented that once in the nucleus, Dif binds to kappa B-like sequence motifs present in promoter regions of immunity genes. These results suggest that mammalian and insect immunity share a common evolutionary origin.

Caspase-mediated processing of the Drosophila NF-kappaB factor Relish.

The NF-κB-like transcription factor Relish plays a central role in the innate immune response of Drosophila. Unlike other NF-κB proteins, Relish is activated by endoproteolytic cleavage to generate a DNA-binding Rel homology domain and a stable IκB-like fragment. This signal-induced endoproteolysis requires the activity of several gene products, including the IκB kinase complex and the caspase Dredd. Here we used mutational analysis and protein microsequencing to demonstrate that a caspase target site, located in the linker region between the Rel and the IκB-like domain, is the site of signal-dependent cleavage. We also show physical interaction between Relish and Dredd, suggesting that Dredd indeed is the Relish endoprotease. In addition to the caspase target site, the C-terminal 107 aa of Relish are required for endoproteolysis and signal-dependent phosphorylation by the Drosophila IκB kinase β. Finally, an N-terminal serine-rich region in Relish and the PEST domain were found to negatively regulate Relish activation.

Activation of the Drosophila NF‐κB factor Relish by rapid endoproteolytic cleavage

The Rel/NF‐κB transcription factor Relish plays a key role in the humoral immune response in Drosophila. We now find that activation of this innate immune response is preceded by rapid proteolytic cleavage of Relish into two parts. An N‐terminal fragment, containing the DNA‐binding Rel homology domain, translocates to the nucleus where it binds to the promoter of the Cecropin A1 gene and probably to the promoters of other antimicrobial peptide genes. The C‐terminal IκB‐like fragment remains in the cytoplasm. This endoproteolytic cleavage does not involve the proteasome, requires the DREDD caspase, and is different from previously described mechanisms for Rel factor activation.

Induction and regulation of antimicrobial peptides in Drosophila

Activation of the innate immune response involves recognition of the infectious agent and the subsequent activation of cellular and humoral reactions. In insects, a number of immunity genes are activated at the level of transcription leading to the synthesis of antimicrobial peptides. Genetic analyses in Drosophila have identified several signal transduction pathways that promote activation of these immunity genes. Recent data suggest that the insect immune system is able to discriminate between a bacterial and a fungal infection, and responds by higher levels of activation of the appropriate peptides to repel the infection. These and other recent data on transcription factors and regulation of antimicrobial genes are integrated into a model to suggest how differential activation of antifungal and antibacterial peptides can occur in response to fungal and bacterial infection.

Cooperative control of Drosophila immune responses by the JNK and NF-κB signaling pathways

Jun N‐terminal kinase (JNK) signaling is a highly conserved pathway that controls both cytoskeletal remodeling and transcriptional regulation in response to a wide variety of signals. Despite the importance of JNK in the mammalian immune response, and various suggestions of its importance in Drosophila immunity, the actual contribution of JNK signaling in the Drosophila immune response has been unclear. Drosophila TAK1 has been implicated in the NF‐κB/Relish‐mediated activation of antimicrobial peptide genes. However, we demonstrate that Relish activation is intact in dTAK1 mutant animals, and that the immune response in these mutant animals was rescued by overexpression of a downstream JNKK. The expression of a JNK inhibitor and induction of JNK loss‐of‐function clones in immune responsive tissue revealed a general requirement for JNK signaling in the expression of antimicrobial peptides. Our data indicate that dTAK1 is not required for Relish activation, but instead is required in JNK signaling for antimicrobial peptide gene expression.

Serpent regulates Drosophila immunity genes in the larval fat body through an essential GATA motif

Insects possess a powerful immune system, which in response to infection leads to a vast production of different antimicrobial peptides. The regulatory regions of many immunity genes contain a GATA motif in proximity to a κB motif. Upon infection, Rel proteins enter the nucleus and activate transcription of the immunity genes. High levels of Rel protein‐mediated Cecropin A1 expression previously have been shown to require the GATA site along with the κB site. We provide evidence demonstrating that the GATA motif is needed for expression of the Cecropin A1 gene in larval fat body, but is dispensable in adult fat body. A nuclear DNA‐binding activity interacts with the Cecropin A1 GATA motif with the same properties as the Drosophila GATA factor Serpent. The GATA‐binding activity is recognized by Serpent‐specific antibodies, demonstrating their identity. We show that Serpent is nuclear in larval fat body cells and haemocytes both before and after infection. After overexpression, Serpent increases Cecropin A1 transcription in a GATA‐dependent manner. We propose that Serpent plays a key role in tissue‐specific expression of immunity genes, by priming them for inducible activation by Rel proteins in response to infection.

The imd gene is required for local Cecropin expression in Drosophila barrier epithelia.

Surfaces of higher eukaryotes are normally covered with microorganisms but are usually not infected by them. Innate immunity and the expression of gene‐encoded antimicrobial peptides play important roles in the first line of defence in higher animals. The immune response in Drosophila promotes systemic expression of antimicrobial peptides in response to microbial infection. We now demonstrate that the epidermal cells underlying the cuticle of larvae respond to infected wounds by local expression of the genes for the antimicrobial peptide cecropin A. Thus, the Drosophila epidermis plays an active role in the innate defence against microorganisms. The immune deficiency (imd) gene was found to be a crucial component of the signal‐induced epidermal expression in both embryos and larvae. In contrast, melanization, which is part of the wound healing process, is not dependent on the imd gene, indicating that the signalling pathways promoting melanization and antimicrobial peptide gene expression can be uncoupled.

THE DORSAL-RELATED IMMUNITY FACTOR, DIF, IS A SEQUENCE-SPECIFIC TRANS-ACTIVATOR OF DROSOPHILA CECROPIN GENE EXPRESSION

A new member of the Rel family of transcription factors, the dorsal‐related immunity factor, Dif, was recently cloned and suggested to be involved in regulating the immune response in Drosophila. Despite its classification as a Rel family member, the Dif cDNA‐encoded product has not been proven previously to be a transcription factor. We now present evidence that the Dif gene product trans‐activates the Drosophila Cecropin A1 gene in co‐transfection assays. The transactivation requires a 40 bp upstream element including an insect kappa B‐like motif. A dimer of the kappa B‐like motif 5′‐GGGGATTTTT inserted into a minimal promoter conferred high levels of reporter gene expression by Dif, while a multimer of several mutated versions of this motif was not activated, demonstrating the sequence specificity of Dif. Full trans‐activation by Dif requires the C‐terminal part of the protein. The morphogen dorsal (dl) can also activate the Cecropin A1 promoter, but to a lesser extent and in a less sequence‐specific manner than Dif. Simultaneous overexpression of Dif and dl in co‐transfection assays revealed that dl possesses a dominant negative effect on Dif transactivation. This study establishes that Dif is a sequence‐specific transcription factor and is probably a key activator of the immune response in Drosophila.

Genome-Wide RNA Interference in Drosophila Cells Identifies G Protein-Coupled Receptor Kinase 2 as a Conserved Regulator of NF-κB Signaling

Because NF-κB signaling pathways are highly conserved in evolution, the fruit fly Drosophila melanogaster provides a good model to study these cascades. We carried out an RNA interference (RNAi)-based genome-wide in vitro reporter assay screen in Drosophila for components of NF-κB pathways. We analyzed 16,025 dsRNA-treatments and identified 10 novel NF-κB regulators. Of these, nine dsRNA-treatments affect primarily the Toll pathway. G protein-coupled receptor kinase (Gprk)2, CG15737/Toll pathway activation mediating protein, and u-shaped were required for normal Drosomycin response in vivo. Interaction studies revealed that Gprk2 interacts with the Drosophila IκB homolog Cactus, but is not required in Cactus degradation, indicating a novel mechanism for NF-κB regulation. Morpholino silencing of the zebrafish ortholog of Gprk2 in fish embryos caused impaired cytokine expression after Escherichia coli infection, indicating a conserved role in NF-κB signaling. Moreover, small interfering RNA silencing of the human ortholog GRK5 in HeLa cells impaired NF-κB reporter activity. Gprk2 RNAi flies are susceptible to infection with Enterococcus faecalis and Gprk2 RNAi rescues Toll10b-induced blood cell activation in Drosophila larvae in vivo. We conclude that Gprk2/GRK5 has an evolutionarily conserved role in regulating NF-κB signaling.

The GATA factor Serpent is required for the onset of the humoral immune response in Drosophila embryos

Innate immunity in Drosophila is characterized by the inducible expression of antimicrobial peptides. We have investigated the development and regulation of immune responsiveness in Drosophila embryos after infection. Immune competence, as monitored by the induction of Cecropin A1-lacZ constructs, was observed first in the embryonic yolk. This observation suggests that the yolk plays an important role in the humoral immune response of the developing embryo by synthesizing antimicrobial peptides. Around midembryogenesis, the response in the yolk was diminished. Simultaneously, Cecropin expression became inducible in a large number of cells in the epidermis, demonstrating that late-stage embryos can synthesize their own antibiotics in the epidermis. This production likely serves to provide the hatching larva with an active antimicrobial barrier and protection against systemic infections. Cecropin expression in the yolk required the presence of a GATA site in the promoter as well as the involvement of the GATA-binding transcription factor Serpent (dGATAb). In contrast, neither the GATA site nor Serpent were necessary for Cecropin expression in the epidermis. Thus, the inducible immune responses in the yolk and in the epidermis can be uncoupled and call for distinct sets of transcription factors. Our data suggest that Serpent is involved in the distinction between a systemic response in the yolk/fat body and a local immune response in epithelial cells. In addition, the present study shows that signal transduction pathways controlling innate and epithelial defense reactions can be dissected genetically in Drosophila embryos.