Two distinct roles are described for Dorsal, Dif and Relish, the three NF-kappaB/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-kappaB/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. It is proposed that this buffering of gene expression levels serves to keep the neuro-epithelium constantly poised for neurogenesis (Ayyar, 2007).
The results suggest a dual role for the NF-kappaB/Rel proteins of Drosophila in the formation of SOPs. First, they could be recruited directly to the sc promoter and regulate transcription. The SOP enhancer of sc, required for auto-regulation of sc in the SOPs, contains α boxes (ACTAGA), consensus sequences for NF-kappaB/Rel. Evidence has been obtained for a role of these sequences in both activation and repression of sc. Expression of Rel-VP16, a potent transcriptional activator form of Relish, is able to ectopically activate a reporter gene containing the intact sc SOP enhancer but not one in which the α3 box is mutated. So activation in this experimental situation requires the presence of an intact α3 site. The experiment does not rule out indirect effects, so further work is required to verify whether activation is direct. It is suggested the NF-kappaB/Rel proteins participate in activation and repression of transcription of sc, a hypothesis consistent with dl, Dif and Rel mutant phenotypes of additional as well as missing bristles. Second, unexpected role is described of Rel in mRNA turnover of sc, ase and sens, neuronal genes required to specify and/or maintain the neuronal fate of SOP cells. In Rel mutants, transcripts of sc, ase and sens accumulate due to increased transcript stability. Therefore in the wild type, Relish promotes rapid mRNA turnover, presumably indirectly through an unidentified transcriptional target. A similar phenotype is observed in Toll-8 mutants, which furthermore, interact genetically with Rel mutants. Transcripts for Rel are reduced in the Toll-8 mutant suggesting a role for Toll-8 in maintaining the levels of Rel transcript. This might be the reason for the genetic interaction (Ayyar, 2007).
A number of differences are apparent between mutants of the three NF-kappaB/Rel-encoding genes of Drosophila. Mutants triply homo- or hetero-zygous have a normal complement of bristles, while single homo- or hetero-zygous animals have either additional or missing bristles. This suggests possible opposing functions for these genes. Furthermore bristle phenotypes due to loss or gain of function differ in detail between the three mutants. Together these results point to the importance of the stoichiometric relationships between the three NF-kappaB/Rel proteins and raise the possibility that different Dorsal/Dif/Relish homo- or hetero-dimers may have distinct binding sites and therefore different targets. This merits further investigation (Ayyar, 2007).
If NF-kappaB/Rel proteins both activate and repress sc, then they are expected to activate in SOP cells and repress in cells of the proneural clusters not chosen to be SOPs. Two possible ways that this could occur are discussed. First, activation in the SOP may rely on high levels of proneural protein and low levels of NF-kappaB/Rel protein; conversely repression may require low levels of proneural and high levels of NF-kappaB/Rel protein. Notch-mediated lateral inhibition results in high levels of Sc in the SOP and lower levels in surrounding cells. Toll-8 expression is excluded from SOP cells suggesting, that, if Toll-8 affects NF-kappaB/Rel activity, there would be lower levels of NF-kappaB/Rel in SOPs. NF-kappaB has been shown to activate transcription even without stimulus if IkappaB levels are low enough to allow NF-kappaB-dependent gene expression in the basal state. Interestingly, it has been shown that low levels of Dorsal can act synergistically with bHLH proteins to activate target genes in the embryo. This depends on direct association of Dorsal and bHLH proteins and cooperative binding to closely linked binding sites for the two respective proteins. Furthermore cooperative binding for Sc and Dorsal has been demonstrated. In the sc SOP enhancer one of the alpha boxes is indeed close to an E box, so perhaps high levels of Sc and low levels of NF-kappaB/Rel combine to activate transcription in the SOP. Two observations are consistent with this hypothesis: Rel-VP16 was able to ectopically activate sc-SOPE-lacZ only at sites where ac and sc are expressed and, after over-expression of NF-kappaB/Rel proteins, bristles are generally missing on the lateral notum (where Toll-8 levels are high), whereas ectopic bristles are found on the medial notum (where Toll-8 levels are low) (Ayyar, 2007).
A second means by which NF-kappaB/Rel proteins could act differently in SOP and in non-SOP cells, may be the presence/absence of co-factors. It has been shown that Dorsal can be converted from an activator to a repressor by association with the co-repressor Groucho. This bi-functionality is attributable to the fact that Dl only weakly interacts with Gro. During embryogenesis both Cut and Dead ringer bind an AT-rich silencer sequence, AT2, present in target genes of Dorsal and both Dorsal and Dead ringer bind the co-repressor Groucho and recruit it to DNA. A similar AT-rich sequence (the β box) is present in the sc SOP enhancer. Furthermore repression of sc by the E(spl) proteins, targets of Notch signalling in non-SOP cells, is already known to require the activity of Groucho (Ayyar, 2007).
Transcripts for sc, ase and sens (and GFP) accumulate in Rel and Toll-8 mutants as a result of increased transcript stability. Transcript stability correlates with the presence of a six or seven nucleotide motif in the transcribed sequence of these genes. The motif is present in sc, ase and sens, but not ac the transcription of which is unaffected in Rel mutants. The motif is almost identical to the heptamer in MyoD and Sox9 that is associated with transcript stability after inhibition of NF-kappaB/Rel signalling in C2C12 cells. A sc mutant with a truncated sc transcript lacking one of the two motifs present in the coding sequence of this gene, has a phenotype similar to Rel and Toll-8 mutants and an increase in sc mRNA. It has been suggested that increased stability of the transcripts rather than increased transcription underlies this phenotype. The presence of the heptamer is noted in a number of genes involved in sensory organ patterning suggesting possible regulation by NF-kappaB/Rel of a battery of genes in the imaginal epithelium. A similar motif is present in other vertebrate targets of NF-kappaB/Rel. Post-transcriptional regulation of target genes by NF-kappaB/Rel could therefore be an ancient feature common to Drosophila and mammals and possibly even jellyfish. It has been suggest that an unknown factor, presumably a transcriptional target of NF-kappaB/Rel, regulates messenger turnover through association with this sequence. In Rel and Toll-8 mutants the accumulated transcripts are not translated. This must be an effect of the mutants because ectopic expression in wild-type flies allows translation and ectopic bristle formation (Ayyar, 2007).
Promotion of a rapid turnover of transcripts of neuronal genes presumably does not take place in the SOPs where high levels of the protein products of these genes are required. Accordingly Toll-8 expression is extinguished in the SOPs after their formation. Factors specific to the SOP presumably allow translation of the transcripts. It is therefore suggested that high levels of Relish provided by Toll-8 in non-SOP cells might be required for post-transcriptional regulation of neuronal genes (Ayyar, 2007).
In wild-type animals expression of neuronal precursor genes such as sens and ase is restricted to SOPs where they are activated by high levels of Ac and Sc. The results suggest that they are in fact expressed over the entire neuro-epithelium but that mRNA turnover is rapid due to NF-kappaB/Rel activity. Activation of ac-sc in proneural clusters would counteract the effects of NF-kappaB/Rel to allow selection of SOPs. After selection of SOPs for the large sensory bristles is finished, Toll-8 expression is maintained in the epithelium, suggesting that high levels of NF-kappaB/Rel are still required for continued transcript turnover. Continuous buffering of neuronal gene expression presumably continues until the next round of neurogenesis that takes place after pupariation when precursors for the small bristles form. Therefore it is hypothesized that NF-kappaB/Rel plays a subtle role in maintaining steady state levels of expression of many genes required for neural development. The maintenance of low levels of expression of neuronal genes would keep the tissue poised for neurogenesis that takes place in repeated rounds. Perhaps low levels of expression of neuronal genes are characteristic of neuro-epithelia in general (Ayyar, 2007).
The hypothesis concerning the dual role of NF-kappaB/Rel in neurogenesis in Drosophila is as follows. The neuro-epithelium of the imaginal discs expresses neuronal genes. Prior to development of SOPs, high levels of Toll-8 maintain high levels of Rel and result in nuclear accumulation of NF-kappaB/Rel. Through an unknown transcriptional target(s), Relish promotes rapid turnover of neuronal transcripts by a post-transcriptional mechanism. This might be mediated by a specific sequence in the coding regions of target genes. Activation of ac and sc in proneural clusters by regulatory proteins of the notal prepattern counteracts the effects of Relish. After singling out of SOPs by Notch-mediated lateral inhibition, Toll-8 expression ceases in the SOPs. Reduced levels of signal uncover a trans-activator function for NF-kappaB/Rel that, synergistically with Sc, helps to maintain high levels of sc expression in the SOP, possibly through direct binding to consensus sequences in the sc SOP enhancer. The NF-kappaB/Rel proteins may also directly repress sc in non-SOP cells of the proneural clusters. It remains to be seen to what extent each of the three proteins participates in these two processes (Ayyar, 2007).
Since co-expression of Relish with Dif or Dorsal can enhance the expression of Drosomycin and defensin, it has been speculated that the proteins can interact directly with the target genes to regulate their expression. Among the immunity genes, the cecropin and diptericin promoters have been the most thoroughly analyzed. Previously, it has been shown that Dif can bind to the kappaB site in the cecropin promoter, whereas Dorsal can bind to a similar site in the diptericin promoter. The binding probably leads to activated transcription of cecropin and diptericin. Furthermore, Dif and Dorsal may preferentially activate cecropin and diptericin, respectively, based on DNA binding and transfection studies. Meanwhile, the results suggest the possibility that the heterodimers formed by these Rel proteins bind to kappaB sites in the promoters of drosomycin and defensin. Based on the conserved sequence of insect kappaB motif, similar kappaB sites have been identified in the promoters of both drosomycin and defensin. It was then tested whether Rel proteins expressed in the stable cell lines can interact directly with these kappaB sites using EMSA. The parental S2* cell extract exhibits very little DNA binding activity. The cell extract that expresses FLAG-tagged Relish has strong binding activity toward the oligonucleotide containing the drosomycin kappaB site. This activity is supershifted by anti-FLAG antibody, showing Relish is the protein factor that binds directly to the kappaB site. Compared with Relish, Dif and Dorsal have relatively lower binding affinity. When Relish is co-expressed with Dif or Dorsal, new complexes are formed that have faster mobility, probably due to the formation of Dif/Relish or Dorsal/Relish heterodimer, which has lower molecular weight. The result indicates that Relish enhances the binding of Dif and Dorsal to the kappaB site. Moreover, the corresponding bands are supershifted by anti-FLAG antibody, demonstrating that FLAG-tagged Relish complexes with Dif and Dorsal in binding to DNA (Han, 1999).
To further confirm that Rel proteins bind to the kappaB sites within the oligonucleotides, competition EMSA was carried out. Whereas 20-fold excess amount of the same wild type oligonucleotide effectively blocks the binding of the Rel proteins to the 32P-labeled wild type oligonucleotide, the oligonucleotide with mutated kappaB motif does not affect the binding, suggesting that the Rel proteins bind specifically to the kappaB motif of drosomycin (Han, 1999).
The binding activity of Rel proteins to the kappaB site of defensin promoter shows similar pattern to that of drosomycin promoter, albeit with lower affinity. Interestingly, this is coincident with lower level of defensin expression in S2* cells and in adult fly. Whether or not the affinity of the Rel proteins toward the target promoter determines the inducibility requires further investigation. When the cellular extracts that contain combinations of Rel proteins were examined, Relish was found to enhance the binding activity of both Dif and Dorsal. The corresponding bands could also be supershifted using the anti-FLAG antibody, showing that the complex indeed contains Relish. The result therefore demonstrates that Rel proteins can interact directly with the defensin promoter (Han, 1999).
The Cecropin A1 gene promoter is known to have a functional kappaB-site, and is thus a potential target for Relish regulation. The effect of Relish overexpression was tested on a CecA1-lacZ reporter gene construct after cotransfection of the mbn-2 blood cell line. In addition to a full-length Relish cDNA, the effect of a truncated 'Rel-only' construct that lacks the IkappaB-like domain was tested. Mammalian p50 is produced by the proteolytic degradation of the ankyrin domain of p105. The Rel-only contruct was designed to be similar to a p105 fragment that produces a stable p50 when transfected into mammalian cells. Overexpression of the full-length Relish gene stimulates expression from the CecA1-lacZ fusion reporter 3-fold over the maximally lipopolysaccharide-induced control, while the Rel-only construct increases expression as much as 10-fold. Relish can thus stimulate cecropin transcription, directly or indirectly, and the sequences present in the Rel-only construct are sufficient for this effect. The lesser effect seen with the full-length construct may be due to the presence of an inhibitory IkappaB-like domain. Alternatively, this construct may be less efficiently translated, since the cDNA clone contains a short open reading frame 5' of the start site that is likely to reduce the level of protein expression. Similar false starts are found at the 5' end of Dif cDNA clones, and these have been suggested to possibly serve a translational regulation function (Dushay, 1996).
Infection of Drosophila by Gram-negative bacteria triggers a signal transduction pathway (the IMD pathway) culminating in the expression of genes encoding antimicrobial peptides. A key component in this pathway is a Drosophila IkappaB kinase (DmIKK) complex, which stimulates the cleavage and activation of the NF-kappaB transcription factor Relish. Activation of the DmIKK complex requires the MAP3K dTAK1, but the mechanism of dTAK1 activation is not understood. In human cells, the activation of TAK1 and IKK requires the human ubiquitin-conjugating enzymes Ubc13 and UEV1a. This study demonstrates that the Drosophila homologs of Ubc13, (Bendless) and UEV1a, are similarly required for the activation of dTAK1 and the DmIKK complex. Surprisingly, the Drosophila caspase DREDD and its partner dFADD are required for the activation of DmIKK and JNK, in addition to their role in Relish cleavage. These studies reveal an evolutionarily conserved role of ubiquitination in IKK activation, and provide new insights into the hierarchy of signaling components in the Drosophila antibacterial immunity pathway (Zhou, 2005).
A cell culture system was established to study the IMD and Toll signaling pathways in S2 cells. The IMD pathway is activated by treating the cells with Gram-negative peptidoglycan (which is present in crude preparations of lipopolysaccharides), whereas activation of the Toll pathway is achieved by treating the cells with the Spätzle ligand. Active Spätzle is produced from a cell line stably transfected with a plasmid containing the copper-inducible metallothionein promoter driving the expression of active Spätzle C-106. When these cells are treated with copper, active SPZ is secreted into the medium, and this conditioned media can be used to activate naïve cells. Using the RNAi-mediated gene inactivation method, it was found that SPZ-induced Drosomycin gene activation in S2 cells requires the Drosophila Rel proteins Dif and Dorsal, as well as Toll, dMyD88, Tube, Pelle, and Slimb, as expected. In sharp contrast, these dsRNAs do not block the expression of antimicrobial peptide genes induced by peptidoglycan. Instead, RNAi studies demonstrate that peptidoglycan-induced gene expression requires all known components of the IMD pathway. Therefore this RNAi approach was used to determine the roles of candidate signaling components in the IMD and Toll signaling pathways (Zhou, 2005).
Previous studies have shown that activation of the mammalian IKK complex requires a ubiquitination step. In particular, TRAF6-mediated IKK activation was shown to require a dimeric ubiquitin-conjugating enzyme composed of the Ubc13 and UEV1a proteins. Bendless and dUEV1a are the Drosophila homologs of Ubc13 and UEV1a, respectively. Bendless and dUEV1a, like their mammalian counterparts, associate with each other in vivo. To investigate whether Bendless and dUEV1a are required for antibacterial gene expression in response to peptidoglycan, the RNAi-mediated gene inactivation method was used. S2 cells were transfected with various dsRNAs. After 48 h, cells were first treated with 20-hydroxy-ecdysone for 24 h to enhance their competence to induce antimicrobial genes in response to immune challenge, and then treated with peptidoglycan or SPZ to activate the IMD or Toll signaling pathways, respectively. Total RNA was isolated from these cells and subjected to Northern blotting analysis using cDNA fragments corresponding to Diptericin or Drosomycin as probes, to examine the activation of the IMD and Toll pathways, respectively. Both Bendless and dUEV1a are required for maximal levels of antibacterial peptide gene expression in response to peptidoglycan treatment. In fact, when both Bendless and dUEV1a are both targeted by RNAi, Diptericin induction is reduced to near background levels. (The partial effect of Bendless or dUEV1a RNAi alone is likely because of the fact that RNAi often does not generate a complete null phenotype.) By contrast, the induction of Drosomycin by Toll activation is unaffected by the RNAi-mediated knock-down of Bendless and dUEV1a. Note that the same cells were stimulated with either peptidoglycan or SPZ in. As a control, S2 cells treated with DmIKKgamma or Toll dsRNA showed significantly reduced peptidoglycan-induced Diptericin or SPZ-induced Drosomycin gene expression, respectively. As a control, mRNA and/or protein levels of targeted genes were examined by RT-PCR and/or Western blotting to confirm the effectiveness of RNAi (Zhou, 2005).
To provide further evidence that Bendless is involved in the IMD pathway, a dominant negative mutant Bendless was used to determine whether peptidoglycan-induced antibacterial gene activation can be blocked. Stable S2 cell lines were generated that express either wild-type or C87A Bendless under the control of the metallothionein promoter. The cysteine to alanine mutation at position 87 creates a dominant-negative mutant because this residue, located in the catalytic pocket in ubiquitin-conjugating enzymes, is crucial for the catalytic activity of E2s. These cells were then treated with various combinations of peptidoglycan and copper, and Northern blotting was employed to examine the expression of antibacterial genes including Attacin, Cecropin and Diptericin. Overexpression of wild-type Bendless has no effect on peptidoglycan-induced antibacterial gene activation, since similar levels of antibacterial peptide gene expression were detected in cells treated with or without copper. In contrast, overexpression of Bendless C87A leads to a significant reduction in peptidoglycan-activated expression of antibacterial peptide genes (Zhou, 2005). Bendless flies have been identified which carry a proline to serine substitution at position 97 within the strictly conserved active site region of E2s. In order to determine whether bendless flies are defective in response to Gram-negative bacterial infection, both wild-type and bendless flies were subjected to E. coli infection and Diptericin gene activation was examined by Northern blotting. bendless flies display significantly weaker Diptericin gene activation compared with wild-type flies. These results indicate that the Bendless-dUEV1a E2 complex is required for signaling by the IMD pathway (Zhou, 2005).
Experiments were carried out to determine whether Bendless and dUEV1a are required for peptidoglycan-induced activation of the DmIKK complex. Previous studies have shown that peptidoglycan treatment induces the kinase activity of the endogenous DmIKK complex in S2 cells. Cells were transfected with dsRNAs corresponding to various mRNAs. After 48 h, these cells were treated with peptidoglycan for 15 min, and the endogenous DmIKK complex was immunoprecipitated and subjected to in vitro kinase assays using recombinant Relish protein as substrate. Bendless or dUEV1a dsRNA treatment leads to a significant decrease in peptidoglycan-induced DmIKK kinase activity, suggesting that Bendless and dUEV1a are required for peptidoglycan-induced DmIKK activation. As a control, DmIKKgamma dsRNA treatment completely abolished peptidoglycan-induced DmIKK activation. It is concluded that the ubiquitin-conjugating enzymes Bendless and dUEV1a are specifically involved in the Drosophila IMD pathway, and they play a role upstream of the DmIKK complex (Zhou, 2005).
Overexpression of IMD in Drosophila results in the activation of antibacterial genes in the absence of bacterial infection. IMD overexpression, under control of the copper-inducible metallothionein promoter, can also strongly activate expression of the Diptericin gene in S2 cells. This stable cell line therefore provides a useful tool to perform an epistatic analysis to determine the position of Bendless/dUEV1a complex relative to IMD in the Drosophila antibacterial signaling pathway. The IMD stable cells were first transfected with dsRNAs derived from various genes and then stimulated with copper or peptidoglycan, and IMD- and peptidoglycan-induced Diptericin gene activation was examined. Overexpression of IMD, via the addition of copper, leads to strong activation of the Diptericin gene. In fact, copper-induced IMD expression is as potent as peptidoglycan in driving diptericin expression. Cells transfected with LacZ dsRNA show a similar Diptericin expression profile compared with cells that were mock-treated. Consistent with the observation that the DmIKK complex functions downstream of IMD, cells transfected with DmIKKgamma dsRNA are severely defective in both IMD- and peptidoglycan-induced Diptericin expression. Furthermore, cells treated with dsRNAs derived from Bendless and dUEV1a genes display a significant reduction in both peptidoglycan- and IMD-mediated Diptericin gene activation. These results indicate that the ubiquitin conjugating enzymes Bendless and dUEV1a function downstream of IMD in this signaling pathway (Zhou, 2005).
Thus, by differentially activating the IMD and Toll signaling pathways in Drosophila S2 cells, this study shows that Bendless (dUbc13) and dUEV1a are required for the Drosophila IMD signaling pathway. Using RNAi to target Bendless and/or dUEV1a significantly reduces the levels of peptidoglycan-induced antibacterial peptide gene expression and activation of the Drosophila IKK complex. This mechanism of IKK activation is highly conserved; in mammals Ubc13 and UEV1a are required for TNFalpha-, IL-1β-, and TCR-mediated IKK and NF-kappaB activation (Zhou, 2005).
This ubiquitin-dependent kinase activation does not involve proteasome-mediated degradation. Proteasome inhibitors do not block IKK activation, in flies or humans. Moreover, ubiquitination without degradation has been shown to activate the human IKK complex, and a similar Drosophila activity has been identified, The primary sequence of the mammalian Ubc13/UEV1a and those of the Drosophila Bendless/dUEV1a are highly conserved (90% similarity for Ubc13, 79% for UEV1a). The crystal structures of the yeast and human Ubc13/UEV1a (Mms2) have shown clearly that this E2 complex can make only K63-linked polyubiquitin chains. Thus, it is likely that the Drosophila Bendless/dUEV1a E2 catalyzes the formation of K63-linked ubiquitin chains (Zhou, 2005).
In a cell-free system, human TRAF6 was shown to be an E3 ligase that auto-ubiquitinates in conjunction with Ubc13/UEV1a. This results in the activation of TRAF6 and, in turn, the activation of TAK1. Activated TAK1 phosphorylates key serine residues in the activation loop of IKKβ, resulting in the activation of IKKβ. It is suspected that similar mechanisms are involved in the Drosophila IMD pathway. This study demonstrates that the Drosophila TAK1 homolog functions downstream of Bendless and dUEV1a. Furthermore, a Drosophila homolog of TAB2/TAB3 is also required for the IMD pathway. Interestingly, the C-terminal zinc finger domain of TAB2, which is conserved in the Drosophila protein, has recently been shown to bind specifically to K63-polyubiquitin chains. Strikingly, it has found that a galere/dTAB2 mutant, which is defective in the IMD pathway, carries a mutation in this zinc finger domain (Zhou, 2005).
The Drosophila E3 ligase, analogous to human TRAF2 or TRAF6, which functions with Bendless and dUEV1a in the activation of dTAK1 and DmIKK remains to be identified. In Drosophila, the dTRAF2 protein is the closest homolog of mammalian TRAF6, and it is the only Drosophila TRAF protein that contains the RING domain, typical of E3 ligases. However, RNAi knockdown and dominant-negative studies suggest that dTRAF2 is not involved in either the IMD or the Toll signaling pathways in S2 cells. In fact this gene is expressed at undetectably low levels in S2 cells. In one previous study dTRAF2 was reported to interact physically and functionally with Pelle, a key signaling component in the Toll signaling pathway that controls the antifungal immune response. However, these studies were based on overexpression experiments and in vitro binding assays, which might not reflect the physiological role of dTRAF2. In contrast, a recent publication demonstrated that dTRAF2 mutants are not fully able to induce antimicrobial peptide genes following E. coli infection. However, these studies did not clearly determine whether dTRAF2 is involved in the Toll or IMD pathways. The data suggest that dTRAF2 is not a critical component of the IMD pathway in S2 cells. Further studies, in cells and in flies, are necessary to elucidate the role of dTRAF2 in Drosophila immunity (Zhou, 2005).
The possibility is considered that other Drosophila RING-containing proteins might be involved in Drosophila immunity. However, RNAi knockdown studies with 10 different RING domain-encoding genes failed to block either the Toll or IMD pathways. Finally, since the structure of the RING domain of Rad5 has been successfully modeled to fit into the structure of the dimeric ubiquitin-conjugating enzyme complex Ubc13/UEV1a, it was reasoned that the potential ubiquitin ligase involved in the IMD pathway might physically interact with Bendless and dUEV1a. Therefore yeast two-hybrid screens were performed using Bendless and dUEV1a as baits in an effort to identify their protein interaction partners. A Drosophila RING protein, CG14435, was identified in such screens. CG14435 interacts robustly with both Bendless and dUEV1a in yeast two-hybrid assays. Furthermore, the CG14435-Bendless and CG14435-dUEV1a interaction was confirmed by co-immunoprecipitation of overexpressed proteins in S2 cells. However, RNAi-based studies suggest that CG14435 is not involved in the Drosophila innate immunity signaling pathways. It has been shown that bendless flies display defective synaptic connectivity and abnormal morphology within the visual system, suggesting Bendless functions in a variety of developmental processes. In addition, the Saccharomyces cerevisiae homologs of Bendless and dUEV1a have been implicated in DNA damage repair. Therefore it is possible that CG14435 is involved in some cellular processes other than immunity which require Bendless and dUEV1a. Further studies are necessary to elucidate the physiological role of CG14435 and to identify the ubiquitin ligase activity required for ubiquitination-dependent DmIKK activation (Zhou, 2005).
As in the TRAF6 pathway, dTRAF2 and/or other E3 ligases that function with Bendless and dUEV1a in the IMD pathway may be the target(s) of K63 polyubiquitination. Another possible target of Bendless/dUEV1a-mediated ubiquitination is the Drosophila IKKgamma subunit (also known as NEMO in mammals) of the IKK complex. In mammals, it has recently been shown that NEMO is K63 polyubiquitinated by the Ubc13/UEV1a complex in response to Bcl10 expression or T-cell activation. Other possible targets of ubiquitination by Bendless and dUEV1a in the IMD pathway include the Drosophila TAB2 homolog and IMD. Recently, it was shown that the two mammalian homologs of TAB2 and TAB3 were ubiquitinated or associated with other ubiquitinated proteins. In addition, the mammalian RIP1, which is homologous to IMD protein especially in its death domain, has recently been shown to be K63 polyubiquitinated and associated with TAB2 in a TNFalpha-dependent manner. In any case, K63 polyubiquitin chains likely function to recruit the Drosophila TAK1/TAB2 complex, via the TAB2 K63 polyubiquitin binding domain, to either (or both) the upstream activators, such as IMD, and/or the downstream target of dTAK1 kinase activity, the Drosophila IKK complex (Zhou, 2005).
As expected, the epistatic analyses presented in this study demonstrate that IMD functions upstream of all other components in the pathway except the receptor PGRP-LC, and is required for IKK activation. Moreover, Bendless and dUEV1a function downstream of IMD and upstream of dTAK1, as predicted from the model for Ubc13 and UEV1a in mammals. dTAK1 is required for activation of the Drosophila IKK complex and likely functions as the IKK kinase (Zhou, 2005).
Although it is established that dFADD and DREDD are required for the IMD pathway, previous experiments have suggested that they function downstream of the DmIKK complex. For example, DREDD overexpression in flies leads to Diptericin gene expression in the absence of Gram-negative bacterial infection, and DREDD-mediated Diptericin gene activation requires neither the DmIKK complex nor dFADD. Also, recent studies have shown that DREDD interacts with Relish, and that a caspase-cleavage site within Relish is required for peptidoglycan-induced Relish activation. Therefore, it was speculated that DREDD functions downstream of the DmIKK complex by directly cleaving DmIKK-phosphorylated Relish. This possibility is consistent with the observation that DREDD and dFADD are required for dTAK1Delta-mediated Diptericin gene activation. Surprisingly, dFADD and DREDD are required for peptidoglycan-induced DmIKK activation, arguing that DREDD and dFADD function upstream in the pathway. In addition, DREDD is also required for peptidoglycan-induced JNK activation, but neither DREDD nor dFADD are required for dTAK1- or dTAK1Delta-mediated DmIKK activation, suggesting that dFADD and DREDD act at a step upstream of dTAK1 in response to peptidoglycan. Based on these observations, it is proposed that dFADD and DREDD play dual roles in the Drosophila antibacterial signaling pathway. On the one hand, dFADD transduces signals from IMD to DREDD, resulting in DREDD activation and enabling Relish cleavage; in contrast, dFADD and DREDD contribute to peptidoglycan-induced DmIKK activation through a mechanism that remains to be elucidated. DREDD may function similarly to human Caspase-8, which is a DED-containing apical caspase similar to DREDD. Caspase-8 has recently been shown to be required for NF-kappaB activation in response TCR-signaling. This role of Caspase-8 requires the enzymatic activity of full-length protein Caspase-8 and is involved in recruiting the IKK complex to the upstream signaling complex of CARMA1, Bcl10, and MALT1. Interestingly MALT1 is also a caspase-like gene (sometimes referred to as a paracaspase), and it is thought to function as an E3-ligase accessory factor with Ubc13 and UEV1a in TCR-mediated NF-kappaB activation. DREDD may similarly function as E3-ligase accessory factors with Bendless and dUEV1a as the E2, in the IMD pathway (Zhou, 2005).
The following scheme is proposed for the Drosophila antibacterial signaling pathway. Peptidoglycan treatment or Gram-negative bacterial infection leads to the activation of the membrane-bound peptidoglycan-recognition protein, PGRP-LC. Activated PGRP-LC in turn transduces signal to IMD. IMD, in turn, interacts with dFADD and subsequently DREDD. It is proposed that IMD, dFADD and DREDD form a complex that contributes to dTAK1 activation, perhaps as part of an E3 ligase. This complex is likely to function in conjunction with Bendless/dUEV1a to activate dTAK1 and then DmIKK. Once activated, the Drosophila IKK complex phosphorylates Relish, which is subsequently cleaved. The N-terminal Relish cleavage product, an NF-kappaB transcription factor, then translocates to the nucleus where it activates antimicrobial peptide gene expression. In addition to their role in IKK activation, DREDD and dFADD are also proposed to function downstream in this pathway, in the signal-induced cleavage of phospho-Relish (Zhou, 2005).
The founding member of the Inhibitor of Apoptosis (IAPs) protein family was originally identified as a cell death inhibitor. However, recent evidence suggest that IAPs are multifunctional signalling devices that influence diverse biological processes. In order to investigate the in vivo function of Drosophila IAP2 diap2 null alleles were generated. diap2 deficient animals develop normally and are fully viable suggesting that diap2 is dispensable for proper development. However, these animals are 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) signalling cascade, a NF-kappaB-dependent pathway that shares striking similarities with the one of mammalian tumour necrosis factor receptor 1 (TNFR1). diap2 mutant flies fail to activate NF-kappaB-mediated expression of antibacterial peptide genes and, consequently, rapidly succumb to bacterial infection. Genetic epistasis analysis places diap2 downstream of or in parallel to imd, Dredd, Tak1 and Relish. Therefore, DIAP2 functions in host-immune response to Gram-negative bacteria. In contrast, it was found that the Drosophila Traf-family member Traf2 is dispensable to resist Gram-negative bacterial infection. Taken together, these genetic data identify DIAP2 as an essential component of the Imd signalling cascade protecting the organism from infiltrating microbes (Leulier, 2006).
In vivo, DIAP2 is indispensable for Imd-mediated expression of antibacterial peptide genes. Like known mutants of the Imd pathway, flies with a mutation in the diap2 gene fail to induce expression of Attacin-A, Cecropin-A1, Defensin, Diptericin, Drosocin and Metchikowin, and mount an efficient immune reaction in response to infection by Gram-negative bacteria. Consequently, diap2 mutant flies succumb to Gram-negative bacterial infection. In contrast, such flies mount a normal Toll-dependent immune response and are resistant to infection by fungi and Gram-positive bacteria. The diap2 null mutant phenotype, therefore, demonstrates that DIAP2 is an essential component of the Imd pathway. Thus, these data are consistent with recent RNAi studies that have implicated diap2 in the Imd pathway. DIAP2 is a member of the evolutionarily conserved IAP protein family. IAPs are classified by the presence of the Baculovirus IAP Repeat (BIR) domain through which they interact with various 'client' proteins. Genetic analysis of the Drosophila IAP DIAP1 has provided some of the most compelling insights into the in vivo function of this protein family. DIAP1, the first and most extensively studied Drosophila IAP, is essential for cell survival and acts as a potent caspase inhibitor. Mutations that abrogate physical association of DIAP1 with caspases cause widespread and unrestrained caspase activation leading to cell and organismal death. In contrast to diap1, diap2 null-mutants do not show an apparent cell death phenotype and develop normally. This is unexpected because both these IAPs interact with caspases and IAP-antagonists with similar affinities. Moreover, when overexpressed, DIAP2 can rescue diap1-RNAi-mediated apoptosis, suggesting that DIAP2 can functionally substitute for DIAP1 in its ability to regulate caspases. Nevertheless, diap2 mutant animals do not show any apparent apoptosis related phenotypes during development. However, these animals appear to be sensitised to Reaper-mediated killing in the eye. The lack of any apparent gross developmental phenotype may be due to sufficiently high levels of DIAP1 protein that may thwart unscheduled caspase activation in response to loss of DIAP2. In this respect it is noteworthy that during embryonic development the levels of diap1 mRNA dramatically exceed those of diap2 (17 fold difference). Moreover, similarly to cIAP2 knock-out mice, where a cell death phenotype is revealed only after LPS challenge, phenotypic manifestation may only become apparent under certain conditions or in selective tissues. In agreement with this notion, RNAi-mediated depletion of DIAP2 has no effect on cell viability in unchallenged tissue culture cells, but significantly sensitizes S2 cells to stress-induced apoptosis (Leulier, 2006).
Although IAPs have originally been identified as apoptosis inhibitors, recent evidence suggests that IAPs are multifunctional signalling devices that, depending on the protein they interact with, influence diverse biological processes. In this respect, it is noteworthy that IAPs also carry C-terminal RING finger domains providing them with E3 ubiquitin-protein ligase, and hence, signalling activity. Thus, in addition to inhibiting apoptosis, IAPs also fulfil functions that operate independently of their ability to control caspases and cell death. Therefore, BIR-containing proteins are more precisely referred to as BIRCs rather than IAPs. Consistent with the notion that BIRCs are multifunctional proteins, the mammalian c-IAP1 and c-IAP2 bind to caspases as well as RIP1 and TRAF320 2, two components of the TNF receptor signalling complex. c-IAP1 or c-IAP2, or both, can promote ubiquitylation and degradation of TRAF2, RIP1 and NF-kappaB kinase (IKKgamma)/NF-kappaB essential modulator (NEMO). Hence, these BIRC proteins are thought to modulate the response to TNF. More recently, another BIRC protein was identified as a important regulator of innate immune surveillance in mammals. BIRC1e (NAIP5) was found to control the intracellular pathogen Legionella pneumophila, a Gram-negative microbe that causes severe bacterial pneumonia known as Legionnaires' disease. BIRC1e protects infected host macrophages by restricting intracellular replication of this pathogen (Leulier, 2006).
The Drosophila BIRC protein DIAP2 is similarly required for innate immune responses and the resistance to Gram-negative bacterial infection. diap2 null mutants become highly susceptible to Gram-negative bacteria and fail to induce antibacterial peptide gene expression. Intriguingly, the Imd pathway, which is required for antibacterial peptide gene expression in response to Gram-negative microbes, shares significant similarities with the TNFR1 signalling cascade. The notion that the BIRC proteins c-IAP1, c-IAP2 and DIAP2 are core components of the TNFR1- and Imd-pathway, respectively, further reinforces the parallels between the mammalian TNFR1 pathway and the one of Imd of Drosophila, pointing to an evolutionary conservation of these pathways in NF-kappaB activation. Moreover, both pathways seem to rely on ubiquitin-mediated protein modifications. As in human cells, where activation of TAK1 and IKK requires the E2 ubiquitin-conjugating enzyme complex Ubc13/UEV1A, Drosophila Ubc13(Bendless)/UEV1A are similarly required for activating Tak1 and the Drosophila IKK complex. Moreover, recent RNAi data from cultured cells suggest that Drosophila Tab contributes to Imd signalling, although this still awaits in vivo validation. Therefore, similar to the TNFR1 pathway, ubiquitin-mediated protein modification is likely to activate the Tak1/Tab complex via Tab's ability to bind to Lys63-linked polyubiquitin chains, thereby recruiting Tak1 to activator platforms. In contrast to the E2 ubiquitin-conjugating enzyme complex, little is known about the nature of the E3 ubiquitin-ligase of the Imd pathway. While TRAF2 is crucial for Ubc13/UEV1A-mediated ubiquitylation in the mammalian TNFR1 pathway, it seems that for Imd signalling Traf2, the TRAF2/6 orthologue in flies, is not a critical component. Traf2 null mutation did not completely block NF-kappaB activation in Drosophila. Moreover, the data clearly indicate that Traf2 mutant flies are fully competent to mount an immune response that resists Gram-negative bacterial infection. Hence, Traf2 appears not to be essential for an effective Imd-mediated immune response. Since the Drosophila genome encodes at least three TRAF family members, it is possible that the loss of Traf2 function is complemented by other TRAF family members. Alternatively, other signalling pathways that bypass Traf2 to transmit the infection signal to NF-kappaB may exist in Drosophila. In agreement with this notion, RNAi-mediated knock-down of all three Drosophila TRAFs also did not abrogate Imd signalling in S2 cells. Thus, an E3 ubiquitin ligase different to, or in addition of Traf2 may be responsible for Imd signalling in Drosophila. Since DIAP2 carries a RING finger domain, it represents a likely candidate (Leulier, 2006).
The genetic epistatic analysis places diap2 downstream or in parallel of imd, Dredd, Tak1 and Relish. Over-expression of imd, Dredd, Tak1 and Relish failed to induce Diptericin expression in diap2 mutant animals; in wild-type animals, enforced expression of these genes (in the absence of any infection) resulted in reproducible Diptericin induction. Intriguingly, Diptericin induction following enforced expression of imd and Dredd is also blocked in Tak1 mutant animals, indicating that both DIAP2 and Tak1 are required downstream of Dredd. In contrast, kenny and ird5 seem not to be required for Diptericin induction when Dredd is over-expressed. A recent report indicates that Relish cleavage and nuclear translocation on its own is not sufficient for Diptericin expression, and that, at least in vivo, a further cooperative input from the JNK signalling pathway is required. According to this scenario the Imd signalling pathway bifurcates at the level of Tak1, with Tak1 activating the NF-kappaB as well as JNK signalling branch, both of which are required for expression of antibacterial peptide genes in the fat body. In light of this model, the observation that diap2 acts genetically downstream of imd, Dredd, Tak1 and Relish may indicate that DIAP2 functions at the level of Tak1. This view is in agreement with recent reports from Drosophila tissue culture cells, which suggest that DIAP2 is required for Tak1-mediated JNK activation. In this respect, DIAP2 functions at the same epistatic position as the putative E3 ubiquitin ligase of the Imd pathway. Future biochemical experiments will be required to test whether DIAP2 is indeed the E3 ubiquitin ligase that functions together with Ubc13/UEV1A to stimulate Tak1 (Leulier, 2006).
Although the underlying mechanism for the impaired induction of antibacterial peptide gene expression by loss of DIAP2 remains to be defined, the genetic observations made in this study are likely to have relevance not only for innate immune responses in Drosophila but also for TNFR1 signalling in mammals. While in flies DIAP2 is indispensable for Imd signalling, genetic studies in mice have, so far, failed to uncover a physiological role for c-IAP1 and c-IAP2 in TNFR1 signalling. Since c-iap1 knock out mice carry significantly elevated levels of c-IAP2 protein, it is feasible that the increased c-IAP2 levels functionally compensate for the loss of c-IAP1. Consistently, mammalian IAPs have been reported to be under strict homeostatic control by regulating each other's protein levels, which provides a mechanistic explanation for the crosstalk among IAPs. Thus, in mammals, double knock out mice lacking both c-iap1 and c-iap2 genes will be required to study the role of c-IAP1 and c-IAP2 in TNFR1 signalling. Therefore, Drosophila, where redundancies and compensatory mechanisms are less problematic, provides an ideal model system to study caspase-independent functions of IAPs in an in vivo setting (Leulier, 2006).
The Rel/NF-kappaB transcription factor Relish plays a key role in the humoral immune response in Drosophila. Activation of this innate immune response is preceded by rapid proteolytic cleavage of Relish into two parts. The proteolytic cleavage of Relish depends on Death related ced-3/Nedd2-like protein (Dredd). 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 IkappaB-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 (Stöven, 2000).
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. In a screen for factors that negatively regulate the IMD pathway, two partial loss-of-function mutations were isolated 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 Cullin1, 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. It is concluded that 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 (Khush, 2002).
In wild-type flies Diptericin is tightly controlled by the IMD pathway. Therefore, to identify genes that normally function to repress the IMD pathway, 2,000 yellow, white (y,w) F1 male progeny from male flies mutagenized with ethyl methanesulfonate were screened for constitutive expression of a Green Fluorescent Protein (GFP) reporter gene under the control of the Diptericin promoter. Two males, J6 and G49, expressed Diptericin-GFP, and this gene was constitutively expressed in larvae and adults in homozygous lines derived from these males. Although flies carrying the J6 and G49 mutations are viable and fertile at 25°C, G49 is pupal lethal at 29°C, indicating temperature-sensitive phenotypes associated with this mutation (Khush, 2002).
Using recombination mapping, the J6 and G49 mutations were shown to be tightly linked to the y locus on the proximal tip of the X chromosome. To further localize the two mutations, deletions were used to determine that J6 falls in the area defined by the overlap of Df(1)74k24.1, Df(1)svr, and Df(1)su(s)83, placing it in cytological region 1B10 near the Dredd gene. Two lethal P-element insertions in the Bloomington stock center collection, l(1)G0389 and l(1)G0109, which map near this region, were shown to not complement the constitutive Diptericin expression in the J6 and G49 lines. By sequencing DNA flanking the P elements in the two insertion lines, both elements were ascertained to lie within 200 bp of each other in the 5′ untranslated region of the SkpA gene. To confirm that J6 and G49 are mutations in SkpA, a wild-type SkpA transgene on the second chromosome was shown to suppress the constitutive Diptericin expression phenotype in G49 flies. The J6 and G49 lines were shown to each contain a point mutation in the SkpA gene that generates a single amino acid change in the SkpA protein: J6, renamed SkpAJ6, converts threonine 98 to an isoleucine, and G49, renamed SkpAG49, replaces glutamic acid 101 with a lysine. These alleles are hypomorphic mutations of SkpA since the P-element insertions are pupal lethal at 25°C. SkpAG49 is pupal lethal at 29°C, and homozygous SkpAG49 adults transferred to 29°C express Diptericin at similar levels as flies heterozygous for SkpAG49 and either the P-element insertions or deletions that remove SkpA. At 29°C, therefore, SkpAG49 behaves like a null mutation, which probably reflects the significant change from the negatively charged glutamic acid to the positively charged lysine in this allele (Khush, 2002).
The SkpA gene encodes a protein that is highly similar to Skp1 proteins in humans and yeast. Skp1 proteins are components of SCF ubiquitin ligases that target substrates to the proteasome, and crystal structures of human Skp1 complexed with the F-box protein Skp2 and the cullin protin Cul1 have been solved. SkpAJ6 and SkpAG49 both affect a conserved region of SkpA that corresponds to helix 5 of Skp1; helix 5 forms part of the core interface between Skp1, the F-box region of Skp2, and the amino-terminal domain of Cul1, with some amino acids in this helix making direct contact with residues in Skp2 and Cul1. This suggests that the SkpAJ6 and SkpAG49 mutations disrupt interactions between SkpA and the F-box protein and cullin components of an SCF complex. Protein interaction studies indicate that SkpA functions with the F-box protein Slimb and the Cullin-like protein Cullin1 (Cul1) in a Drosophila SCF complex. In support of this model, slimb1 and dcul1l(2)02074 mutant larvae, as well as larvae carrying the DTS5 mutation, a dominant-negative mutation that affects the β6 subunit of the 26S proteasome, were shown to express Diptericin at levels comparable to those in the SkpA mutants. To further test the DTS5 phenotype, the UAS-Gal4 system was used to overexpress a UAS-DTS5 transgene in larval fat bodies: DTS5 overexpression induces Diptericin to levels that are comparable to those generated by bacterial infection with Erwinia carotovora carotovora 15 (Ecc15). Flies heterozygous for mutations at both the SkpA and slimb loci were generated: these flies constitutively express Diptericin, indicating a synergistic interaction between SkpA and slimb. The constitutive Diptericin expression in the slimb1, dcul1l(2)02074, and DTS5 mutants and the interaction between SkpA and slimb together suggest that an SCFSkpA/Cul1/Slimb ubiquitin ligase represses Diptericin expression by targeting a regulatory factor for degradation by the 26S proteasome (Khush, 2002).
To determine if the constitutive Diptericin expression in the SCF complex mutants is mediated through the IMD pathway, Diptericin levels were examined in larvae homozygous for mutations in either SkpA, or slimb and various genes of the IMD pathway: SkpAJ6;imd1 and SkpAG49;dtak11 double mutants display constitutive Diptericin expression, although Diptericin levels are slightly reduced in the SkpAG49;dtak11 larvae. Mutations in DmIkkγ, DmIkkβ, and Relish, however, completely block Diptericin expression in the SkpAJ6 background, and a Dredd mutation completely blocks Diptericin expression in the slimb1 background. The constitutive Diptericin expression observed in SkpA and slimb mutants, therefore, does not require IMD and dTak1, but it is dependent on the DmIKK complex, Dredd, and Relish. These results imply that, in wild-type flies, the SCFSkpA/Cul1/Slimb negatively regulates the IMD pathway by targeting one of these factors, or an additional unidentified component of the IMD pathway, for degradation by the proteasome. In contrast to fat body cells, the IMD pathway is the primary regulator of all antimicrobial genes, including Drosomycin, in surface epithelial tissues. A Drosomycin-GFP transgene is constitutively expressed in tracheal cells but not in fat body cells of slimb1 mutant larvae; this expression pattern further demonstrates that the IMD pathway, but not the Toll pathway, is constitutively activated when the SCFSkpA/Cul1/Slimb complex is compromised (Khush, 2002).
Although the genetic results do not allow differentiation between the DmIKK complex, Dredd, Relish, or other unidentified downstream components of the IMD pathway as targets of the ubiquitin-proteasome pathway, the mammalian Relish homolog, P105, is regulated by an SCF complex that contains the Slimb homolog β-TrCP/E3RSIκB. Consequently, RNA-mediated interference (RNAi), an effective technique for specifically inhibiting targeted proteins, was used in cultured Drosophila S2 cells to test for interactions between the SCFSkpA/Cul1/Slimb complex and Relish. Initially, SkpA and Slimb activity were blocked in S2 cells via RNAi; then, transient expression of a full-length Relish protein, modified by an N-terminal FLAG tag, was induced in the same S2 cells and the effects of the SkpA and slimb RNAi treatments on FLAG-Relish protein stability was monitored using Western blots and anti-FLAG antibodies (Khush, 2002).
Reducing Slimb activity, in the absence of LPS stimulation, visibly increases steady-state levels of both full-length Relish and the active N-terminal Rel-homology domain; levels of both polypeptides are further increased by inhibiting Slimb and SkpA simultaneously. This effect is specific since RNAi of the SkpA homologs, SkpB and SkpD, does not increase Relish levels. Dredd RNAi does increase Relish levels at day 1, but this is probably because Dredd inhibition blocks Relish processing. Previous studies show that Relish processing in S2 cells is induced by lipopolysaccharide (LPS) and requires Dredd activity. As expected, therefore, RNAi of Dredd blocks LPS-induced Relish processing. Simultaneous RNAi of SkpA and Slimb in the presence of LPS, however, results in higher steady-state levels of the Rel-homology domain up to 4 days after Relish induction. Higher levels of the Rel-homology domain after SkpA and Slimb RNAi could be caused by increased processing of full-length Relish. However, because full-length Relish levels also mount, the explanation is favored that Rel-homology domain turnover is reduced. Although the Slimb and SkpA RNAi treatments appear to inhibit Relish turnover, Relish levels do eventually diminish. This suggests that RNAi efficiency decreases with time, possibly due to degradation of the transfected double-stranded RNA. These RNAi experiments indicate that the constitutive antimicrobial gene expression in SkpA and slimb mutant flies is caused by higher Relish levels, and they suggest that the SCFSkpA/Cul1/Slimb complex represses the IMD pathway by promoting the degradation of both full-length and processed Relish proteins (Khush, 2002).
If the constitutive antimicrobial gene expression in flies carrying mutations that affect the SCFSkpA/Cul1/Slimb complex or proteasome is due to higher Relish levels, this would imply some level of steady-state Relish activation. Low levels of the Rel-homology domain have been reported in nuclear extracts from unstimulated S2 cells, and these low levels indicate that Relish is constitutively processed. Increasing Relish levels in larvae and adults via the Gal4-UAS system is sufficient to induce low levels of Diptericin expression. These results indicate that Relish is constitutively processed and activated to some level, supporting the hypothesis that Relish activity, in the absence of infection, is countered by ubiquitination and degradation (Khush, 2002).
A Drosophila IkappaB kinase complex containing DmIKKß and DmIKKgamma, homologs of the human IKKß and IKKgamma proteins, has been identified. This complex is required for the signal-dependent cleavage of Relish, a member of the Rel family of transcriptional activator proteins, and for the activation of antibacterial immune response genes. In addition, the activated DmIKK complex, as well as recombinant DmIKKß, can phosphorylate Relish in vitro. Thus, it is proposed that the Drosophila IkappaB kinase complex functions, at least in part, by inducing the proteolytic cleavage of Relish. The N terminus of Relish then translocates to the nucleus and activates the transcription of antibacterial immune response genes. Remarkably, this Drosophila IkappaB kinase complex is not required for the activation of the Rel proteins Dif and Dorsal through the Toll signaling pathway, which is essential for antifungal immunity and dorsoventral patterning during early development. Thus, a yet to be identified IkappaB kinase complex must be required for Rel protein activation via the Toll signaling pathway (Silverman, 2000).
To identify the signaling components required for the Drosophila immune response a reverse genetic approach, taking advantage of the Drosophila Genome Project, was undertaken. A cDNA sequence with homology to the kinase domain of the human IKK genes was identified in the BDGP EST database. Examination of the amino acid sequence of the encoded protein, which has been designated DmIKKß (Drosophila melanogaster IKKß), displays significant similarity to the N-terminal region of hIKKalpha and hIKKß but is more similar to hIKKß. The amino acid sequence of the C terminus of DmIKKß is only weakly related to the corresponding regions of IKKalpha and IKKß. A predicted coiled-coil can be detected in a region corresponding to the predicted leucine zipper coiled-coil of hIKKalpha and hIKKß. The DmIKKß gene maps to chromosomal location 89B as determined with the BDGP P1 filter array (Silverman, 2000).
The mammalian IKKalpha and IKKß proteins are found in a high molecular weight IkappaB kinase complex that includes the structural component IKKgamma or NEMO. To investigate the possibility that DmIKKß is also a component of a similar kinase complex, a yeast two-hybrid screen was performed using DmIKKß as bait. A Drosophila larval cDNA library was screened, and a total of 85 independent positive clones were analyzed. All of these clones were found to contain overlapping inserts from a cDNA that encodes a Drosophila protein with homology to hIKKgamma primarily in its C terminus. This gene, referred to as DmIKKgamma hereafter, maps to chromosomal location 60E as determined with the BDGP P1 filter array. Secondary structure predictions of the protein encoded by DmIKKgamma suggest the existence of several coiled-coil regions. The overall amino acid sequence homology between DmIKKgamma and hIKKgamma suggests a putative structural and functional relationship between the two proteins, especially in their C-terminal halves. DmIKKß and DmIKKgamma interact to form a complex both in vivo and in vitro (Silverman, 2000).
To determine whether DmIKKß and DmIKKgamma are involved in the activation of antibacterial genes, an LPS-inducible Drosophila cell line was engineered to express different versions of these genes. When S2* cells are treated with LPS, the expression of antibacterial peptide genes, such as Diptericin, Cecropin, and Attacin, are induced. Thus, S2* cells were stably transfected with plasmids that express wild-type or potentially dominant negative versions of DmIKKß and DmIKKgamma under the control of the copper-inducible metallothionein promoter. Four cell lines were generated that express one of the following proteins: DmIKKß wild type, DmIKKß K50A, DmIKKgamma wild type, or DmIKKgamma 201-387. DmIKKß K50A was chosen because similar mutations in hIKKalpha or hIKKß, which change a conserved lysine in the ATP binding domain, create dominant negative proteins, whereas the DmIKKgamma201-387 was designed because a similarly truncated hIKKgamma acts as a dominant negative mutant in human cells. Full-length hIKKgamma can also act as a dominant negative when expressed at high levels, presumably by titrating limiting components required for functional complex assembly. DmIKKß and DmIKKgamma interact to form a complex both in vivo and in vitro (Silverman, 2000).
After the addition of copper, DmIKK proteins are rapidly produced at high levels as detected by immunoblotting. To determine whether these DmIKK proteins block LPS induction of antibacterial peptide expression in S2* cells, the stable cell lines were first treated with copper to induce expression of the DmIKKs and then stimulated with LPS. DmIKKß K50A blocks the LPS induction of the Diptericin, Cecropin, and Attacin genes. Similarly, either full-length or truncated versions of DmIKKgamma are potent inhibitors of antibacterial peptide gene expression. These data clearly show that DmIKKß K50A, DmIKKgamma full-length, or DmIKKgamma201-387 potently inhibit Diptericin, Cecropin, and Attacin gene induction. By contrast, the expression of wild-type DmIKKß slightly decreases the level of expression of these genes. It is concluded that DmIKKß and DmIKKgamma are required for the activation of antibacterial gene expression in response to LPS (Silverman, 2000).
Genetic studies have shown that the Drosophila Relish gene is essential for the antibacterial immune response. Bacterial infection or LPS treatment activates the endoproteolytic cleavage of Relish. Once cleaved, the N-terminal RHD of Relish translocates into the nucleus, where it activates the transcription of antibacterial genes. Considering that the overexpression of dominant negative DmIKKß or DmIKKgamma blocks the induction of antibacterial peptide genes, it is likely that DmIKKß (and the DmIKK complex) functions in the signaling pathway leading to the cleavage of Relish. In order to test this possibility, the DmIKK overexpressing cell lines were employed to follow the fate of Relish protein cleavage. The stable cell lines and the parental cells were first treated with copper and then induced with LPS for 15 min. LPS treatment of the parental cells, or any of the stable lines that were not treated with copper, results in Relish cleavage. Full-length Relish (~110 kD) is cleaved to generate fragments of ~68 kD and ~49 kD, corresponding to the N- and C-terminal fragments, respectively. Expression of DmIKKß K50A, DmIKKgamma, or DmIKKgamma201-387 blocks the cleavage of Relish. Overexpression of wild-type DmIKKß causes a slight accumulation of full-length Relish (Silverman, 2000).
To obtain additional evidence that DmIKKß and DmIKKgamma are required for LPS-induced antibacterial gene expression, the inhibitory effect of double-stranded RNA (dsRNA), also referred to as RNAi, was exploited. The presence of gene-specific dsRNA molecules in Drosophila or Caenorhabditis elegans embryos has been shown to destabilize the cognate mRNAs. dsRNA corresponding to the DmIKKgamma or DmIKKß genes was synthesized in vitro and transfected into the LPS-inducible S2* cell line. Transfection of gene-specific dsRNA causes a significant reduction in the amount of the corresponding mRNA. For example, DmIKKgamma dsRNA lowers DmIKKgamma mRNA to nearly undetectable levels, while DmIKKß or LacZ dsRNA has no effect on DmIKKgamma mRNA levels. DmIKKgamma protein levels are also greatly reduced only in those cells transfected with DmIKKgamma dsRNA; however, it is important to note that although DmIKKgamma protein is reduced approximately 10-fold, it is still detectable after RNAi treatment. Importantly, dsRNA-mediated interference of either DmIKKß or DmIKKgamma greatly inhibits the LPS-induced expression of antibacterial genes, such as Attacin, Cecropin, and Diptericin. The LPS-induced cleavage of Relish is also inhibited by either DmIKKß or DmIKKgamma dsRNA. Full-length Relish protein persists after the induction with LPS only in those lanes transfected with DmIKK dsRNA. Although the inhibition of Relish cleavage seen with DmIKK RNAi is not as dramatic as that observed with the dominant negative DmIKKs, it is clear that full-length Relish is not as efficiently cleaved in those cells with reduced levels of DmIKKß or DmIKKgamma. In both the dominant negative and the RNAi experiments, a stronger inhibition is always observed at the transcriptional level, as compared to that seen at the protein level. This suggests that small perturbations in the amount of the nuclear translocated Relish can have dramatic effects on the level of transcriptional activation. Small differences in the amounts of transcription factors have been shown to exhibit all-or-none effects on promoters that require the cooperative assembly of transcription enhancer complexes. dsRNA-mediated interference does not create a null allele. Thus, the reduced amount of Relish cleavage and antibacterial gene expression still observed in the RNAi-treated cells is most likely due to the remaining DmIKKs found in these cells. Both the dominant negative and RNAi experiments show that DmIKKß and DmIKKgamma are required for LPS-induced Relish endoproteolytic cleavage and transcriptional activation of antibacterial peptide genes in vivo (Silverman, 2000).
The Toll signaling pathway, which leads to Cactus degradation and activation of Dif and Dorsal, is necessary for the antifungal immune response as well as early embryonic patterning. The DmIKKß/gamma complex could be involved in the Toll signaling pathway in addition to its role in the antibacterial pathway. To test this possibility directly, the RNAi technique was used with a Drosophila cell line specifically engineered to assay the Toll signaling pathway. Schneider S2* cells were stably transfected with a torso-pelle fusion gene controlled by the metallothionein promoter, creating the S2*tpll cell line. Previous studies have demonstrated that fusing the transmembrane domain of torso to the pelle gene creates a constitutively active pelle kinase that can stimulate the Toll signaling pathway in fly embryos or in tissue culture cells. In the S2*tpll cell line, addition of copper leads to the activation of the Toll antifungal pathway. Drosomycin transcription is induced, but Diptericin transcription is unaffected. Transfection of dsRNA into the S2*tpll cells leads to specific loss of the corresponding mRNA and protein. DmIKKgamma dsRNA causes a substantial reduction in the level of DmIKKgamma protein. However, neither DmIKKß nor DmIKKgamma dsRNA blocks the torso-pelle-mediated activation of Drosomycin transcription. These experiments strongly argue that DmIKKß and DmIKKgamma are not required for the Toll-mediated antifungal pathway but are required for the LPS-induced antibacterial pathway (Silverman, 2000).
The finding that dominant negative DmIKKs or DmIKK RNAi can block Relish cleavage and Relish-dependent gene activation suggests that Relish may be a bona fide target of the DmIKK complex. To test this possibility, experiments were carried out to determine whether DmIKKß or the DmIKK complex can phosphorylate Relish protein in vitro. Flag-tagged Relish was immunoprecipitated with Flag antibodies from a Schneider cell line that expresses very high levels of the epitope-tagged protein. Immunoprecipitated Relish was then used as a substrate with recombinant DmIKKß in a kinase assay using [gamma-32P]ATP. A band the size of Relish was labeled with 32P by the recombinant kinase. As a control, extracts from the parental Schneider cell line, which does not express Flag-Relish, were also used in a Flag immunoprecipitation. When these immunoprecipitates were used as substrates for DmIKKß, phosphorylation of Relish was not observed. To further demonstrate that the phosphorylated band is Relish, a similar experiment was performed using anti-Relish antibodies, instead of Flag antibodies, to precipitate Relish. Again, a band corresponding to Relish was phosphorylated in a DmIKKß-dependent manner. It is concluded that DmIKKß can directly phosphorylate Relish (Silverman, 2000).
To determine whether this DmIKKß phosphorylation of Relish is specific, the ability of other, related kinases to phosphorylate Relish was tested. For these experiments, the specificity of DmIKKß was compared to that of recombinant human IKKß and IKKepsilon. While DmIKKß readily phosphorylates Relish, phosphorylation was not observed with either hIKKß or hIKKepsilon. When these same recombinant human IKKs were used in a GST-IkappaBalpha kinase assay, they were both active. Thus, the ability to phosphorylate Relish is not shared between the Drosophila and human IkappaB kinases. Preliminary experiments have shown that the DmIKKß can phosphorylate the linker region between the RHD and the Ankyrin domain. Surprisingly, DmIKKß can also phosphorylate Cactus and IkappaBalpha, and this phosphorylation occurs within the N-terminal regulatory domain, which is required for signal-dependent degradation by the proteasome. Thus, the ability to specifically phosphorylate IkappaB proteins is shared by the Drosophila and human IkappaB kinases, whereas only the Drosophila kinase can phosphorylate Relish. The mammalian IkappaB kinase complex has been shown to phosphorylate p105; however, this phosphorylation leads to its degradation rather than its processing (Silverman, 2000).
To test the possibility that the activated DmIKK complex can phosphorylate Relish, an immunocomplex kinase assay was performed. First, the DmIKK complex was precipitated from an LPS-inducible Schneider cell line using an anti-DmIKKgamma antibody. This antibody recognizes the endogenous DmIKKgamma that is expressed in this cell line. Although the anti-DmIKKß antibodies are less sensitive (DmIKKß cannot be detected in crude extracts), the DmIKKß protein can be detected after immunoprecipitation with anti-DmIKKgamma antibodies. Immunoprecipitation experiments were performed with extracts prepared from cells treated with LPS or left untreated; no differences were detected, with or without LPS, in the levels of DmIKKß or DmIKKgamma expressed and precipitated. However, LPS causes a specific increase in the level of Relish kinase activity that was detected in the immunoprecipitated DmIKK complex. Thus, the DmIKK complex that is precipitated with anti-DmIKKgamma antibodies contains an LPS-inducible Relish kinase activity. This observation supports the view that DmIKKß and DmIKKgamma form (part of) an LPS-inducible kinase complex that phosphorylates Relish, activating its cleavage in response to LPS (Silverman, 2000).
Loss-of-function mutants in the Drosophila homolog of the mammalian I-kappa B kinase (IKK) complex component IKK gamma have been generated. Drosophila IKK gamma is required for the Relish-dependent immune induction of the genes encoding antibacterial peptides and for resistance to infections by Escherichia coli. However, it is not required for the Toll-DIF-dependent antifungal host defense. The results indicate distinct control mechanisms of the Rel-like transactivators DIF and Relish in the Drosophila innate immune response and show that Drosophila Toll does not signal through a IKK gamma-dependent signaling complex. Thus, in contrast to the vertebrate inflammatory response, IKK gamma is required for the activation of only one immune signaling pathway in Drosophila (Rutschmann, 2000).
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 IkappaB kinases (IKKs). The ird5 phenotype and sequence suggest that the gene is specifically required for the activation of Relish, a Drosophila NF-kappaB family member (Lu, 2001).
Mutations in ird5 prevent induction of a diptericin-lacZ reporter gene in response to infection and also prevent transcriptional induction of the endogenous diptericin gene. E. coli-induced expression of all seven classes of antimicrobial peptide genes in ird5 mutant larvae were examined by RNA blot hybridization, including the genes encoding antibacterial (Diptericin, Cecropin A, Defensin, Attacin, Drosocin, and Metchnikowin) and antifungal (Drosomycin) peptides. In wild-type larvae, all the antimicrobial genes are strongly induced after bacterial challenge. In contrast, in larvae homozygous for either ird5 allele, there is no detectable induction of the diptericin, cecropin A, defensin, drosocin, or metchnikowin genes. The attacin gene is induced in the mutants to ~30% of normal levels, while drosomycin is induced to normal levels. The same effects on the induction of antimicrobial peptide genes are seen in ird51/Df and ird52/Df animals, suggesting that both alleles cause a complete loss of gene function (Lu, 2001).
Mutations in three other genes, imd, Relish, and Death related ced-3/Nedd2-like protein, have been shown to prevent normal induction of antibacterial peptide genes in adult Drosophila. The pattern of antimicrobial peptide gene induction in ird5 mutants was compared with that in imd and Relish mutants in both larvae and adults. Mutations in all three genes have very similar effects on antimicrobial gene induction in larvae: diptericin and cecropin A are not induced; attacin induction is reduced and drosomycin induction is normal. In adult animals, the antimicrobial gene expression phenotypes of ird5 and Relish mutants are very similar: diptericin induction is blocked, cecropin A and attacin induction is reduced, and drosomycin induction is normal. The antimicrobial gene expression phenotype of imd adults is slightly different, with some residual diptericin expression. Mutations in Dredd, a Drosophila caspase, prevent normal induction of diptericin and attacin and allow induction of drosomycin. These comparisons suggest that ird5, Dredd, Relish, and probably imd act in the same signaling pathway to control the induction of antibacterial peptide genes in response to infection (Lu, 2001).
To assess the importance of the ird5 gene in controlling the growth of invading bacteria, bacterial survival and growth were compared in wild-type, ird5, imd, and Relish animals. In wild-type larvae, most of the E. coli injected into the animal are killed by 6 h after infection. At this same time point, there are four to 15 times as many E. coli in ird5 mutant larvae as in wild-type animals. The effects of the ird5 mutations are more striking in experiments with adults: at 24 h after infection, there are 20-350 times as many bacteria per animal in ird5 mutants compared to wild type. The bacterial growth phenotype of ird5 mutants is similar to that seen in Relish mutant larvae and adults and somewhat stronger than that of imd mutants. This is consistent with the stronger effects of ird5 and Relish on the antimicrobial peptide genes: Mutations in either ird5 or Relish prevent normal induction of diptericin, cecropin, drosocin, attacin, and metchnikowin, while induction of metchnikowin is induced in imd mutants (Lu, 2001).
The mutations responsible for the failure to induce the diptericin-lacZ reporter gene for both ird5 alleles were mapped between the visible markers cu (86D1-4) and sr (90D2-F7) on the right arm of the third chromosome. The deficiency Df(3R)sbd45(89B4-10) fails to complement the immune response defect of either ird5 allele. Further deficiency-complementation tests and male recombination mapping narrowed the ird5 interval to 89B4-9, between pannier and Stubble. Two molecular-defined genes in this interval were considered as candidates responsible for the ird5 phenotype, Akt and a gene defined by an EST that is related to mammalian IkappaB kinases (IKKs). Mutant alleles of Akt cause recessive lethality, and ird5/Akt[l(3)89Bq] heterozygous animals are viable and showed normal induction of the diptericin-lacZ reporter gene, indicating that the ird5 phenotypes are not caused by mutations in Akt (Lu, 2001).
Mammalian IKKbeta is required for activation of NF-kappaB in response to inflammatory signals such as TNF-alpha and IL-1; the IKK homolog was therefore considered as a candidate gene for ird5. A full-length cDNA was cloned for the IKK homolog, termed here DmIkkbeta. The same gene has also identified molecularly as encoding a kinase activated by LPS in a Drosophila cell line (Kim, 2000; Medzhitov, 2000; Silverman, 2000). Based on genomic DNA sequence, DmIkkbeta is located between pannier and mini-spindles. Two size classes of transcripts, 2.7 and 4.2 kb, were detected from the DmIkkbeta gene; the cDNA corresponds to the 2.7-kb transcript. Both transcripts are expressed at higher levels after infection. The complete open reading frame of DmIkkbeta was sequenced from the ird51 and ird52 chromosomes. A single C-to-T nucleotide substitution was found in ird51 that would change a glutamine codon (CAA) at amino acid 266 of the open reading frame to a stop codon (TAA) within the conserved kinase domain. No sequence changes were identified in the open reading frame in ird52; however, neither DmIkkbeta transcript was detectable in ird52 homozygotes. This analysis indicates that both ird5 alleles are associated with mutations that should abolish DmIkkbeta activity (Lu, 2001).
The ird5 immune response phenotype shows striking specificity: all of the antibacterial peptide genes are strongly affected by the ird5 mutations, but the antifungal peptide gene drosomycin is induced normally in ird5 mutants. The specific immune response phenotype of ird5/DmIkkbeta in vivo contrasts with the global effects on antimicrobial peptide genes seen in cell lines when a dominant negative form of the same gene is expressed in cultured cells (Kim, 2000). The ird5/DmIkkbeta mutant phenotype implies that, in vivo, ird5 is not an essential component of the Toll pathway, which is required for the induction of drosomycin. The ird5/DmIkkbeta gene is therefore a component of an independent signaling pathway, which could be activated by another member of the Drosophila Toll-like receptor family (Lu, 2001).
Mammalian IKKalpha and IKKbeta phosphorylate serine residues in the N-terminal domain of IkappaB; these serine residues target IkappaB for degradation, thereby allowing the nuclear localization and activation of NF-kappaB. The ird5/DmIkkbeta sequence suggests that the protein encoded by this gene phosphorylates an IkappaB-like protein. There are two known Drosophila IkappaB-like proteins that could act as inhibitor proteins in the immune response: Cactus, and the C-terminal ankyrin repeat domain of Relish. In cactus mutants, drosomycin is expressed constitutively, but the antibacterial peptide genes are not, which indicates that Cactus is not involved in the pathways that regulate the antibacterial peptide genes. Furthermore, ird5/DmIkkbeta homozygous mutant females are fertile, demonstrating that this gene is not required for degradation of Cactus during dorsal-ventral patterning in the embryo (Lu, 2001).
The ird5/DmIkkbeta phenotype is similar to the phenotype of Relish mutants. For both genes, homozygous mutant flies are viable and fertile, indicating that the two genes are not essential for development. Mutations in either Relish or ird5/DmIkkbeta completely prevent induction of diptericin and cecropin but allow some induction of attacin and drosomycin. Mutations in either gene produce comparable effects on bacterial growth. These results argue that ird5/DmIkkbeta and Relish act in the same signaling pathway and suggest that Ird5/DmIkkbeta activates Relish-containing dimers. Relish activation requires proteolytic cleavage of Relish protein into an N-terminal Rel domain that translocates to the nucleus and a C-terminal ankyrin repeat domain that remains in the cytoplasm. Recent biochemical experiments have shown that DmIkkbeta can phosphorylate Relish protein (Silverman, 2000), which is consistent with the model that phosphorylation of Relish by DmIkkbeta leads to targeted proteolysis and activation of Relish (Lu, 2001).
Although ird5/DmIkkbeta is expressed maternally, ird5 mutant females are fertile, demonstrating that the gene is not required for embryonic dorsal-ventral patterning. However, a small fraction of embryos (~0.5%) produced by homozygous ird51 or ird51/ird52 females show a weakly dorsalized phenotype, suggesting that ird5/DmIkkbeta does have a minor role in the maternal pathway that activates Dorsal. It is suggested that there is another kinase in the early embryo that is primarily responsible for phosphorylation and degradation of Cactus. The normal induction of drosomycin in ird5/DmIkkbeta mutants suggests that there will also be another kinase activated by the Toll pathway in the immune response -- perhaps the same kinase that acts downstream of Toll to activate Dorsal in the embryo. The genome sequence indicates that there is one additional IkappaB kinase gene in Drosophila. Future experiments will test whether this gene plays a role in embryonic patterning and the antifungal immune response (Lu, 2001).
The data suggest that different Drosophila Rel dimers are activated by homologous but distinct signaling pathways. Given the similarities of innate immune response pathways in Drosophila and mammals, it is likely that similar pathway-specific signaling components will mediate the activities of the members of the mammalian Rel proteins (Lu, 2001).
NF-kappaB/Rel proteins function as homodimers or heterodimers, which recognize specific DNA sequences within target promoters. The activity of different Drosophila Rel-related proteins in modulating Drosophila immunity genes was examined by expressing the Rel proteins in stably transfected cell lines. How different combinations of these transcriptional regulators control the activity of various immunity genes was also examined. The results show that Rel proteins are directly involved in regulating the Drosophila antimicrobial response. Furthermore, the drosomycin and defensin expression is best induced by the Relish/Dif and the Relish/Dorsal heterodimers, respectively, whereas the attacin activity can be efficiently up-regulated by the Relish homodimer and heterodimers. These results illustrate how the formation of Rel protein dimers differentially regulate target gene expression (Han, 1999).
The parental S2 cell line used in this series of experiments can be induced by lipopolysaccharide (LPS) to express members of a subset of antimicrobial genes and these are therefore designated as S2* cells. In these cells, attacin, cecropin, and diptericin are inducible, but drosomycin induction is not detectable. Comparing with the induction levels of attacin and cecropin, defensin is less inducible. The differential expression of the antimicrobial genes is consistent with a multiple pathway model in which either LPS activates multiple receptors leading to the induction of individual immunity genes or a single LPS receptor can activate an intracellular pathway that branches at a certain point to regulate different immunity genes. The S2* cells may lack some, but not all, of the regulatory molecules that act downstream of the branch point, resulting in a partial inducibility by LPS (Han, 1999).
Although different pathways have been proposed to regulate various immunity gene expression, the utilization of specific Rel proteins in each pathway is not known even though most of these genes can be regulated by NF-kappaB-binding elements. To correlate the function of Rel proteins with the regulation of immune response, the mRNA expression of Rel protein genes in S2* cells was examined after LPS challenge. The mRNA for all three Rel protein genes is detectable in S2* cells, albeit at different levels. relish mRNA has high basal expression level, and the expression can be further up-regulated by 4-fold after 1 h of LPS treatment. Dif and dorsal mRNA levels are low and less responsive to LPS treatment. The different expression levels of Dif, dorsal, and relish mRNA may account for the inducibility of only a subset of immunity genes and suggest that Relish can function as a primary transcription factor in controlling attacin and cecropin expression (Han, 1999).
Stable cell lines that overexpress different Rel proteins, including Dif, Dorsal, and Relish, were established and used to analyze immunity gene regulation. S2* cells were transfected with different combination of plasmids that contained the Rel protein coding sequences under the control of metallothionein promoter. Stably transfected cells were selected. The transfected constructs were examined using FLAG or hexahistidine (His) epitope. Western blot results show that all three proteins are expressed, individually or in combination, to significant levels. The FLAG-tagged Relish expression is relatively uniform in cells designed to express Relish. Expression of His-tagged Dif and Dorsal shows some variation. Despite the variation, the interaction of different Rel proteins seems to be a more important determinant in gene regulation as demonstrated by examining immunity gene induction (Han, 1999).
The expression of anti-microbial genes was examined in the presence of exogenous Rel proteins prior to or after LPS treatment. After 18 h of incubation with CuSO4 to induce the Rel proteins, the cells were challenged with LPS, and RNA samples were analyzed by Northern blot. The expression of Dif or Dorsal alone prior to LPS incubation causes a modest up-regulation of drosomycin gene expression. Dif and Dorsal together have a similar modest effect on drosomycin induction. 1-6 h of LPS treatment further up-regulates drosomycin expression. This Dif or Dorsal dependent up-regulation is specific, because Relish itself does not cause S2* cells to express drosomycin (Han, 1999).
A functional interaction between Relish and Dif or Relish and Dorsal was detected by co-expressing two Rel proteins in the S2* cells. When Relish is co-expressed with Dif, drosomycin induction is enhanced significantly. This increased expression is observed before LPS challenge. Treatment of the cells with LPS further increases drosomycin expression by 2-fold. Even though there is a significant amount of Relish RNA in the parental S2* cell, Dif functions better with more exogenous Relish expression. It is possible that Dif and Relish form an unstable heterodimer, and the formation of the heterodimer requires higher concentrations of Dif and Relish than that produced by the endogenous genes. The result nonetheless suggests that the formation of Dif/Relish dimer is the rate-limiting step for full activation of drosomycin. Although the Relish and Dorsal combination also increases the expression and the inducibility of drosomycin, the effect of Dif/Relish on drosomycin expression is 4-fold higher than that of Dorsal/Relish. It is possible that the promoter of drosomycin has higher affinity toward Dif/Relish heterodimer. Alternatively, additional protein factors are involved in activating drosomycin expression, and these proteins coordinate better with Dif/Relish heterodimer. In summary, these results demonstrated that Relish functionally interacts with Dif and Dorsal to control drosomycin expression and that drosomycin expression is most efficiently activated by Dif/Relish heterodimer (Han, 1999).
The induction of defensin prefers the Relish/Dorsal heterodimer over other combinations, which is different from that of drosomycin. Surprisingly, the induction of the defensin gene is down-regulated by the expression of Dif or Dorsal alone. The Dif/Dorsal combination shows similar repressive effect. These results suggest that the Dif or Dorsal homodimers bind to the defensin promoter in a nonproductive conformation, thereby blocking the activation by an endogenous factor such as Relish. In contrast, Expression of Relish in combination with Dorsal and Dif restores the inducibility of defensin upon LPS treatment. Unlike the induction of drosomycin by Dif/Relish and Dorsal/Relish, defensin is not expressed prior to LPS treatment even in the presence of the heterodimers. One interpretation is that an additional protein factor, activated by LPS, coordinates with Rel proteins to activate defensin expression. The results, therefore, show that although the expression of Dif or Dorsal alone represses defensin expression, the co-expression of Dorsal and Relish best enhances defensin gene induction, and the co-expression of Dif and Relish can also mediate the induction (Han, 1999).
Dif, when expressed alone, is sufficient to activate cecropin and diptericin expression before LPS treatment. The result is consistent with the previous reports that Dif can activate cecropin and diptericin expression in transfection assays. However, the co-expression of Dif or Dorsal, in combination with Relish, does not show additional effect on the expression levels of cecropin and diptericin induced by LPS. Taken together, most combinations of Rel proteins can mediate cecropin and diptericin induction, and Dif homodimer is an efficient activator for these two genes (Han, 1999).
The Relish protein contains both Rel domain and IkappaB domain. It is likely that the IkappaB domain of Relish can complex with the Rel domain (RelD) to prevent the activation of Relish when expressed as a full-length protein. To study the function of Relish without the interference of the inhibitory domain, RelD (from amino acid 4 to 600) was expressed in a stable cell line. When RelD is expressed in S2* cells, attacin and cecropin are already activated prior to LPS treatment. In contrast, full-length Relish protein expression does not significantly activate attacin expression prior to LPS treatment. Furthermore, RelD does not affect drosomycin expression. In the presence of LPS, the expression of attacin and cecropin in the RelD-expressing cells is further elevated by only 2-fold. This suggests that RelD, in the absence of other Rel factors, can activate transcription of specific target genes. The result is in agreement with the observation that the ability of the parental S2* cells to up-regulate Relish by LPS is coincidental with the expression of attacin and cecropin. It may be that the formation of the processed Relish (RelD-like product) is the rate-limiting step, which is regulated by LPS. Therefore, the result supports the notion that Relish can function as the primary transcription factor in controlling attacin and cecropin gene induction. Because RelD alone can function to activate attacin and cecropin but not drosomycin expression, it further supports the idea that different combinations of Rel proteins have preferred target genes in vivo (Han, 1999).
Based on the functional interaction of Drosophila Rel proteins, it was expected that part of the protein function is achieved by the formation of heterodimers. To analyze further the interaction of Relish with Dif or Dorsal, co-immunoprecipitation experiments were performed. Anti-FLAG antibody was used to pull down FLAG-tagged Relish from extracts of cells that express Relish. The reaction is specific because the antibody does not recognize Dif or Dorsal protein, which contains the HIS epitope. From the samples that had Relish co-expressed with Dif or Dorsal, the His-tagged Dif and Dorsal can be co-immunoprecipitated as examined by Western blot. Quantitative analysis showed that 1-2% of total Dif or Dorsal can be co-immunoprecipitated along with Relish. It is possible that higher percentage of heterodimers are formed in the cells but escape detection. Furthermore, such amounts of heterodimer formation may be sufficient to account for the biological activities observed when assaying for the immunity gene expression. Nevertheless, the results demonstrate the specific interactions between Relish and Dif or Relish and Dorsal (Han, 1999).
Insects can effectively and rapidly clear microbial infections by a variety of innate immune responses including the production of antimicrobial peptides. Induction of these antimicrobial peptides in Drosophila has been well established to involve NF-kappaB elements. Evidence is presented for a molecular mechanism of Lipopolysaccharide (LPS)-induced signaling involving Drosophila NF-kappaB, Relish, in Drosophila S2 cells. LPS induces a rapid processing event within the Relish protein releasing the C-terminal ankyrin-repeats from the N-terminal Rel homology domain (RHD). Examination of the cellular localization of Relish reveals that the timing of this processing coincides with the nuclear translocation of the RHD and the retention of the ankyrin-repeats within the cytoplasm. Both the processing and the nuclear translocation immediately precede the expression of antibacterial peptide genes cecropin A1, attacin, and diptericin. Over-expression of the RHD but not full-length Relish results in an increase in the promoter activity of the cecropin A1 gene in the absence of LPS. Furthermore, the LPS-induced expression of these antibacterial peptides is greatly reduced when RELISH expression is depleted via RNA-mediated interference. In addition, loss of Cactus expression via RNAi has revealed that Relish activation and nuclear translocation is not dependent on the presence of Cactus. Taken together, these results suggest that this signaling mechanism involving the processing of RELISH followed by nuclear translocation of the RHD is central to the induction of at least part of the antimicrobial response in Drosophila, and is largely independent of Cactus regulation (Cornwell, 2001).
In mammals, TAK1, a MAPKKK kinase, is implicated in multiple signaling processes, including the regulation of NF-kappaB activity via the IL1-R/TLR pathways. TAK1 function has been studied primarily in cultured cells, and its in vivo function is not fully understood. Null mutations have been isolated in the Drosophila Tak1 gene that encodes Tak1, a homolog of TAK1. Tak1 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. Genetic studies also indicate that Tak1 functions downstream of the Imd protein and upstream of the IKK complex in the Imd pathway that controls the Rel/NF-kappaB like transactivator Relish. In addition, 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. This genetic demonstration of Tak1's role in the regulation of Drosophila antimicrobial peptide gene expression suggests an evolutionary conserved role for TAK1 in the activation of Rel/NF-kappaB-mediated host defense reactions (Vidal, 2001).
The Toll signaling pathway, which was first identified as a regulator of embryonic dorsal-ventral patterning, is one regulator of antimicrobial peptide gene expression in Drosophila. Upon infection, the Spaetzle (Spz) protein is cleaved to generate a ligand for the Toll transmembrane receptor protein; Toll binding by Spz stimulates the degradation of the IkappaB homolog, Cactus, and the nuclear translocation of the Rel proteins Dorsal and Dorsal-like immunity factor (Dif). A second pathway regulating antimicrobial peptide gene expression in flies was initially identified by a mutation in the immune deficiency (imd) gene that results in susceptibility to Gram-negative bacterial infection and an impairment of antibacterial peptide gene expression. imd encodes a homolog of the mammalian Receptor Interacting Protein (RIP) (P. Georgel, in preparation). The imd gene encodes a death domain-containing protein. In mammals, RIP appears to function in an adaptor complex associated with the tumor necrosis factor (TNF) receptor, and genetic placement of imd suggests that IMD has a conserved function in flies as part of a receptor-adaptor complex that responds to Gram-negative bacterial infection. Molecular studies have isolated four additional factors that appear to define the Imd pathway: Relish, a third Drosophila Rel protein; two members of a Drosophila IkappaB kinase (IKK) complex, that is, the kinase DmIKKß and a structural component DmIKKgamma and Dredd, a caspase. Relish is a homolog of the mammalian P100 and P105 compound Rel proteins that contain both Rel domains and inhibitory ankyrin domains. A receptor for the Imd pathway has not been identified, but infection triggers Relish cleavage and nuclear translocation of the Rel domain (Stöven et al. 2000). Relish cleavage requires DmIKKß activity, indicating that, like the mammalian IKK complex that functions in the IL1-R and TNF-R pathways, the Drosophila IKK complex regulates Rel protein activity. However, and in contrast to P100 and P105 processing, Relish cleavage is not blocked by proteasome inhibitors but does require a functional Dredd gene, suggesting that Dredd may cleave Relish directly after infection (Stöven, 2000). Like imd, mutations in DmIKKß, DmIKKgamma, Dredd, and Relish affect antibacterial peptide gene expression after infection and induce susceptibility to Gram-negative bacterial infections. However, mutations in these genes do not induce susceptibility to fungal infections, demonstrating that the immune responses regulated by the Imd pathway are required to resist Gram-negative bacterial but not fungal infections (Vidal, 2001 and references therein).
Some significant conclusions of recent studies on the regulation of Drosophila antimicrobial peptide gene expression are that the Toll and Imd pathways do not share any components and that each pathway regulates specific Rel proteins. The only evidence of interactions between the two pathways is the observation that both pathways are required to fully induce some of the antimicrobial peptide genes, suggesting that these genes respond to combinations of Rel proteins controlled by the two pathways. The influence of each pathway on the expression of each antimicrobial peptide gene is apparent in flies carrying mutations that affect either the Toll or the Imd pathway: Drosomycin is mainly controlled by the Toll pathway; Diptericin and Drosocin can be fully activated by the Imd pathway; and full Metchnikowin, Defensin, Cecropin A and Attacin activation requires both pathways (Vital, 2001 and references therein).
None of the antimicrobial peptide genes are induced in imd;Toll double mutant flies, demonstrating that Imd and Toll are two essential pathways that regulate antimicrobial gene expression pathways. Despite an increased understanding of the regulation of antimicrobial peptide gene expression in flies, various intermediates in the Toll and Imd pathways remain uncharacterized: for example, neither the kinase that targets Cactus for degradation in the Toll pathway nor the receptor-adaptor complex that regulates the Imd pathway have been identified. Following the observations that null mutations affecting the Imd pathway are not required for viability, a search for additional members of the Imd pathway was initiated by screening for nonlethal mutations that induce susceptibility to Gram-negative bacterial infection in adult flies. Null mutations in the Drosophila transforming growth factor activated kinase 1 gene (Tak1) encoding the Drosophila homolog of the mammalian mitogen-activated protein kinase kinase kinase (MAPKKK) TAK1 induce high susceptibility to Gram-negative bacterial infection and block antibacterial peptide gene expression. These results indicate that Drosophila Tak1 codes for a new component of the Imd pathway (Vidal, 2001).
To identify Drosophila genes that mediate defense reactions to bacterial infection, ~2500 lines carrying ethyl methanesulfonate (EMS)-induced mutations on the X chromosome were tested for susceptibility to bacterial infection: male adult flies were pricked with a needle dipped into a pellet of the Gram-negative bacterial species Erwinia carotovora carotovora 15 (E. carotovora 15) and screened for mutants that failed to survive infection. Using this assay, nine recessive, homozygous viable mutations were isolated that render flies highly susceptible to E. carotovora 15 infection: Less than 10% of the mutated flies survived 48 h postinfection, whereas more than 90% of the wild-type flies survived. These nine mutations fall into two complementation groups: B118, which represents five of the mutations, and D10, corresponding to Tak1, which represents the other four mutations. The B118 group corresponds to the caspase encoding gene Dredd (Vidal, 2001),
To compare the D10 phenotype with the phenotypes generated by mutations in other genes that regulate Drosophila immune responses, the susceptibility of D10 and other mutant lines to infection by four microorganisms was assayed: flies were pricked with the Gram-negative bacteria Escherichia coli (E. coli), the Gram-positive bacteria Micrococcus luteus (M. luteus), or the fungus Aspergillus fumigatus, and flies were naturally infected with the entomopathogenic fungus Beauveria bassiana (B. bassiana). The D10 phenotype is similar to the imd and Relish phenotypes; flies carrying the D10, imd, and Relish mutations are susceptible to Gram-negative bacterial infection and resistant to Gram-positive bacterial and fungal infections, although D10 flies, like imd flies, exhibit slightly lower susceptibility to Gram-negative bacterial infection compared to Relish mutants. In contrast, mutations in the spz gene render flies susceptible to fungal infections, and only flies carrying mutations in both spz and imd are susceptible to Gram-positive bacterial infection. This survival analysis demonstrates that the D10 gene product, like Imd and Relish, is required to resist Gram-negative bacterial infection (Vidal, 2001).
The Toll pathway is required for the full induction of the antifungal peptide genes and a subset of the antibacterial peptide genes. Mutations that block the Toll pathway reduce the expression of these genes; conversely, mutations that block the Imd pathway reduce the expression of genes with antibacterial activity. To determine how the D10 mutation affects antimicrobial peptide gene expression, the levels of Diptericin, Cecropin A, Defensin, and Attacin, which encode antibacterial peptides, Drosomycin, which encodes an antifungal peptide, and Metchnikowin, which encodes a peptide with both antibacterial and antifungal activity, were monitored in flies homozygous for two D10 alleles. In addition, the D10 phenotype was compared with all of the previously identified mutations affecting the Imd pathway and with a spz mutation that blocks the Toll pathway (Vidal, 2001).
Pricking adult flies with a mixture of Gram-positive and Gram-negative bacteria activates the expression of all the antimicrobial peptide genes; in the D10 mutants, however, mixed Gram-negative/Gram-positive infections induce significant levels of only Drosomycin and Metchnikowin. Quantitative measurements of three independent RNA blot experiments show that in D10 flies, Drosomycin is induced to wild-type levels; Metchnikowin is induced to 70% of wild-type levels; Cecropin A, Defensin, and Attacin are induced to <25% of wild-type levels, and Diptericin is induced to <5% of wild-type levels. This pattern of antimicrobial peptide gene expression in the D10 mutants is similar to the patterns displayed in mutants of the Imd pathway, although the D10 mutations, like imd, have slightly weaker effects on antimicrobial peptide gene expression compared to the Dredd, DmIKKß, DmIKKgamma, and Relish mutations. This weaker phenotype in D10 and imd flies correlates well with their lower susceptibility to E. coli infection (Vidal, 2001).
Because of its chromosomal location and the functions of its mammalian homologs, Tak1 was chosen as a candidate gene mutated in D10 flies. Several different experiments to determine whether the D10 alleles correspond to mutations in Tak1. Tested f wasirst, the ability of a 15 Kb genomic fragment that contains Tak1 to rescue the immune response deficiency in D10 flies. D10 adults carrying the Tak1 transgene (P[Tak1+]) both express Diptericin and resist Gram-negative bacterial infection at levels comparable to wild-type flies, demonstrating that this genomic fragment rescues the D10 phenotypes. Overexpression of the Tak1 cDNA using the UAS/GAL4 system partially rescues Diptericin expression in the D101 mutant. The Tak1 genomic coding sequence was tested. The four D10 alleles all contain mutations within the Tak1 kinase domain. This led to the renaming of the D10 alleles Tak11 to Tak14: Tak11 and Tak14 were generated by missense mutations in conserved residues, Tak12 was generated by a point mutation that creates a stop codon, and Tak13 contains a deletion of 31 base pairs that also results in a premature stop codon. All four Tak1 alleles inhibit Diptericin induction by Gram-negative bacterial infection to the same degree, and this inhibition is not enhanced in flies heterozygous for each allele and a deficiency spanning Tak1. In addition, flies homozygous for the four alleles are equally susceptible to Gram-negative bacterial infection. The apparent null phenotype manifested by the four Tak1 alleles indicates that the Tak1 kinase domain is essential for Tak1 function in the Imd pathway. This observation is supported by results from experiments with a kinase dead form of Tak1, Tak1-K46R, which acts as a dominant negative inhibitor of Tak1. Tak1-K46R expression driven by the UAS/GAL4 system blocks Diptericin expression after mixed bacterial infection, confirming that Tak1 is required for the Drosophila antibacterial immune response. The results of the rescue experiments, sequencing data, and the dominant negative Tak1 mutant phenotypes together demonstrate that D10 encodes the MAPKKK Tak1 (Vidal, 2001).
The loss-of-function Tak1 mutations display immune response phenotypes that are very similar to the phenotypes generated by mutations in imd, DmIKKß, DmIKKgamma, Dredd, and Relish, suggesting that these genes function together in the Imd pathway. Previous studies indicated that DmIKKgamma and DmIKKß directly regulate Relish activity (Silverman, 2000); however, the positions of Imd, Tak1, and Dredd in the Imd pathway were not determined. To avail of a genetic approach for ordering the Imd pathway, both Dredd and Tak1 were overexpressed via the UAS/GAL4 system. In lines carrying a heat shock (hs)-GAL4 driver and either the Dredd or Tak1 cDNAs under the control of a UAS promoter, heat shock induces Diptericin expression to about 15%-20% of the level observed in adults 6 h after bacterial infection. This result indicates that the overexpression of these two genes is sufficient to activate the antibacterial pathway in the absence of infection. By using this UAS/GAL4 system to overexpress Tak1 and Dredd in various mutant backgrounds, the epistatic relationships were tested between Tak1 and Dredd, and the other genes of the Imd pathway. Mutations in DmIKKß and DmIKKgamma, but not imd, block Diptericin induction by Tak1 overexpression, indicating that Tak1 functions downstream of Imd and upstream of the IKK complex. However, the Diptericin expression induced by Dredd overexpression is not affected by mutations in imd or DmIKKgamma. For genetic reasons, Northern blot analysis could not be used to test the effect of mutations in Relish and DmIKKß, which are on the third chromosome, on the UAS-Dredd-induced Diptericin expression. Therefore, the UAS-Dredd transgene was overexpressed through a female, adult fat body driver (yolk-GAL4) and Diptericin expression was monitored with a Diptericin-LacZ reporter gene. LacZ titration assays have demonstrated that ß-galactosidase activity is induced in lines overexpressing UAS-Dredd in the absence of infection. The Dredd-mediated Diptericin-LacZ induction is strongly reduced in Relish but not in DmIKKß mutants, confirming the Northern blot results showing that Dredd functions downstream of the IKK complex and demonstrating that Dredd regulates Diptericin expression through Relish (Vidal, 2001).
Natural fungal infections highlight the ability of Drosophila to discriminate between pathogens and activate specific immune response pathways that lead to adapted immune responses. Although only the Imd pathway is required to resist Gram-negative bacterial infection, the Toll pathway is still activated to some degree in flies pricked with Gram-negative bacteria. This suggests that injury (and associated contamination) contributes to a nonspecific immune response. E. carotovora 15 naturally infects the Drosophila larval gut and triggers both the local and systemic expression of antimicrobial peptide genes. In contrast to infection by pricking, natural E. carotovora 15 infection induces Diptericin expression more strongly than Drosomycin expression. To compare the contribution of the Toll and Imd pathways to antimicrobial peptide induction after natural E. carotovora 15 infection, Diptericin and Drosomycin expression were quantified in larvae from various mutant lines after natural E. carotovora 15 infections. The Diptericin expression induced by E. carotovora 15 natural infection is entirely dependent on the genes of the Imd pathway, confirming that Diptericin is exclusively regulated by the Imd pathway. Interestingly, the level of Diptericin induction is higher in spz mutants than in wild-type larvae. In addition, Drosomycin induction after E. carotovora 15 natural infection of larvae is also dependent on the Imd pathway: mutations blocking the Imd pathway have stronger effects than the spz mutation on Drosomycin expression induced by natural infection. These data contrast somewhat with previous observations that the imd mutation does not block Drosomycin induction by E. carotovora 15 infection. However, the earlier analysis of Drosomycin induction by E. carotovora 15 was based on the qualitative analysis of a Drosomycin-lacZ reporter gene; it is thought that that the quantitative analysis of Drosomycin expression by Northern blot is a more accurate determination of Drosomycin expression patterns. In conclusion, the current results indicate that Drosomycin gene induction after natural Gram-negative bacterial infection is largely mediated by the Imd pathway (Vidal, 2001).
Natural infections by E. carotovora 15 also trigger the local expression of antimicrobial peptide genes in various epithelial tissues, and both Diptericin expression in the anterior midgut and Drosomycin expression in the trachea are dependent on the imd gene. By assaying the expression of Diptericin-lacZ and Drosomycin-GFP reporter genes in naturally infected Tak1 and Dredd mutant larvae, it has been shown that both genes are required for Diptericin and Drosomycin expression in epithelial tissues after natural E. carotovora. These data confirm the predominant role of the Imd pathway in antimicrobial peptide regulation after natural E. carotovora 15 infection and suggest that Gram-negative bacterial recognition in flies preferentially activates the Imd pathway (Vidal, 2001).
Two Drosophila tumor necrosis factor receptor-associated factors (TRAFs), Traf1 and Traf2, are proposed to have similar functions with their mammalian counterparts as a signal mediator of cell surface receptors. However, as yet their in vivo functions and related signaling pathways are not fully understood. Traf1 is shown to be an in vivo regulator of c-Jun N-terminal kinase (JNK) pathway in Drosophila. Ectopic expression of Traf1 in the developing eye induces apoptosis, thereby causing a rough-eye phenotype. Further genetic interaction analyses reveal that the apoptosis in the eye imaginal disc and the abnormal eye morphogenesis induced by Traf1 are dependent on JNK and its upstream kinases, Hep and TGF-ß activated kinase 1. In support of these results, the Traf1-null mutant shows a remarkable reduction in JNK activity with an impaired development of imaginal discs< and a defective formation of photosensory neuron arrays. In contrast, Traf2 was demonstrated as an upstream activator of nuclear factor-kappaB (NF-kappaB). Ectopic expression of Traf2 induces nuclear translocation of two Drosophila NF-kappaBs, Dif and Relish, consequently activating the transcription of the antimicrobial peptide genes diptericin, diptericin-like protein, and drosomycin. Consistently, the null mutant of Traf2 shows immune deficiencies in which NF-kappaB nuclear translocation and antimicrobial gene transcription against microbial infection were severely impaired. Collectively, these findings demonstrate that Traf1 and Traf2 play pivotal roles in Drosophila development and innate immunity by differentially regulating the JNK- and the NF-kappaB-dependent signaling pathway, respectively (Cha, 2003).
Microbial infection studies have demonstrated the ability of Drosophila to detect pathogens and activate specific signaling pathways, Toll or Imd pathways, which lead to adapted immune responses. In recent years, several families of antimicrobial peptides and their coding genes have been successfully identified: cecropins, attacins, diptericin, defensin, drosomycin, drosocin, and diptericin-like protein (dptlp). Understanding the molecular mechanisms underlying how microbial infection induces expression of these antimicrobial peptides has been the main question to answer in this field. Meanwhile, Traf2 have been identified as a downstream adaptor for Toll receptor (Shen, 2001) and Toll activation leads to immune responses. Therefore, it was suspected that DTRAFs would be involved in this defense mechanism (Cha, 2003).
Three representative antimicrobial genes, diptericin, dptlp, and drosomycin, were chosen as probes to determine the activity of the antimicrobial defense system. To examine whether Drosophila Traf1 and Traf2 have the ability to induce the transcription of diptericin, dptlp, and drosomycin, Traf1 or Traf2 was ectopically expressed in third-instar larvae by using hs-GAL4 driver, and the expression levels of diptericin, dptlp, and drosomycin were monitored by Northern blot analyses (Cha, 2003).
The transcription of diptericin, dptlp, and drosomycin was increased by ectopic expression of Traf2 in the absence of microbial infection. However, the expression levels of diptericin, dptlp, and drosomycin were not altered by Traf1 overexpression. In addition, Traf2-induced expression of diptericin and dptlp is completely inhibited in a relish (rel, Drosophila NF-kappaB)-null mutant background, whereas drosomycin expression is partially inhibited by the same mutation. The partial inhibition of the drosomycin expression by rel mutation suggests that the involvement of another Drosophila NF-kappaB, such as Dif, in antimicrobial response gene transcription. These results strongly suggest that Traf2, but not Traf1, functions downstream of microbial sensory receptors, Toll or Imd, and upstream of the NF-kappaBs to regulate Drosophila immune responses (Cha, 2003).
To further confirm the results, transgenic fly lines that have a GFP or a LacZ reporter gene fused to the drosomycin or the diptericin promoter, respectively, were used, allowing observation of the reporter gene activity, which reflects the drosomycin or diptericin gene expression level. The drosomycin-GFP reporter activity is dramatically increased in the microbe-infected larva compared to the uninfected control. As expected, Traf2 overexpression alone in the absence of microbial infection strongly induces drosomycin-GFP reporter gene activity. Further dissection analyses show that drosomycin-GFP and diptericin-LacZ reporter activities are highly induced in the fat body, which is a representative target tissue for immune responses in Drosophila. However, Traf1 overexpression fails to induce the reporter activities in both whole larvae and their fat bodies, further confirming the noninvolvement of Traf1 in the immune responses of Drosophila (Cha, 2003).
In order to confirm that the Traf2-induced immune responses are mediated by Dif and Relish, which are Drosophila NF-kappaBs specifically activated by Toll and Imd pathways, respectively, the subcellular localization of Dif and Relish was determened by using their specific antibodies (Cha, 2003).
Dif and Relish are dispersed in the cytoplasm of fat body cells in the absence of microbial infection. In contrast to this, either the microbial infection or overexpression of Traf2 fully induces the nuclear translocation of both Dif and Relish, demonstrating that both Dif and Relish participate in the Traf2-mediated immune responses. However, the subcellular localization of Dif and Relish is not altered by Traf1 induction, further confirming that Traf1 is not involved in the NF-kappaB signaling pathway. These data clearly demonstrated that Traf2, but not Traf1, has the capability to induce transcriptional activation of immune response genes by specifically activating NF-kappaBs (Cha, 2003).
The Traf2-null mutant, Traf2ex1, was generated by P-element excision method. RT-PCR analysis shows that the homozygous Traf2ex1 mutant fails to produce Traf2 mRNA. Intriguingly, the mutant flies manage to develop into adults and show no morphological defects. To determine whether the Traf2ex1 mutant shows a deficiency in immune responses, the transcriptional induction level of diptericin and drosomycin was examined after microbial infection. The null mutation of Traf2 drastically disrupts the transcriptional induction of diptericin and drosomycin when compared to the wild-type control. However, Traf1-null mutation (Traf1ex1) has no effect on the induction of diptericin and drosomycin gene expressio