In Drosophila, the immune deficiency (Imd) pathway controls antibacterial peptide gene expression in the fat body in response to Gram-negative bacterial infection. The ultimate target of the Imd pathway is Relish, a transactivator related to mammalian P105 and P100 NF-kappaB precursor. Relish is processed in order to translocate to the nucleus, and this cleavage is dependent on both Dredd, an apical caspase related to caspase-8 of mammals, and the fly Ikappa-B kinase complex (dmIKK). dTAK1, a MAPKKK, functions upstream of the dmIKK complex and downstream of Imd, a protein with a death domain similar to that of mammalian receptor interacting protein (RIP). Finally, the peptidoglycan recognition protein-LC (PGRP-LC) acts upstream of Imd and probably functions as a receptor for the Imd pathway. Interference with dFADD (FlyBase designation: BG4) function by double-stranded RNA inhibition demonstrates that dFADD is a novel component of the Imd pathway. dFADD double-stranded RNA expression reduces the induction of antibacterial peptide-encoding genes after infection and renders the fly susceptible to Gram-negative bacterial infection. Epistatic studies indicate that dFADD acts between Imd and Dredd. These results reinforce the parallels between the Imd and the TNF-R1 pathways (Leulier, 2002).
dFADD is a gene encoding a death domain protein with an overall structure similar to that of mammalian Fas-associated death domain-containing protein (FADD), an adaptor that is believed to interact with the TNF-R1 complex through homophilic death domain interactions with the TNF-R-associated death domain-containing protein (TRADD). FADD then recruits pro-caspase-8 through homophilic death effector domain associations. Consequently, dFADD is an obvious candidate for linking the death domain protein Imd and the Dredd apical caspase in the Imd pathway. Inducible expression of dFADD double-stranded RNA has been used to determine if dFADD functions in the Imd pathway. This approach, which exploits the UAS/GAL4 binary system to drive expression of double-stranded RNA in a defined tissue is a form of RNA interference (RNAi) that has previously been shown to block the expression of defined genes (Leulier, 2002).
Transgenic flies carrying either UAS-dTAK1-IR or UAS-dFADD-IR have been generated. Both constructs consist of two 500 bp-long inverted repeats (IR) of the gene, separated by an unrelated DNA sequence that acts as a spacer, to give a hairpin-loop-shaped RNA. These transgenic flies were crossed to flies carrying various GAL4 drivers in order to activate transcription of the hairpin-encoding transgene in the progeny. Three GAL4 lines were used in this study: daughterless-GAL4 (da-GAL4), which expresses GAL4 strongly and ubiquitously; hs-GAL4, which directs expression of GAL4 ubiquitously after heat shocks; and yolk-GAL4, which expresses the yeast transactivator in the fat body of female adults (Leulier, 2002).
dTAK1-deficient flies do not express the antibacterial peptide-encoding gene Diptericin upon immune challenge and are highly susceptible to infection by Gram-negative bacteria. A similar phenotype is generated by mutations affecting the other components of the Imd pathway. Interestingly, the expression of UAS-dTAK1-IR induced by either the hs-GAL4 or the yolk-GAL4 drivers produces an immune deficiency phenotype similar to dTAK1 mutants: UAS-dTAK1-IR flies fail to express antibacterial-encoding genes after infection and are highly susceptible to Gram-negative bacterial infection. However, the UAS-dTAK1-IR expression phenotype is weaker than the dTAK1 null mutant phenotype, both in terms of survival and affect on anti-microbial peptide gene expression, suggesting that the inducible expression of RNAi mimics a partial loss-of-function mutation of the target gene. In agreement with what was observed in dTAK1 null mutants, expression of UAS-dTAK1-IR using the ubiquitous driver da-GAL4 does not lead to detectable developmental defects. This contrasts with the results obtained by expression of a dominant-negative construct of dTAK1, which leads to ectopic developmental defects. Taken together, these results demonstrate the suitability of the RNAi approach for functional studies of the antimicrobial response (Leulier, 2002).
To address dFADD's role in the regulation of antimicrobial gene expression, the UAS-dFADD-IR transgene was expressed using the three GAL4 insertions. Flies that express dFADD-IR ubiquitously through da-GAL4 show no detectable defects, suggesting that dFADD is not essential for development. These flies do, however, have phenotypes similar to those generated by mutations affecting the Imd pathway. The expression of antibacterial peptide genes Diptericin and Attacin are strongly reduced after septic injury, while the expression of the antifungal gene Drosomycin remains inducible. In addition, these flies exhibit a high susceptibility to Gram-negative bacterial infection but resistance to fungal infection. This phenotype is identical to that generated by the UAS-dTAK1-IR construct and is similar to (although slightly weaker than) those generated by null mutations in dTAK1, kenny, ird5, Dredd, Relish, and imd. These results demonstrate that, like the other components of the Imd pathway, dFADD is required for a full antibacterial response (Leulier, 2002).
Overexpression of the imd gene leads to constitutive transcription of antibacterial peptide genes, and this induction requires the Dredd caspase. Expression of both dTAK1-IR and dFADD-IR strongly reduces the Imd-mediated induction of antibacterial peptide-encoding genes, indicating that, genetically, dFADD and dTAK1 function downstream of Imd. This result was confirmed by demonstrating that the dTAK11 mutation also blocks the constitutive Diptericin expression induced by imd overexpression. Overexpressing Dredd via the UAS/GAL4 system also leads to Diptericin expression in the absence of infection, which can be monitored with a Diptericin-lacZ transgene. lacZ titration assays demonstrate that the Diptericin reporter gene expression induced by overexpressing the UAS-Dredd transgene is not affected by the coexpression of dFADD-IR. Consequently, these epistatic studies place dFADD function upstream of the Dredd caspase. This result is in agreement with cell culture experiments showing that dFADD binds to Dredd through its N-terminal prodomain and promotes the proteolytic processing of Dredd (Leulier, 2002).
Recent studies have shown that the Drosophila homolog of MyD88, dMyD88, is an essential component of the Toll pathway. In addition, dMyD88 has been shown to bind in vitro to dFADD, pointing to a possible interaction between dFADD and the Toll pathway. Expression of dFADD-IR does not, however, block the constitutive Drosomycin expression induced by the dominant, gain-of-function Toll10b mutation, and dFADD RNAi does not block Drosomycin induction by infection. This result indicates that, like the other components of the Imd pathway, dFADD is not required for Toll pathway function (Leulier, 2002).
Altogether, this analysis indicates that dFADD is a novel component of the Imd pathway that links Imd to Dredd. Biochemical studies show that dFADD contacts Dredd via homotypic dead effector domain interaction, and it is possible that dFADD interacts with Imd via its death domain. Consequently, dFADD, Dredd, and Imd may be components of a multiprotein adaptor complex functioning downstream of the receptor of the Imd pathway. Genetic studies suggest that the Imd pathway bifurcates downstream of Imd, with one branch leading to caspase activation via dFADD and the second branch leading to activation of the IKK complex via activation of dTAK1; both of these events are required for Relish processing (Leulier, 2002).
Studies using loss-of-function mutations in the genes encoding components of the Imd pathway did not provide clear evidence for a role of this cascade in developmentally regulated apoptosis. However, recently, it has been shown that the overexpression of imd with the da-GAL4 driver in flies induces an early larval lethality that can be partially rescued by coexpression of the viral caspase inhibitor P35, suggesting that Imd can also promote apoptosis. Interestingly, it was noted that the lethality induced by imd overexpression is totally suppressed in Dredd mutants but only marginally reduced in dTAK1 mutants, suggesting that this effect is mediated through the dFADD/Dredd arm but not the dTAK1-dmIKK arm of the Imd pathway (Leulier, 2002).
In conclusion, the implication of dFADD in the Drosophila Imd pathway strengthens the parallels between the Imd and TNF-R1 pathways: both cascades regulate NF-kappaB via RIP-MAPKKK-IKK intermediates and promote caspase activation through the FADD adapter. In Drosophila, these two processes are required to activate Relish, while, in mammals, current models suggest that the TNF-R1 pathway leads to either NF-kappaB activation or programmed cell death activation. Additional experiments are still required to demonstrate a clear role for the Imd pathway in the regulation of apoptosis. Finally, this study validates the use of the inducible expression of double-stranded RNA to address the in vivo function of genes that mediate the Drosophila antimicrobial response (Leulier, 2002).
The Imd signaling cascade, similar to the mammalian TNF-receptor pathway, controls antimicrobial peptide expression in Drosophila. A large-scale RNAi screen was performed to identify novel components of the Imd pathway in Drosophila S2 cells. In all, 6713 dsRNAs from an S2 cell-derived cDNA library were analyzed for their effect on Attacin promoter activity in response to Escherichia coli. Seven gene products required for the Attacin response in vitro were identified, including two novel Imd pathway components: inhibitor of apoptosis 2 (Iap2) and transforming growth factor-activated kinase 1 (TAK1)-binding protein (TAB). Iap2 is required for antimicrobial peptide response also by the fat body in vivo. Both these factors function downstream of Imd. Neither TAB nor Iap2 is required for Relish cleavage, but may be involved in Relish nuclear localization in vitro, suggesting a novel mode of regulation of the Imd pathway. These results show that an RNAi-based approach is suitable to identify genes in conserved signaling cascades (Kleino, 2005).
Drosophila has developed a highly sophisticated immune defense, which is required for living in a natural environment that is rich in bacteria and fungi. In contrast to mammals, Drosophila has no adaptive, that is, antibody-mediated immunity, which makes it a good model for studying the pattern recognition receptors and signaling pathways of innate immunity. In Drosophila, there are two major pathways that respond to microbes: the Imd and the Toll pathways. Both of them are strikingly well conserved throughout evolution. Thus, novel findings from work on Drosophila immune response can fuel discoveries in the mammalian systems (Kleino, 2005).
In Drosophila, evolutionarily conserved peptidoglycan recognition proteins (PGRPs) are of paramount importance for microbial recognition. Several Drosophila PGRPs are necessary for normal resistance to bacteria . Secreted PGRP-SA is essential for induction of immune response genes via the Toll pathway in response to certain Gram-positive bacteria in vivo. In contrast, PGRP-LC is the first component of the Imd pathway (Choe, 2002; Gottar, 2002; Rämet, 2002). It is located on the cell membrane where it appears to act as a pattern recognition receptor for bacteria either alone or together with other PGRPs (Takehana, 2004). Recently, intracellular domain of PGRP-LC was shown to bind directly to the Imd, which is the next known component downstream of PGRP-LC (Choe, 2005). Imd contains a death domain with homology to the mammalian receptor-interacting protein 1. The signal is propagated via transforming growth factor-activated kinase 1 (TAK1) to Drosophila homologs for IKKgamma and IKKalpha/ß (Key and Ird5, respectively) (Rutschmann, 2000; Lu, 2001). Whether TAK1 phosphorylates the Drosophila IKKs directly is uncertain and the mechanism of TAK1 activation is elusive. TAK1 has also been shown to play a role in the regulation of the c-Jun N-terminal kinase (JNK) pathway (Park, 2004). Finally, the signal leads to the activation of the Drosophila NF-kappaB homolog Relish, involving its phosphorylation by the IKK complex (Silverman, 2000) and cleavage by a caspase currently believed to be Dredd (Leulier, 2000: Stöven, 2000; Stöven, 2003), which forms a complex with BG4, a homolog to mammalian Fas-associated death domain protein (FADD; Leulier, 2002). The phosphorylated and cleaved Relish is then translocated to the nucleus, where it binds to DNA leading to synthesis of antimicrobial peptides (Kleino, 2005 and references therein).
Normal response to most Gram-negative bacteria in Drosophila depends on the Imd pathway, which is very similar to the TNF receptor signaling pathway in mammals. In order to determine whether there are still unknown components in the Imd signaling pathway, a large-scale RNAi-based screen was carried out in Drosophila S2 cells using a luciferase-reporter-based quantitative assay. The activity of the pathway was assayed using Attacin-luciferase (Att-luc) reporter. Transfection efficiency and cell viability were monitored using Act5C-ß-gal reporter. The Imd signaling pathway was activated with heat-killed Escherichia coli. At first, tests were carried out to see if dsRNA targeting a known component of the pathway caused a decrease in Att-luc activity. Relish (Rel) RNAi blocked the Imd pathway activity in a dose-dependent manner. 10 ng of Rel dsRNA per 5.0 x 105 S2 cells in 500 microl of medium reduced the luciferase activity by >50% and more than 0.1 microg of Rel dsRNA blocked the luciferase activity almost completely. Therefore, RNAi very effectively silences the expression of the targeted gene in this assay, which thus can be used to identify essential components of the Imd pathway (Kleino, 2005).
6713 dsRNAs from an S2 cell-derived cDNA library were assayed for their effect on the Imd signaling pathway in S2 cells using Att-luc reporter as a read-out. Most dsRNA treatments had little or no effect. Seven genes decreased Att-luc activity by >80% without decreasing Act5C-ß-gal activity by more than 40%, indicating that viability and the translation machinery were unaffected. These genes included three (PGRP-LC, imd and Rel) out of eight known components of the Imd pathway. Rel was identified three times. Novel genes identified were kayak, longitudinals lacking (lola), inhibitor of apoptosis 2 (Iap2) and CG7417. The CG7417 protein is a homolog to the mammalian TAK1-binding proteins 2 and 3 (TAB2 and TAB3), hereafter called TAB. Interestingly, a dsRNA treatment silencing Rel, TAB, PGRP-LC, imd or lola also strongly decreased the Drosomycin reporter (Drs-luc) activity induced by the constitutively active form of Toll (Toll10b). Therefore, it appears that a low level of Rel activity is required also for normal Drs response via the Toll pathway in S2 cells (Kleino, 2005).
Kayak is a known component of the JNK signaling pathway; RNAi targeting kayak caused an 88 +/- 7% decrease in Att-luc activity. This is in accordance with recent results, which indicate that JNK is essential for normal antimicrobial peptide release in S2 cells (Kallio, 2005). RNAi targeting lola caused 87 +/- 5% decrease in Att-luc activity. Lola is a nuclear factor that is required for axon growth in the Drosophila embryo and normal phagocytosis of bacteria in S2 cells (Rämet, 2002). Lola has not been indicated to play a role in the synthesis of antimicrobial peptides. In the reporter assay, lola RNAi decreased Att-luc activity slightly less than known components of the Imd pathway. Of note, RNAi silencing of lola also decreased Drs-luc activity induced by Toll10b in S2 cells (Kleino, 2005).
In all, 35 dsRNA treatments representing 22 genes caused a greater than three-fold increase in the Att-luc activity in response to heat-killed E. coli after ecdysone treatment in S2 cells. These genes could be divided into the following categories based on the putative function of their encoding protein: (1) genes involved in microtubule organization or actin cytoskeleton regulation (par-1, Rab-protein 11, multiple ankyrin repeats single KH domain [mask], alpha-Tubulin at 84B, CG6509 and PDGF- and VEGF receptor related [Pvr]); (2) helicases and other genes involved in DNA replication (Helicase 89B, Rm62, kismet, mutagen-sensitive 209 and double parked); (3) signaling molecules (daughter of sevenless, CG32782 and Ecdysone-induced protein 75B); (4) transcription factors (E2F transcription factor and Zn-finger homeodomain 1) and (5) uncharacterized genes. Of note, kismet was identified eight times, Pvr six times and E2F transcription factor twice in this screen. The mechanisms for how these genes affect signaling through the Imd pathway remain to be studied. Of note, none of these dsRNA treatments notably induced the Imd pathway without E. coli (Kleino, 2005).
Two novel components of the Imd pathway, Iap2 and TAB, were identified that appear to be absolutely necessary for induction of Att-luc activity in S2 cells in response to heat-killed E. coli. dsRNA targeting either Iap2 or TAB causes a drastic, 98 +/- 1% decrease in Att-luc activity. Iap2 or TAB RNAi has no effect on cell growth as determined by cell counts, indicating that the result is not due to increased cell death. To verify that the observed phenotypes were caused by decreased expression of Iap2 and TAB, targeted RNAi with gene-specific primers was carried out. Specific dsRNA treatments targeting either Iap2 or TAB drastically decreases the Att-luc activity. TAB RNAi also decreases the Drs reporter activity via the Toll10b-induced Toll pathway. Whether the effect of Iap2 or TAB RNAi was ecdysone dependent was examined. If ecdysone was not used, Att-luc induction was clearly (35 +/- 2%, N=3) weaker, but also this induction was blocked by RNAi targeting either Iap2 or TAB, indicating an ecdysone-independent mechanism (Kleino, 2005).
To ascertain that the results were not due to an artifact related to the use of a reporter construct, the expression level of Cecropin A1 (CecA1), another well-characterized antimicrobial gene regulated by the Imd pathway, was analyzed by semiquantitative RT-PCR. A 6-h exposure to heat-killed E. coli increased the mRNA level of CecA1. This increase could be blocked entirely by RNAi targeting either Rel or TAB. In addition, induction was reduced by RNAi targeting Iap2. Corresponding results were obtained also for Att D and Diptericin (Dpt). dsRNA treatments targeting either Rel or TAB totally blocked the induction, while the effect of Iap2 RNAi was somewhat more moderate (Kleino, 2005).
To investigate whether these in vitro findings are of in vivo relevance, the inducible expression of Iap2 dsRNA was used in Drosophila in vivo. The UAS/GAL4 binary system to drive expression of dsRNA in a defined tissue has been previously used to block the expression of defined genes. To this end, transgenic flies were generated carrying the UAS-Iap2-IR. This construct has two 500 bp long inverted repeats (IR) of the gene, separated by an unrelated DNA sequence that acts as a spacer, to give a hairpin-loop-shaped RNA. These transgenic flies were crossed to flies carrying various GAL4 drivers in order to activate transcription of the hairpin-encoding transgene in the progeny. Iap2 has been shown to be required for the regulation of apoptosis in Drosophila (F. Leulier, personal communication to Kleino, 2005), and overexpression of UAS-Iap2-IR with the ubiquitous and strong daughterless-GAL4 (da-GAL4) driver leads to lethality at the pupal stage. To address the role of Iap2 in antimicrobial gene expression, the UAS-Iap2-IR transgene was expressed using the C564-GAL4 driver that expresses GAL4 in the adult fat body. Flies were kept at 25°C to avoid the induction of apoptosis in the fat body. Flies that express Iap2-IR ubiquitously through C564 showed no detectable defects. However, the expression of the antibacterial peptide gene Dpt was strongly reduced after infection with the Gram-negative bacteria Erwinia carotovora. This phenotype was similar, although weaker, than those generated by BG4-IR RNAi. Importantly, the expression of Drs remained inducible in Iap2-IR; C564 flies, indicating that Iap2 did not block the Toll pathway and that the fat body remained functional (Kleino, 2005).
To map the locations of Iap2 and TAB in the Imd signaling cascade, known components of the cascade were overexpressed including a constitutively active form of Relish (Rel DeltaS29-S45), wild-type Relish or wild-type Imd. All these caused an activation of Att expression in S2 cells. Att induction caused by expression of either Relish construct could not be blocked by RNAi targeting either imd, TAB or Iap2, indicating that both TAB and Iap2 are located upstream of Relish. In contrast, Att induction caused by overexpression of Imd is blocked by RNAi targeting either Rel, imd, TAB or Iap2, indicating that both TAB and Iap2 lie downstream of Imd in the hemocyte-like S2 cells (Kleino, 2005).
To assess whether Iap2 is located downstream of Imd in the fat body in vivo, the UAS-imd construct with a heat-shock-GAL4 (hs-GAL4) driver was overexpressed; this induced expression of the Dpt gene in the absence of infection. Although there is some constitutive Dpt expression in these flies, the level of Dpt increases after heat shock. Using these flies, Dpt expression was reduced by coexpression of UAS-Iap2-IR by 44 +/- 8% (N=2) in these flies. Total RNA was extracted from unchallenged adult flies, collected 6 or 16 h after a heat shock (37°C, 1 h) and RT−PCR analysis was used to monitor the expression level of Dpt. This indicates that Iap2 functions, genetically, downstream of Imd in the fat body in vivo (Kleino, 2005).
To map the exact location of Iap2 in the Imd signaling cascade, Iap2 was overexpressed in S2 cells, which resulted in a minimal but reproducible induction of Att expression. This induction was completely blocked by dsRNAs targeting the known components of the Imd pathway, except dsRNAs targeting either imd or TAK1. These results indicate that Iap2 lies downstream of TAK1 in the Imd signaling pathway. To ascertain efficacies of the dsRNA treatments used, effect of the dsRNA treatments on E. coli-induced Att response was simultaneously measured. All of the dsRNA treatments strongly decreased the Att response, suggesting that the expression of targeted genes was effectively silenced. Of note, it was not possible to stimulate the Imd pathway with the expression vector containing the full-length cDNA of TAB (Kleino, 2005).
Upon Imd pathway activation, the NF-kappaB homolog Relish becomes phosphorylated by the IKK complex and thereafter cleaved by a caspase putatively thought to be Dredd. Finally, Relish is translocated to the nucleus. To study the role of TAB and Iap2 on Relish cleavage, Drosophila hemocyte-like mbn-2 and S2 cells were stimulated with commercial lipopolysaccharide (LPS) known to contain a bacterial component that activates the Imd pathway, followed by Western blotting with Relish antibody (alpha-C; Stöven, 2000). In unstimulated, GFP dsRNA-treated mbn-2 cells, most of Relish is uncleaved (Relish-110), whereas upon LPS stimulus, Relish cleavage is induced. As expected, in Dredd and key dsRNA-treated cells Relish cleavage was blocked. Interestingly, TAB, Iap2 or TAK1 dsRNA did not affect Relish cleavage. Similar results were obtained also in S2 cells. This points to a novel mechanism of regulation of Relish activity. There was no Relish detected in Rel dsRNA-treated cells, indicating that the half-life of REL-49 (C-terminal Relish cleavage product) is less than the duration of the dsRNA treatment. Of note, REL-49 was observed also in all Dredd dsRNA-treated cells. It is possible that after RNAi knockdown, there is a small amount of Dredd left, sufficient to cleave REL-110 in unstimulated cells. Alternatively, there is some constitutively cleaved Relish in cell lines and this cleavage is Dredd independent (Kleino, 2005).
To investigate whether Iap2 or TAB play a role in the nuclear localization of the activated Relish protein, dsRNA-treated S2 cells were stained with alpha-RHD antibody (Stöven, 2000). In GFP dsRNA-treated cells, Relish is translocated into the nucleus upon LPS stimulus. As expected, there is no nuclear staining of Relish in Dredd or key dsRNA-treated, LPS-stimulated S2 cells. Importantly, the nuclear translocation of Relish appears to be affected in both Iap2 and TAB dsRNA-treated cells compared to GFP dsRNA-treated controls. This suggests that cleavage of Relish is not sufficient for translocation of Relish to the nucleus but another, yet to be characterized signal that is propagated via Iap2 and TAB is required. Alternatively, a different staining pattern could be due to decreased stability of nuclear Relish or slower kinetics. Of note, compared to key and Dredd dsRNA-treated cells, very faint nuclear staining can be seen in Iap2 dsRNA-treated cells. Surprisingly, Relish nuclear localization was normal in TAK1 dsRNA-treated cells. This implies a possibility that the role of TAK1 in the Imd pathway signaling is downstream of translocation of Relish into the nucleus. Altogether, these results show that the regulation of Relish activity is more complex than previously thought. The involvement of TAB and Iap2 in nuclear localization but not cleavage of Relish indicates a novel mode of regulation in the Imd pathway (Kleino, 2005).
Iap2 codes for a 498 amino-acid (aa) protein that has three N-terminal BIR (baculovirus IAP repeat) domains and a C-terminal RING-finger (Really Interesting New Gene) domain. Drosophila Iap2 is well conserved throughout phylogeny and has high sequence similarity with many mammalian Iap2s, such as human, rat and mouse (E values 9 x 10-66, 2 x 10-66 and 3 x 10-66, respectively). Interestingly, the CARD (caspase recruitment domain), identified in apoptotic signaling proteins, is present in the mammalian homologs but missing from Drosophila. It has been shown that RING domain containing proteins, including IAPs, bind E2 ubiquitin-conjugating enzymes catalyzing the transfer of ubiquitin from E2 to a substrate, therefore acting as E3 ligases. Ubiquitination can lead to either proteasomal degradation, or, in the case of non-K48-linked polyubiquitination, to multiple outcomes such as activation or relocalization of the substrate protein. Human c-Iap2 is expressed most strongly in immune tissues including spleen and thymus and has been proposed to associate with TRAFs through its BIR domains (Rothe, 1995). However, in a luciferase assay, neither TRAF1 nor TRAF2 dsRNA treatment reduced the Imd pathway activity, indicating that TRAFs are not essential for Imd pathway activity in Drosophila S2 cells (Kleino, 2005).
The Drosophila TAB codes for an 831 aa protein that has an N-terminal CUE domain (97-139 aa) and a C-terminal zinc-finger (ZnF) domain (765-789 aa). Only two other Drosophila genes code for a CUE domain: CG2701, and CG12024. Their function is unknown. TAB is the only Drosophila protein with both CUE and ZnF domains. These domains are homologous to the respective domains in mammalian TABs. The CUE domain carries a ubiquitin-binding motif, whereas the ZnF domain has an alpha-helical coiled-coil region. It has been shown that Drosophila TAK1 binds TAB (CG7417) in a two-hybrid protein interaction system (Giot, 2003). In humans, the C-terminal coiled-coil domain of TAB3 mediates the association with TAK1, and it is also required for stimulation of TAB3 ubiquitination by TRAF6 (Ishitani, 2003). Apart from these two domains, there is very little sequence similarity, suggesting that these domains are functionally important. Indeed, it has been shown that the ZnF domain and the CUE domain, to a lesser extent, of human TAB2 and TAB3 are important to NF-kappaB activation (Kanayama, 2004). The exact molecular mechanism by which Iap2 and TAB modulate signaling via the Imd pathway in Drosophila remains to be studied (Kleino, 2005).
This study identified two novel components of the Imd signaling cascade: Iap2 and TAB. Both of these have mammalian homologs, further indicating high conservation of this signaling cascade. TAB is an 831 aa protein that has conserved CUE and ZnF domains. As in mammals, it is plausible that TAB regulates TAK1 activity also in Drosophila, since TAB was the only protein to bind TAK1 in a two-hybrid protein interaction system (Giot, 2003). Both Iap2 and TAB are located downstream of Imd. Interestingly, Relish is cleaved appropriately without Iap2 or TAB, but there appears to be an effect to the transportation of Relish to the nucleus. Therefore, it is speculated that there is another, previously unidentified level of regulation needed for Relish activation. Since the RING domain-containing Iap2 is a putative E3 ligase, it is hypothesized that this regulation could involve ubiquitination of Relish -- or another protein regulating the activity of Relish -- by Iap2. Possible interaction of Iap2 with the other Imd pathway components remains to be studied (Kleino, 2005).
Surprisingly, Relish nuclear localization is normal in TAK1 dsRNA-treated cells. This implies a possibility that the role of TAK1 in the Imd pathway signaling is downstream of translocation of Relish into the nucleus. These results are in line with recent results from Delaney (in preparation, reported by Kleino, 2005), which indicate that Relish activation is intact in TAK1 mutant flies. Therefore, TAK1 may control the activity of another transcription factor -- possibly via the JNK pathway -- required for normal antimicrobial peptide response in Drosophila. This is in line with identification of Kayak as an important factor for Att response in this study and with earlier results indicating that the JNK pathway is required for normal Att response in S2 cells (Kallio, 2005). Of note, the effect of dsRNA treatments targeting JNK pathway components is more modest compared to TAK1 RNAi in this experimental setting. This is in line with the earlier results of Silverman (2003), who showed that in S2* cells TAK1 RNAi totally blocks Dpt, Cecropin and Att response to LPS, whereas RNAi targeting JNK pathway components hemipterous and basket have a more moderate effect. Nevertheless, the regulatory interplay that has been detected between the Imd and the JNK pathway in the Drosophila innate immune response (Boutros, 2002: Park, 2004) is likely to attract more attention in the future (Kleino, 2005).
This present study underlines the convenience of RNAi-based screening in S2 cells. Importantly, two novel components of the Imd pathway have been identified. The exact roles Iap2 and TAB play in the activation of Relish remain to be solved. In addition, these findings will likely focus attention to investigate the importance of Iap2 in mammalian TNF receptor signaling. This methodology can be readily applied to study other conserved signaling cascades (Kleino, 2005).
Innate immunity in vertebrates and invertebrates is of central importance as a biological programme for host defence against pathogenic challenges. To find novel components of the Drosophila immune deficiency (IMD) pathway in cultured haemocyte-like cells, RNA interference library was screened for modifiers of a pathway-specific reporter. Selected modifiers were further characterized using an independent reporter assay and placed into the pathway in relation to known pathway components. Interestingly, the screen identified the Inhibitor of apoptosis protein 2 (IAP2) as being required for IMD signalling. Whereas loss of DIAP1, the other member of the IAP protein family in Drosophila, leads to apoptosis, IAP2 is dispensable for cell viability in haemocyte-like cells. Cell-based epistasis experiments show that IAP2 acts at the level of Tak1 (transforming growth factor-ß-activated kinase 1). The results indicate that IAP gene family members may have acquired other functions, such as the regulation of the tumour necrosis factor-like IMD pathway during innate immune responses (Gesellchen, 2005).
The results indicated that IAP2 is required for the IMD-Rel branch and cell-based epistasis mapped it downstream of IMD and upstream of Relish. Since the IMD pathway branches at the same level or downstream of Tak1 into Rel- and JNK-dependent signalling, whether IAP2 is also required for activation of the JNK branch was examined. Thus, the expression of the IMD/JNK-specific target genes Puckered and Matrix metalloproteinase 1 (Mmp-1) was examined by qPCR. Depletion of IAP2 by RNAi disrupts Mmp-1 and puc induction after innate immune stimuli to a level similar to that of knockdown of known factors specific for the IMD−JNK branch (Mkk4/hep). These experiments, together with the epistasis experiments, support a model whereby IAP2 acts, similarly to Tak1, downstream of IMD and upstream or at the level of the branching point of the IMD−Rel and IMD−JNK signalling arms (Gesellchen, 2005).
This study has identified several new components of the IMD innate immune pathway. The experiments implicate several signalling factors in the control of IMD-dependent responses in haemocyte-like cells, including a GTPase-activating protein, a homologue of the mammalian Tak1-binding protein, and several proteins involved in RNA binding and processing. Their role in Drosophila immune response in vivo remains to be characterized. Strikingly, the screen identifies IAP2, a member of the Inhibitor of Apoptosis Protein family, as being required for Drosophila innate immune signalling. IAP2 is specifically involved in the IMD signalling pathway, since it disrupts the induction of the IMD-Rel and IMD-JNK pathway target genes and is not required for other immune-induced pathways, such as Toll and JAK-STAT. Cell-based epistasis analysis and qPCR experiments monitoring the IMD-JNK branch suggest a function of IAP2 downstream of IMD and upstream or at the same level as Tak1. Although most previously characterized IAPs were shown to act as inhibitors of caspases, it is unlikely that the role of IAP2 is to inhibit DREDD, the caspase implicated in IMD signalling. If this were correct, depletion of IAP2 should lead to an enhancement of pathway activity after immune stimulus or to a constitutive expression of target genes without an immune stimulus, which is not the case. Since human Tak1 has been shown to be activated by polyubiquitination, and it has recently been shown that ubiquitination is required for the activation of Tak1 and the IKK complex in Drosophila, it was speculated that IAP2 may have a role in Tak1 ubiquitination through its RING domain. Whether mammalian IAPs have a role in innate immune responses remains to be established (Gesellchen, 2005).
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).
In the Drosophila gut, reactive oxygen species (ROS)-dependent immunity is critical to host survival. This is in contrast to the NF-kappaB pathway whose physiological function in the microbe-laden epithelia has yet to be convincingly demonstrated despite playing a critical role during systemic infections. A novel in vivo approach was used to reveal the physiological role of gut NF-kappaB/antimicrobial peptide (AMP) system, which has been 'masked' in the presence of the dominant intestinal ROS-dependent immunity. When fed with ROS-resistant microbes, NF-kappaB pathway mutant flies, but not wild-type flies, become highly susceptible to gut infection. This high lethality can be significantly reduced by either re-introducing Relish expression to Relish mutants or by constitutively expressing a single AMP to the NF-kappaB pathway mutants in the intestine. These results imply that the local 'NF-kappaB/AMP' system acts as an essential 'fail-safe' system, complementary to the ROS-dependent gut immunity, during gut infection with ROS-resistant pathogens. This system provides the Drosophila gut immunity the versatility necessary to manage sporadic invasion of virulent pathogens that somehow counteract or evade the ROS-dependent immunity (Ryu, 2006).
The intestinal NF-kappaB activation and subsequent local AMP induction are key elements of gut immunity in Drosophila. Some earlier studies in mammals have also described the in vivo protective role of mammalian AMPs against certain invasive pathogenic infections occurring in the barrier epithelia including the intestine and the skin. In Drosophila gut immunity, it has been shown that ROS-mediated antimicrobial response is essential for host survival during gut infection. In addition to oxidant-dependent immunity, phagocytosis by macrophages also plays an important role in a gut infection model. The present study revealed that in the Drosophila gastrointestinal tract, NF-kappaB/AMP-dependent innate immunity is normally dispensable but provisionally crucial in case the host encounters ROS-resistant microbes. Although the precise mechanism by which ROS-resistant microbes induce epithelial cell damages remains to be investigated, it can be speculated that high numbers of local microbes may produce metabolites toxic to the gut epithelia. Alternatively, it is also possible that excess chronic inflammation due to persistent microbes may cause host gut pathology similar to host immune effector-induced metabolic collapse observed in a Salmonella-infected Drosophila model (Ryu, 2006).
It should be noted that yeast and E. coli are not pathogens for the fly in normal situations and that manipulations to render these microbes ROS resistant may not directly reflect natural infection pathways in the animal. However, since ROS are known to be involved in many of the complex interactions between the invading microorganisms and the host, this approach will likely be a relevant method in understanding the integrative relationship between gut immunity and microbial pathogenesis. Arthropod gut immunity during host–pathogen interactions is particularly interesting because the majority of deadly arthropod-transmitted pathogens/parasites causing illnesses such as malaria, plague, typhus and lyme disease have evolved to use the host's gut as a route of transmission. Within the context of pathogen survival strategies, microbial pathogens must evade or counteract innate immune effectors such as ROS and AMPs in order to disseminate and cause diseases. In a constant competition for survival, the pathogen and the host have developed strategies to overcome the other. Along with the highly efficient microbicidal ROS, the Drosophila gastrointestinal tract has been shown to express at least seven different IMD/NF-kappaB-dependent AMPs, including Drosomycin, each exhibiting a distinct spectrum of in vitro antimicrobial activity. In this context, it is proposed that the different spectra of microbicidal activity encompassed by ROS and AMPs may provide the necessary versatility to the Drosophila gastrointestinal innate immune system to ward off microbial infections. Furthermore, these findings suggest that the diversification of intestinal innate immune effectors into ROS and AMP systems might have been driven by selective pressures exerted on the Drosophila gastrointestinal tract by its constant interactions with a series of different microbial species that employ different immune-evasion strategies (Ryu, 2006).
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 sequence of imd1 differs from wild-type imd by a single nucleotide substitution, changing Ala31 to Val31. Although this substitution could seem minor, the mutation fully accounts for the immune deficiency phenotype of imd1 flies. This is indeed demonstrated by observations that the introduction of a wild-type copy of imd into mutant imd1 flies is sufficient to restore (1) immune inducibility of all the antibacterial peptides; (2) binding of protein extracts from immune-challenged flies to NF-kappaB-responsive elements; and (3) survival to bacterial infections. This inference is further supported by the observation that transfection of wild-type imd into S2 cells leads to expression of antibacterial peptide genes, whereas the mutated imd1 form fails to induce this expression (Georgel, 2001).
The Drosophila innate immune system discriminates between pathogens and responds by inducing the expression of specific antimicrobial peptide-encoding genes through distinct signaling cascades. Fungal infection activates NF-kappaB-like transcription factors via the Toll pathway, which also regulates innate immune responses in mammals. The pathways that mediate antibacterial defenses, however, are less defined. Loss-of-function mutations are reported in the caspase encoding gene dredd, which block the expression of all genes that code for peptides with antibacterial activity. These mutations also render flies highly susceptible to infection by Gram-negative bacteria. These results demonstrate that Dredd regulates antibacterial peptide gene expression, and it is proposed that Dredd, Immune Deficiency and the P105-like rel protein Relish define a pathway that is required to resist Gram-negative bacterial infections (Leulier, 2000).
To identify genes that control Drosophila antibacterial immune responses, a screen was carried out for mutations on the X chromosome that affect the expression of the antibacterial peptide gene diptericin after bacterial infection. Among 2500 EMS mutagenized lines, five viable, recessive mutations (named B118, F64, L23, D55, D44) were isolated of a gene that is required for the expression of a diptericin-GFP reporter gene in larvae after bacterial infection. In addition, Northern blot analysis shows that adults homozygous for each of the five alleles do not express the diptericin gene after bacterial injection. The B118 allele was mapped to cytological region 1B9-1B13 on the proximal tip of the X chromosome and a small deficiency, Df(1)R194, was identified that does not complement B118. Deficiency Df(1)R194 spans four previously identified genes: rpL36, l(1)1Bi, dredd and su(s). Several results demonstrate that B118 is a mutation in dredd: (1) B118 is allelic to a viable P element insertion (EP-1412) inserted 50 bp upstream of dredd coding sequences; (2) the two genes flanking dredd, su(s) and l(1)1Bi complement B118; (3) a small deficiency, Df(1)dreddD3, which was generated by imprecise P element excision, and which removes dredd and affects the 5' upstream sequences of su(s), blocks diptericin expression after bacterial infection, and (4) a P element insertion, P[dredd+], containing 7.6 kb of genomic DNA, including dredd but not su(s) and l(1)1Bi , fully restores diptericin expression in B118 flies. All five dredd EMS mutations block diptericin expression after infection to the same degree as Df(1)dreddD3, indicating that they are probably null alleles. The P element insertion in line EP-1412 generates a strong hypomorphic dredd mutation since a small amount of diptericin expression is detectable after infection (Leulier, 2000).
dredd encodes an apical caspase and is an effector of the apoptosis activators reaper, grim and hid. One or more dredd transcripts are specifically enriched in cells programmed to die and dredd overexpression induces apoptosis in SL2 cells. In mammals, the closest dredd homologs are caspases 8 and 10, which mediate apoptosis induced by members of the tumor necrosis factor receptor family. Caspases are produced as inactive zymogens termed pro-caspases; when activated, mature caspases catalyze the proteolytic cleavage of death substrates that are associated with apoptosis. The isolation of mutations in dredd that block diptericin expression after infection demonstrate that Dredd also regulates immune responses. In addition, a dredd-lacZ reporter gene is constitutively expressed in all adult and larval tissues including the fat body, the major immuno-responsive tissue in insects. Infection does not, however, appear to increase dredd expression levels (Leulier, 2000).
The five dredd alleles all contain point mutations that affect different regions of the dredd protein. Alleles B118, D55 and F64 generate either premature stop codons or frameshift changes in the Dredd prodomain. D44 has a missense mutation in sequences encoding the first death effector domain (DED), a region thought to mediate protein-protein interactions. In the protein encoded by allele L23, a tryptophan (W) in the caspase domain is replaced by an arginine (R) residue. The strong phenotype of alleles D44 and L23 indicates that Dredd domains affected in these alleles are essential for Dredd function in immunity (Leulier, 2000).
The isolation of dredd mutations that block diptericin expression enabled the characterization of dredd's role in mediating Drosophila antimicrobial host defense as well as dredd's relationship to other genes that function in this response. Pricking adult flies with a mixture of Gram-positive and Gram-negative bacteria activates the expression of all the genes that encode antimicrobial peptides in Drosophila. In the dreddB118 mutant, however, mixed Gram-positive/Gram-negative infections only induce the expression of the antifungal gene drosomycin and the gene coding for Metchnikowin, which has both antifungal and antibacterial activity; diptericin, cecropin A, attacin A and defensin are expressed at <5% of wild-type levels and metchnikowin is expressed at 50% of the wild-type level. Antimicrobial gene expression is similarly affected in flies homozygous for relE20, a strong or null mutant allele of relish and imd, although most of the antibacterial genes are expressed at slightly higher levels in imd flies. By contrast, a mutation in the spz gene, which blocks Toll activation, reduces drosomycin induction by mixed Gram-negative/Gram-positive bacterial infection and reduces the induction of some of the antibacterial genes (defensin, attacin, cecropin A). These data demonstrate that mutations in dredd are phenotypically similar to mutations in imd and relish, and that these three genes regulate all Drosophila antibacterial peptide gene expression (Leulier, 2000).
To define further the roles of imd, dredd and relish in activating metchnikowin and drosomycin after different types of bacterial infection, metchnikowin and drosomycin expression were quantified in different mutant backgrounds 6 h after infection with either Gram-negative Escherichia coli or Gram-positive Micrococcus luteus bacteria. The dreddB118 and relE20 mutations strongly reduce metchnikowin and drosomycin induction by Gram-negative bacterial infections, while the imd mutation has a weak effect; by contrast, metchnikowin and drosomycin are expressed at close to wild-type levels in the imd, dreddB118 and relE20 mutants after Gram-positive bacterial infection. It is concluded, therefore, that dredd and relish play a greater role in inducing metchnikowin and drosomycin after Gram-negative bacterial infection than after Gram-positive bacterial infection (Leulier, 2000).
The observation that drosomycin and metchnikowin expression is almost completely abolished in imd;Toll double mutants suggests that Gram-positive bacterial infection triggers the expression of metchnikowin and drosomycin via the Toll pathway. In agreement, this analysis shows that mutations in spz affect drosomycin gene expression more strongly after Gram-positive than after Gram-negative bacterial infection, and that the constitutive activation of the Toll pathway in the Tl10b mutant leads to drosomycin expression in the absence of dredd activity. metchnikowin, however, is still expressed to a high level in spz mutants after Gram-positive bacterial infection, indicating that metchnikowin induction by Gram-positive bacterial infection may also be mediated in part by the Imd pathway (Leulier, 2000).
The susceptibility to microbial infection observed in dredd, imd, relish, spz and imd;spz mutants is correlated with the expression pattern of antimicrobial genes in these mutants. dreddB118, relE20 and imd;spzrm7 adults are highly susceptible to bacterial infection by Gram-negative bacteria, and imd adults are slightly less susceptible. These survival results confirm that the activation of defense responses to Gram-negative bacterial infection require imd, dredd and relish. Only the imd;spzrm7 double mutants, however, are highly susceptible to bacterial infection by Gram-positive bacteria, indicating that resistance to Gram-positive bacteria is regulated by both the Toll and Imd pathways. Finally, only spzrm7 and imd;spzrm7 mutants are highly sensitive to natural infection by the entomopathogenic fungus Beauveria bassiana or injection of Aspergillus fumigatus spores, confirming that responses to fungi are largely activated by the Toll pathway (Leulier, 2000).
The dredd immune phenotype is similar to the relish and imd phenotypes; it is predicted that the Imd, Dredd and Relish proteins function in a common signaling pathway that regulates antibacterial peptide gene expression. Based on the respective activites of Dredd as a caspase and Relish as a transcriptional transactivator, it is also hypothesized that Dredd functions upstream of Relish in the control of antimicrobial gene expression. This hypothesis is supported by the observation that Dredd is required for Relish activation via endoproteolytic cleavage. It is believed that the weaker effects of the imd mutation on antibacterial gene expression place the imd gene product at an early stage of the antibacterial cascade where multiple responses, some of which bypass imd, trigger the activation of the pathway. Alternatively, the imd mutation may represent a hypomorphic allele (Leulier, 2000).
Caspases were originally identified as effectors of apoptosis, but there is increasing evidence that caspases also function in other physiological processes. Recent studies suggest that the recruitment of the caspase-8 precursor to the TNF-R1 signaling complex either activates NF-kappaB through a Traf2-, RIP-, NIK- and IKK-dependent pathway or, after proteolytic processing of caspase-8, induces apoptosis. The data indicate that Dredd, a close homolog of caspase-8, may also have dual functions in NF-kappaB signaling and apoptosis in Drosophila. Further biochemical analysis is necessary to determine whether Dredd participates directly in Relish activation or functions further upstream (Leulier, 2000).
Deciphering the mechanisms that enable Drosophila to differentiate between pathogens and mount specific immune responses is essential for understanding innate immunity. Recent studies indicate that the Toll pathway is mainly activated in response to fungal and Gram-positive bacterial infection. Several observations suggest that imd, dredd and relish mediate most of the responses to Gram-negative bacterial infection: (1) these genes regulate the antimicrobial peptide genes that are most highly induced by Gram-negative bacterial infection; (2) dredd and relish control the induction of metchnikowin and drosomycin after Gram-negative bacterial infection, and (3) these three genes are required for resistance to Gram-negative bacterial infection. A model is proposed whereby antimicrobial gene expression in Drosophila adults is regulated by a balance of inputs from the Toll pathway and the Imd pathway, which includes Imd, Dredd and Relish, and that these two pathways are differentially activated by different classes of microorganisms. Identifying the receptors that discriminate between invading microbes and stimulate these pathways presents an exciting challenge in the study of innate immunity (Leulier, 2000).
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 TGF-ß activated kinase 1 (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 (Lemaitre, 1995). imd encodes a homolog of the mammalian Receptor Interacting Protein (RIP) (Georgel, 2001). 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; two members of a Drosophila IkappaB kinase (IKK) complex, that is, the kinase DmIKKß and a structural component DmIKKgamma and Dredd, a caspase. 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 (Vidal, 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 compare the Tak1 (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).
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).
Metchnikowin is a recently discovered proline-rich peptide from Drosophila with antibacterial and antifungal properties. Like most other antimicrobial peptides from insects, its expression is immune-inducible. Evidence is presented that induction of metchnikowin gene expression can be mediated either by the Toll pathway or by the imd gene product. The gene remains inducible in Toll-deficient mutants, in which the antifungal response is blocked, as well as in imd mutants, which fail to mount an antibacterial response. However, in Toll-deficient;imd double mutants, metchnikowin gene expression can no longer be detected after immune challenge. These results suggest that expression of this peptide with dual activity can be triggered by signals generated by either bacterial or fungal infection. Cloning of the metchnikowin gene revealed the presence in the 5' flanking region of several putative cis-regulatory motifs characterized in the promoters of insect immune genes: namely, Rel sites, GATA motifs, interferon consensus response elements and NF-IL6 response elements. Establishment of transgenic fly lines in which the GFP reporter gene was placed under the control of 1.5 kb of metchnikowin gene upstream sequences indicates that this fragment is able to confer full immune inducibility and tissue specificity of expression on the transgene (Levashina, 1998).
Drosophila immunity and embryogenesis appear to be linked by an evolutionarily ancient signaling pathway, which includes the Rel-domain transcription factors Dif and dorsal, respectively, as well as a common inhibitor, Cactus. Previous genetic screens have centered on maternal mutants that disrupt the dorsal pathway. In an effort to identify additional components that influence Rel-domain gene function, a search was conducted for immunodeficiency mutants in Drosophila. One such mutant, which maps near the Black cells (Bc) gene, causes a severe impairment of the normal immune response, including attenuated induction of several immunity genes. Survival assays indicate a positive correlation between the induction of these genes, particularly diptericin, and resistance to bacterial infection. These studies are consistent with the notion that insect anti-microbial peptides work synergistically by binding distinct targets within infecting pathogens. Evidence is also presented that non-specific acquired immunity results from the persistence of bacterial metabolites long after primary infection. The potential usefulness of this study with regard to the identification of conserved components of Rel signaling pathways is discussed (Corbo, 1996).
One of the characteristics of the host defense of insects is the rapid synthesis of a variety of potent antibacterial and antifungal peptides. To date, seven types of inducible antimicrobial peptides (AMPs) have been characterized in Drosophila. The importance of these peptides in host defense is supported by the observation that flies deficient for the Toll or Immune deficiency (Imd) pathway, which affects AMP gene expression, are extremely susceptible to microbial infection. A genetic approach has been developed to address the functional relevance of a defined antifungal or antibacterial peptide in the host defense of Drosophila adults. AMP genes have been expressed via the control of the UAS/GAL4 system in imd;spätzle double mutants that do not express any known endogenous AMP gene. These results clearly show that constitutive expression of a single peptide in some cases is sufficient to rescue imd;spätzle susceptibility to microbial infection, highlighting the important role of AMPs in Drosophila adult host defense (Tzou, 2002).
Antimicrobial peptides (AMPs) are a key component of innate immunity. Their distribution throughout the animal and plant kingdom is ubiquitous, reflecting the importance of these molecules in host defense. In insects, systemic infection induces the synthesis of combinations of AMPs that are secreted from the immune organs, mainly the fat body, an analog of the mammalian liver, into the hemolymph, where the AMPs reach high concentrations. In Drosophila, at least seven types of AMPs (plus isoforms) have been described. Their activities have been either determined in vitro by using peptides directly purified from flies or produced in heterologous systems, or deduced by comparison with homologous peptides isolated in other insect species: (1) Drosomycin and Metchnikowin show antifungal activity; (2) Cecropins have both antibacterial and antifungal activities; (3) Drosocin and Defensin are predominantly active against Gram-negative and -positive bacteria, respectively, and (4) Attacins and Diptericins are similar to peptides from other insects that show antibacterial activity (Tzou, 2002 and references therein).
Analysis of the in vivo roles of each AMP on microbial infection is complicated by the numerous AMP genes present in the fly, as well as the redundant defense mechanisms within the innate immune system. The importance of AMPs, however, is supported by the sensitive phenotype of mutants that do not express AMP-encoding genes. A clear correlation is observed between the lack of expression of antibacterial peptide genes in mutants of the Immune deficiency (Imd) pathway and their susceptibility to Gram-negative bacteria. Conversely, mutations in the Toll pathway block Drosomycin expression and result in susceptibility to fungal infection. Finally, mutants deficient in both the Imd and Toll pathways failed to express any known AMP genes after infection and are extremely susceptible to both fungal and bacterial infections. These evidences of the importance of AMPs in fighting infection, however, are still indirect, because it cannot be exclude that these mutations affect other defense reactions. The Toll pathway, for example, has also been reported to regulate hemocyte proliferation. To study unambiguously the in vivo role of each AMP in Drosophila host defense, imd;spätzle (spz) double mutant flies have been created that are deficient for both the Imd and Toll pathways but that constitutively express different AMPs under the control of a noninducible promoter. These flies express only one AMP on infection and, consequently, a simple survival experiment can be used to monitor the contribution of this peptide in resistance to infection by various microorganisms. This powerful assay allowed the analysis, in vivo, of the spectrum of activity of each peptide and, by combining two different transgenes, any potential synergy among them. These results clearly show that expression of a single peptide, in some cases, is sufficient to rescue the imd;spz susceptibility to microbial infection, highlighting the important role of AMPs in Drosophila adult host defense (Tzou, 2002).
In this assay, the AMP genes are expressed via the UAS/GAL4 system at a level similar to that observed in wild-type induction of the endogenous AMP genes (except Defensin and Diptericin). However, there are still some differences between this assay and the wild-type physiological condition. In the UAS-Pep flies, AMP genes are expressed ubiquitously and constitutively, contrasting to the wild-type flies in which peptides are made mainly by the fat body in an acute phase profile. The accumulation of AMP, therefore, through constitutive gene expression before infection may be critical to confer an effective protection (Tzou, 2002).
This study provides an alternative method for monitoring and comparing the antimicrobial activity of the various Drosophila AMPs. Defensin is the most potent peptide against Gram-positive bacteria, whereas Attacin A and Drosomycin are active against Gram-negative bacteria and fungi, respectively. One copy of UAS-Def is sufficient to protect flies to wild-type level against M. luteus, B. subtilis, and S. aureus. The efficiency of Defensin may explain why the endogenous Defensin gene is transcribed to lower levels than the other AMP genes after infection. One copy of UAS-Drs is sufficient to protect against N. crassa, whereas two copies are required to induce a complete and partial protection against F. oxysporum and A. fumigatus, respectively. These results are consistent with the Minimum Inhibitory Concentration assay of Drosomycin required in vitro to kill these three fungi: 0.3-0.6 µM for N. crassa, 1.2-2.5 µM for F. oxysporum, and 20-40 µM for A. fumigatus. In addition, Diptericin in Drosophila contributes to resistance against some Gram-negative bacteria, although its activity is probably underestimated because of the low levels of Diptericin expression generated by the constructs used in this study. Surprisingly, no clear protective effect of Cecropin A could be detected in this assay, whereas Cecropin A peptide shows strong in vitro activity. The possibility cannot be excluded that in the lines used, Cecropin A is not effectively produced or well processed to the active form. Alternatively, a higher level of Cecropin A expression may be required to generate a protective effect, considering that the Drosophila genome contains three other inducible Cecropin genes (Tzou, 2002).
These results also underline the differential activities of Drosophila AMPs: such is the case of Attacin A and Drosocin in resistance to some Gram-negative bacterial species. Thus the existence of numerous AMPs may help widen the protection against a large number of microorganisms. In the case of Gram-negative bacterial infection, none of the peptides are able to restore a wild-type resistance in imd;spz double mutants. These results and the observation that the Drosophila genome encodes a high number of AMP genes with activity directed against Gram-negative bacteria suggest that the elimination of this class of bacteria may require the global toxicity generated by multiple, rather than one or two, AMPs (Tzou, 2002).
This study does not reveal a striking synergistic activity among any pair of AMPs tested. In some cases, a rather cooperative effect is observed between two AMPs such as Attacin A when coexpressed with either Diptericin or Drosocin in resistance to some Gram-negative bacteria. These observations suggest that the multiple Drosophila AMPs may function in an additive way, rather than synergistically (Tzou, 2002).
Host-pathogen interactions are antagonistic relationships in which the success of each organism depends on its ability to overcome the other. The production of AMPs is a common strategy to eliminate the invading microbes and, consequently, pathogens have evolved strategies to prevail over these defenses. The assay used provides a powerful tool to compare the resistance of various bacteria to different AMPs, because in these experiments, microbes were injected in an environment previously enriched in peptides. The time race between pathogen and the host defense is clearly illustrated by the observation that a preexisting level of Defensin is sufficient to ensure a complete resistance against B. subtilis, a Gram-positive bacterium highly pathogenic for flies. This observation indicates that B. subtilis is sensitive to Drosophila AMP but nevertheless can overtake the Drosophila immune response by its rapid growth. The observation that 'immunizing' flies with nonpathogenic bacteria fully protects Drosophila from a subsequent infection by B. subtilis is consistent with this hypothesis. These results also show that the kinetics of infection by P. aeruginosa or B. bassiana, two highly entomopathogenic microbes, are not delayed in flies expressing AMP genes, suggesting that these microbes have developed some mechanisms to escape the AMP activity. The observation that Drosomycin expression does not confer any protection against B. bassiana is unexpected, because Toll-mediated defense against this pathogen has been reported. This observation suggests that other antifungal peptides (e.g., Metchnikowin) or a yet uncharacterized defense reaction may be required to resist this fungus. Finally, the human pathogen, S. aureus, is also highly pathogenic to Drosophila and shows a better resistance to a high level of Defensin compared with other Gram-positive bacteria. These results underline the correlation between pathogenicity and increased resistance to AMPs (Tzou, 2002).
Microarray studies have shown recently that microbial infection leads to extensive changes in the Drosophila gene expression program. However, little is known about the control of most of the fly immune-responsive genes, except for the antimicrobial peptide (AMP)-encoding genes, which are regulated by the Toll and Imd pathways. Oligonucleotide microarrays have been used to monitor the effect of mutations affecting the Toll and Imd pathways on the expression program induced by septic injury in Drosophila adults. Toll and Imd cascades were found to control the majority of the genes regulated by microbial infection in addition to AMP genes and are involved in nearly all known Drosophila innate immune reactions. However, some genes controlled by septic injury were identified that are not affected in double mutant flies where both Toll and Imd pathways are defective, suggesting that other unidentified signaling cascades are activated by infection. Interestingly, it was observed that some Drosophila immune-responsive genes are located in gene clusters, which often are transcriptionally co-regulated (De Gregorio, 2002).
To identify the target genes of the Toll and Imd pathways in response to microbial infection, the gene expression programs induced by septic injury have been compared in wild-type and mutant adult male flies using oligonucleotide microarrays. In parallel, the survival rate and the expression level of various AMP genes have been monitored after infection by various microorganisms. For the Toll pathway, a strong homozygous viable allele of spz (rm7) was selected. The spz, Tl and pll mutations, alone or in combination with rel, have similar effects on both the survival rate and pattern of AMP gene expression after microbial infection. These findings suggest that the effects of spz mutation on the transcription program induced by infection reflect the role of the entire Toll pathway in the immune response. For the Imd pathway, a null viable allele of relish (E20) was selected. Similarly to the Toll pathway, previous comparative studies did not reveal any striking difference between mutations in relish and null mutations in the genes encoding the other members of the Imd pathway such as kenny, ird5 and dredd, with the sole exception of mutations in dTAK1, which have a slightly weaker phenotype. Again, these data suggest that the effects of rel mutation on the immune response reflect the role of the whole Imd pathway. However, other pathways, including Toll, cannot be excluded from having a minor role in Relish activation (De Gregorio, 2002).
The septic injury experiments were performed using a mixture of Gram-positive and Gram-negative bacteria. This type of infection activates a wide immune response and allows the simultaneous analysis of several categories of immune-responsive genes. However, it has been shown that Toll and Imd pathways are activated selectively by different classes of microorganisms; thus, the use of a bacterial mixture might increase the redundancy of the two pathways in the control of common target genes (De Gregorio, 2002).
The microarray analysis demonstrates that the functions of Toll and Imd pathways in Drosophila immunity can be extended beyond the regulation of AMP genes. The majority of the Drosophila immune-regulated genes (DIRGs) are affected by the mutations in the Toll or Imd pathways. Many of these genes are unknown (see www.fruitfly.org/expression/immunity/ for a complete list); others can be assigned to several immune functions. The susceptibility of the Imd and Toll pathway mutants to different types of microbial infection suggested a dual aspect to the control of the antifungal response by the Toll pathway: a major role for the Toll pathway for the response to Gram-positive bacteria with a minor contribution of Imd, and a predominant role of Imd with a minor contribution of Toll to the resistance against Gram-negative bacteria. In agreement, microarray analysis shows that the Toll pathway controls most of the late genes induced by fungal infection and cooperates with the Imd pathway for the control of genes implicated in several immune reactions such as coagulation, AMP production, opsonization, iron sequestration and wound healing. Interestingly, defensin, which encodes the most effective antimicrobial peptide directed against Gram-positive bacteria, is co-regulated by both the Imd and Toll pathways. The hierarchical cluster analysis of the expression profiles combining the effect of the mutations after septic injury with the response to fungal infection provides a wealth of information that may help to elucidate the function of some of the uncharacterized DIRGs. Until now, the increased susceptibility to infection of Imd- or Toll-deficient flies has been attributed to the lack of expression of AMP genes, and it has been shown recently that the constitutive expression of single AMP genes in imd;spz double mutant flies can increase the survival rate of some types of bacterial infection. The finding that the Toll and Imd pathways are the major regulators of the Drosophila immune response now suggests that other immune defence mechanisms might contribute to the increased susceptibility to infection displayed by mutant flies (De Gregorio, 2002).
The interactions between the Toll and Imd pathways are more complex than merely regulating the same target genes. In agreement with Northern blot analysis, it has been shown that the transcriptional control of relish in response to infection receives a modest input from the Toll pathway, revealing an additional level of interaction between the two cascades. The activation of Toll may increase the level of Relish to allow a more efficient response to bacterial infection. This finding is in agreement with previous observations showing that in mutants where the Toll pathway is constitutively active (Tl10b), all the antibacterial peptides genes, including diptericin, are induced with more rapid kinetics than in wild-type flies. Furthermore, the higher susceptibility to E.coli infection of the rel,spz double mutant compared with the rel single mutants flies indicates that Toll also has a direct, Relish-independent effect on the resistance to infection by Gram-negative bacteria. Northern blot analysis shows that relish induction in response to infection is significantly reduced in dTAK1 and dredd mutants, indicating that the Imd pathway undergoes autoregulation. Interestingly, the Imd pathway can influence the Toll pathway through the control of PGRP-SA, which encodes a recognition protein essential for the activation of the Toll pathway by Gram-positive bacteria. Again, it is interesting to notice that this interaction between the Toll and Imd pathways correlates with the contribution of both pathways to fight infection with Gram-positive bacteria. Interestingly, all the genes encoding components of the Toll pathway required for both antibacterial and antifungal responses (necrotic, spaetzle, Toll, pelle, cactus and Dif) are not controlled by the Imd pathway and are subjected to autoregulation (De Gregorio, 2002).
The Rel/NF-kappaB proteins Dif, Dorsal and Relish, which are the transactivators induced by the Toll and Imd pathways, bind to the kappaB sites present in the promoters of target genes, such as AMP genes, regulating their expression. Therefore, the analysis of the promoters of the DIRGs controlled by Toll or Imd pathways could help to identify all the direct NF-kappaB targets during infection. However, some of the effects of mutations affecting the Toll or Imd pathways that were monitored by microarray analysis might be mediated by the regulation of other transcription factors or signaling cascades. It has been shown recently in larvae that the Tep1 gene is regulated by the JAK-STAT pathway and can be activated by the Toll pathway, suggesting that Toll can control, at least partially, the JAK-STAT cascade. Two genes encoding components of the JNK pathway (puc and d-Jun) are partially regulated by Toll and Imd in response to septic injury (De Gregorio, 2002).
The presence of DIRGs independent of or only partially dependent on both the Imd and Toll pathways suggests the presence of other signaling cascades activated after septic injury. Potential candidates are MAPK and JAK-STAT pathways. Beside their developmental functions, the MAPK pathways have been implicated in wound healing (JNK) and the stress response (MEKK). The JAK-STAT pathway controls the Drosophila complement-like gene TepI. The stimuli that trigger these cascades are not known and it is not clear if these cascades are activated by exogenous or host factors. Interestingly, in vertebrates, the JAK-STAT pathway is activated by cytokines during the immune response. The microarray analysis of mutants in these pathways might help to reveal their exact contribution to the Drosophila immune response. The observation that Toll and Imd pathways control most of the DIRGs raises the question of whether these two pathways are the sole signaling cascades directly activated by microbial elictors, while the other signaling pathways are triggered by other stimuli associated with infection such as wound, stress, cytokine-like factors and Toll and Imd activities (De Gregorio, 2002).
In vertebrates, many genes involved in the immune response are grouped in large chromosomal complexes. The recent completion of the Drosophila genome did not reveal any striking chromosomal organization beside clustering of genes belonging to the same family, probably reflecting recent duplication events. In this study, it was observed that some of the genes responding to microbial infection are located in the same cytological region or are associated in transcriptionally co-regulated genomic clusters. Interestingly, microarray analysis of circadian gene expression in Drosophila has led to the identification of similar clusters of genes. Other microarray analyses might reveal the importance of the genome organization in the definition of adequate transcription programs in response to a variety of stimuli (De Gregorio, 2002).
Surfaces of higher eukaryotes are normally covered with microorganisms but are usually not infected by them. Innate immunity and the expression of gene-encoded antimicrobial peptides play important roles in the first line of defence in higher animals. The immune response in Drosophila promotes systemic expression of antimicrobial peptides in response to microbial infection. The epidermal cells underlying the cuticle of larvae respond to infected wounds by local expression of the genes for the antimicrobial peptide cecropin A. Thus, the Drosophila epidermis plays an active role in the innate defence against microorganisms. The immune deficiency (imd) gene is a crucial component of the signal-induced epidermal expression in both embryos and larvae. In contrast, melanization, which is part of the wound healing process, is not dependent on the imd gene, indicating that the signalling pathways promoting melanization and antimicrobial peptide gene expression can be uncoupled (Tingvall, 2001).
The following model is proposed to explain the transition of CecA1 expression in epidermal tissues of the embryo to the larval fat body. During embryogenesis, CecA1 is induced in the epidermis as a result of direct contact between microbial substances in the privitelline fluid and epidermal cells. When the cuticle is formed during late embryogenesis, this direct contact is broken, and the presence of bacteria or LPS in the larval haemocoel does not promote CecA1 expression in the larval epidermis. During the larval stages, CecA1 expression is restricted to wounded areas of the epidermis, suggesting that either the cuticle needs to be removed to allow direct contact between microbial substances and the epithelial cell layer, or that the wounding is an important signal per se. The conclusion is therefore, that the epidermal cells are immuno-competent from the embryonic stage onwards. The fat body, in contrast, seems to undergo maturation during the first larval instar. Expression of CecA1 can be induced in the fat body of all three larval instars, but not in embryos. Injecting live bacteria into embryos and aging them into larvae shows that a maturation in the immuno-competence of the fat body occurs during first larval instar (Tingvall, 2001).
It was not possible to restore CecA1-driven reporter gene expression in the larval epidermis by inflicting infected wounds in all three larval instars. In addition to the cuticle being a physical barrier it is possible that the larval epidermis at the stage of hatching normally contains cecropins, since a low level of constitutive CecA1 expression is observed in the epidermis of late stage embryos. Some of