InteractiveFly: GeneBrief

immune deficiency : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - immune deficiency

Synonyms - BG5

Cytological map position - 55C9--11

Function - signal transduction

Keywords - immune response

Symbol - imd

FlyBase ID: FBgn0013983

Genetic map position -

Classification - death domain protein

Cellular location - cytoplasmic

NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Chen, L., Paquette, N., Mamoor, S., Rus, F., Nandy, A., Leszyk, J., Shaffer, S. A. and Silverman, N. (2017). Innate immune signaling in Drosophila is regulated by TGFbeta-activated kinase (Tak1)-triggered ubiquitin editing. J Biol Chem [Epub ahead of print]. PubMed ID: 28377500
Coordinated regulation of innate immune responses is necessary in all metazoans. In Drosophila, the Imd pathway detects gram-negative bacterial infections through recognition of DAP-type peptidoglycan and activation of the NF-kappaB precursor Relish, which drives robust antimicrobial peptide (AMP) gene expression. Imd is a receptor-proximal adaptor protein homologous to mammalian RIP1 that is regulated by proteolytic cleavage and K63-polyubiquitination. However, the precise events and molecular mechanisms that control the post-translational modification of Imd remain unclear. This study demonstrates that Imd is rapidly K63-polyubiquitinated at lysine residues 137 and 153 by the sequential action of two E2 enzymes, Ubc5 (Effete) and Ubc13 (Bendless)-Uev1a, in conjunction with the E3 ligase Diap2. K63-ubiquitination activates the TGFβ-activated kinase (Tak1), which feeds back to phosphorylate Imd, triggering the removal of K63-chains and the addition of K48-polyubiquitin. This ubiquitin editing process results in the proteosomal degradation of Imd, which is proposed to function to restore homeostasis to the Drosophila immune response.
Daisley, B. A., Trinder, M., McDowell, T. W., Welle, H., Dube, J. S., Ali, S. N., Leong, H. S., Sumarah, M. W. and Reid, G. (2017). Neonicotinoid-induced pathogen susceptibility is mitigated by Lactobacillus plantarum immune stimulation in a Drosophila melanogaster model. Sci Rep 7(1): 2703. PubMed ID: 28578396
Pesticides are used extensively in food production to maximize crop yields. However, neonicotinoid insecticides exert unintentional toxicity to honey bees (Apis mellifera) that may partially be associated with massive population declines referred to as colony collapse disorder. It was hypothesized that imidacloprid (common neonicotinoid; IMI) exposure would make Drosophila melanogaster (an insect model for the honey bee) more susceptible to bacterial pathogens, heat stress, and intestinal dysbiosis. The results suggested that the immune deficiency (Imd) pathway is necessary for D. melanogaster survival in response to IMI toxicity. IMI exposure induced alterations in the host-microbiota as noted by increased indigenous Acetobacter and Lactobacillus spp. Furthermore, sub-lethal exposure to IMI resulted in decreased D. melanogaster survival when simultaneously exposed to bacterial infection and heat stress (37 degrees C). This coincided with exacerbated increases in TotA and Dpt (Imd downstream pro-survival and antimicrobial genes, respectively) expression compared to controls. Supplementation of IMI-exposed D. melanogaster with Lactobacillus plantarum ATCC 14917 mitigated survival deficits following Serratia marcescens (bacterial pathogen) septic infection. These findings support the insidious toxicity of neonicotinoid pesticides and potential for probiotic lactobacilli to reduce IMI-induced susceptibility to infection.

In the mid 1990s, it became apparent that two distinct pathways control the antifungal and antibacterial responses (Lemaitre, 1995 and 1996). Indeed, it was found that the dorsoventral regulatory gene cassette spätzle/Toll/cactus directs the potent antifungal response in Drosophila adults, whereas the antibacterial defense is largely independent of these genes (Lemaitre, 1996). A mutation was discovered at that time, and referred to as imd (immune deficiency) in which the antibacterial, but not the antifungal response, was compromised (Lemaitre, 1995). Since the initial description of the imd mutation, four additional genes have been shown to participate in the antibacterial defense of Drosophila: (1) ird5, a gene encoding a homolog of mammalian IKKß (Lu, 2001; Silverman, 2000); (2) kenny, a homolog of IKKgamma/NEMO (Rutschmann, 2000; Silverman, 2000); (3) dredd, which is structurally related to vertebrate caspase-8 (Elrod-Erickson, 2000; Leulier, 2000; and (4) Relish, a member of the NF-kappaB family (Hedengren, 1999) (Georgel, 2001 and references therein).

The imd gene has now been identified. This gene encodes a 30 kDa protein with a death domain that exhibits significant similarity with the death domain of mammalian RIP (receptor interacting protein), a protein associating with the TNFalpha receptor 1 and with the Fas receptor. Epistasis experiments indicate that imd acts in the antibacterial defense upstream of the four genes mentioned above. Overexpression of the imd gene in wild-type and imd mutant flies results in forced transcription of all genes encoding antibacterial peptides, but not of that coding for the antifungal peptide Drosomycin. Similar to the dual function of RIP in vertebrates, the data also suggest a possible role for imd in apoptosis. Overexpression of imd induces transcription of the reaper gene and causes the massive appearance of TUNEL-reactive cells in the adult fat body. The effects of ectopic expression of imd on the transcription of antibacterial peptides and on the generation of TUNEL-reactive cells are blocked by coexpression of the baculovirus antiapoptotic protein P35. imd mutant flies are more resistant than control flies to UV irradiation, suggesting that imd may be involved in the apoptotic response to DNA damage (Georgel, 2001).

The induction of antimicrobial peptide genes is correlated in Drosophila to the binding of Rel proteins to kappaB-related responsive elements in their promoter sequences. This type of binding does not occur in imd1 mutant flies (Lemaitre, 1995). The ability of protein extracts from immune-challenged imd1 and imd plus transgenic flies to bind to a labeled oligonucleotide containing a kappaB-related motif from the diptericin promoter was analyzed by gel shift experiments. The binding capacity was rescued in the transgenic flies and was specific for the nucleotide sequence, as evidenced by incubation with a mutated probe (Georgel, 2001).

imd1 flies are susceptible to infection with bacteria (Lemaitre, 1995 and 1996; Leulier, 2000; Rutschmann, 2000). The survival to infection with E. coli and M. luteus (mixture) of imd1 and transgenic flies carrying a wild-type copy of BG5 was examined. As expected, imd1 flies are susceptible to infection in these experiments. However, the transgenic flies have a survival rate similar to that of wild-type flies, demonstrating that BG5 can rescue the imd1 survival phenotype. BG5 will therefore be referred to as the imd gene. The death rates of hemizygous imd1/Df (2R) 2.1 flies, which are similar to those of dredd and kenny alleles (Leulier, 2000; Rutschmann, 2000), are lower than those of imd1 homozygous flies. These data indicate that imd1 is a hypomorphic mutation. The deficiency introduced in this context has no effect by itself, as evidenced by the survival curve of the hemizygous + /Df (2R) 2.1 flies (Georgel, 2001).

Wild-type and imd1 mutant fly lines carrying a UAS-IMD transgene were generated and the effects of overexpression of imd were analyzed using different GAL4 drivers. Expression of imd directed by the hs-GAL4 driver in imd1 mutant flies restores the immune inducibility of all the antibacterial peptides to levels comparable to wild-type. Remarkably, in these flies, the transcription of all the antibacterial peptide genes occurs even in the absence of immune challenge, indicating that overexpression of imd has a dominant effect on the activation of the pathway controlling the antibacterial peptide genes. drosomycin expression is not affected in these experiments; this is consistent with previous observations that drosomycin expression is independent of imd (Lemaitre, 1996). In UAS-IMD/hs-GAL4 flies a constitutive expression of antibacterial peptide genes is observed even in the absence of heat shock. However, no spontaneous expression of these genes was detected in any of the original fly lines carrying the UAS-IMD insertion. It is therefore proposed that the basal level of GAL4 expression from the hsp promoter accounts for the UAS-IMD-dependent transcription of the antibacterial peptide genes. This assumption is supported by the observation that the hs-GAL4 driver is able to sustain a basal level of expression of a UAS-GFP and a UAS-lacZ reporter gene in the absence of heat shock. Overexpression of imd by hs-GAL4 also has a dominant effect on antibacterial genes in wild-type flies (Georgel, 2001).

The effect of overexpression of imd was further analyzed by selecting a tissue-specific driver, yolk-GAL4, which directs transcription of UAS-dependent genes in the fat body of adult females starting 3 to 5 days after hatching. diptericin is strongly expressed in these flies in the absence of immune challenge, but not in unchallenged males of the same genotype (Georgel, 2001).

Expression of most antimicrobial peptide genes is induced by LPS in the Drosophila macrophage-like S2 cell line. These cells are transfected with an imd expression vector and strong expression is observed of attacin- (and drosocin-) luciferase reporter genes in the absence of LPS. In contrast, transfection with mutated imd (imd1) is unable to induce the attacin-luciferase reporter gene. Lack of induction of the attacin reporter gene by the imd1 construct does not reflect the inability of the cells to synthesize the recombinant protein, since the wild-type and mutated IMD proteins are expressed at comparable levels in transfected cells. These results corroborate the in vivo data indicating that the Ala31 to Val31 substitution in the imd1 allele fully accounts for the immune-deficient phenotype (Georgel, 2001).

The imd gene acts upstream of DmIKKgamma and the Caspase Dredd to activate the expression of antibacterial peptide genes. The dominant effect on antibacterial peptide genes of overexpression of imd driven by the hs-GAL4 driver (even in the absence of heat shock) serves to establish the epistatic relationships of imd with other genes in the pathway. In dredd and kenny mutant flies, expression of all antibacterial peptide genes in response to bacterial challenge, as well as survival to infection, is strongly impaired (Elrod-Erickson, 2000; Leulier, 2000; Rutschmann, 2000). The overexpression of imd is unable to confer challenge-independent expression of diptericin in dreddD55 and kenny1 mutant backgrounds. Furthermore, it does not restore immune inducibility of the diptericin gene in these mutants. It is noteworthy that drosomycin expression is not affected in these flies. These results demonstrate that imd acts upstream of both dredd and kenny to activate the antibacterial peptide genes during an immune response. In keeping with these results, transcription of the attacin-luciferase reporter gene induced by overexpression of imd in S2 cells is abolished by an ird5 (IKKß homolog) dominant-negative expression construct (Georgel, 2001).

Transgenic flies were examined in which imd expression was placed under the control of the ubiquitous da-GAL4 driver. It was surprising to observe 100% lethality in these flies during early larval development. The lethality was partially rescued by coexpression of the viral caspase inhibitor P35. This protein inactivates most of the executioner caspases of the death program. It was further noted that overexpression of imd by the fat body-specific driver yolk-GAL4 induced the transcription of a reaper-lacZ reporter in this tissue. Reaper is a key activator of apoptosis in Drosophila. A TUNEL analysis of transgenic flies overexpressing imd also revealed a remarkably large number of labeled nuclei in fat body cells, as compared to controls. This effect was suppressed by coexpression of the antiapoptotic protein P35. Transmission electron microscopy analysis revealed that the fat body cells exhibit the classical morphological aspects of apoptosis, that is, densification and fragmentation of the cytoplasm, membrane blebbing, and stacking of the endoplasmic reticulum (Georgel, 2001).

These observations raised the question as to whether the imd-dependent antibacterial response involves activation of an apoptotic program. However, when injecting gram-negative bacteria into flies, appearance of TUNEL-positive cells are never observed in the fat body nor any induction of the reaper reporter gene, although the antibacterial peptide genes were fully induced. It was reasoned that the imd-dependent control of an apoptotic pathway could be triggered by stimuli different from bacterial infection. One well-established experimental system that induces apoptosis in Drosophila is DNA damage by UV irradiation. Pupae were subjected to increasing doses of UV treatment and a significant mortality in emerging flies was noted at 50,000 µJ/cm2. This lethality is, at least partially, linked to induction of apoptosis by the UV treatment, since it is significantly reduced in flies overexpressing the apoptosis inhibitor P35. Remarkably, when imd mutant pupae are subjected to UV treatment, lethality is also significantly reduced. The reduction in the level of lethality is clearly related to the product of the imd gene, since in flies overexpressing imd an increased sensitivity to UV irradiation is observed. Also, reintroduction of a wild-type copy of the imd gene into imd1 mutant flies restores their sensitivity to UV irradiation to levels comparable to wild-type. Interestingly, dredd mutant flies are also resistant to UV irradiation. In contrast to the clear effect of UV damage on apoptosis-related mortality, this treatment does not induce the expression of diptericin in the absence of a microbial challenge (Georgel, 2001).

The observations that the imd-induced cell death is antagonized by P35 raises the question as to whether P35 also affects the immune induction of antibacterial peptides. Overexpressing p35 in adult fat body cells blocks the challenge-dependent transcription of diptericin. Furthermore, the induction of the diptericin gene by overexpression of imd is also abolished by coexpressing p35. Note that induction of drosomycin by an immune challenge is not affected by P35. These results indicate that P35-sensitive caspases act downstream of imd.

It is fully appreciated that wild-type imd is mandatory for the immune induction of Diptericin and the other antibacterial peptides, but not for that of the antifungal peptide Drosomycin (Lemaitre, 1995, 1996). Four distinct genes have now been characterized that, when mutated, induce a roughly similar phenotype. These genes code for three well-established components within signaling cascades: (1) Relish is a member of the NF-kappaB family of inducible transactivators. Like its mammalian counterpart, p105, it has a C-terminal I-kappaB-like domain, which accounts for its retention in the cytoplasm in the absence of challenge. Nuclear translocation of the Rel homology domain of Relish requires endoproteolytic cleavage by an as yet unidentified protease. (2) Two genes code for the components of a mammalian signalosome equivalent, that is, for homologs of IKKß (ird5) and IKKgamma (kenny). Both genes act upstream of Relish, by a mechanism which has not been established, but which may involve phosphorylation of Relish by IKKß. (3) Finally, the dredd gene encodes a close homolog of mammalian caspase-8. Since Relish is not challenge-dependently cleaved in dredd mutant flies, DREDD must act upstream of this transactivator in the signal transduction pathway (Georgel, 2001).

A major concern in this study was to clarify the relationship between imd and these four other known genes in the pathway. The results clearly show that imd acts upstream of both kenny and dredd. Taken together with other studies they indicate that IMD functions upstream of the DmIKK signaling complex, which in turn activates Relish directly or indirectly. These data establish an imd/kenny/ird5/Relish gene cassette in the control of the antibacterial peptide genes. The presence in IMD of a death domain with similarity to that of the adaptor protein RIP suggests that the IMD protein may be part of a receptor-adaptor complex. By analogy with the role of RIP in TNFR1 signaling, IMD could function by recruiting the signalosome (DmIKKgamma/DmIKKß) to the upstream receptor-adaptor complex (Devin, 2000; Poyet, 2000; Zhang, 2000) (Georgel, 2001 and references therein).

Both the receptor and the ligand(s) activating the IMD pathway remain unknown. This is in sharp contrast with the Toll pathway, which controls the antifungal response of Drosophila and namely the synthesis of the antifungal peptide Drosomycin. In the latter case, genetic analysis indicates that microbial ligands activate an extracellular proteolytic cascade culminating in the cleavage of the cytokine-like polypeptide Spätzle. Cleaved Spätzle in turn interacts with Toll and initiates a signal transduction pathway that leads to the transcription of drosomycin. This does not, however, activate the synthesis of the antibacterial peptides (Lemaitre, 1996). Conversely, overexpression of imd leads to the expression of the antibacterial peptide genes but not that of drosomycin. These data indicate that within the predominant immune-responsive tissue of adult flies, that is, the fat body, the Toll and the IMD pathways are functionally separated and that each controls, via specific Rel transcription factors, a set of given antimicrobial peptide genes. This functional separation of the two signaling pathways within the cells is corroborated by the results that in flies subjected to infection by a bacterial mix (gram-positives and gram-negatives), expression of the antiapoptotic protein P35 abolishes inducibility of the antibacterial peptides, but not that of drosomycin (Georgel, 2001).

Overexpressing imd results in larval lethality, expression of the reaper gene in fat body cells of adult flies, and apoptosis of these cells. Most of these effects could be, at least partially, rescued by coexpressing the antiapoptotic protein P35. The presence of a death domain in imd, however, might bring these results in line with recent studies showing that overexpression of death domain proteins induces apoptosis in mammalian cells. Overexpressing the death domain proteins RIP, FADD, and TRADD has been shown to lead to cell death by recruitment of apical caspases, namely caspase-8 and -10, which in turn activate downstream effector caspases to execute the apoptotic program. RIP, whose death domain is similar to that of IMD, has been proposed to induce cell death by recruiting FADD, which subsequently may activate caspase-8. Drosophila has a FADD homolog (DmFADD) and a caspase-8 homolog (DREDD) (Georgel, 2001). Furthermore, IMD, DmFADD, and DREDD can associate into a multimeric complex in Drosophila S2 cells (R. Medzhitov, personal communication to Georgel, 2001).

At this stage, it cannot be determine whether imd plays a role in developmentally regulated apoptosis. imd is expressed at significant levels during stages of embryogenesis and pupariation, when massive apoptosis is known to occur. However, imd mutants are viable and show no obvious developmental defect, indicating that either imd is not involved in these processes or that redundant control mechanisms could substitute for its role. Interestingly, it has been reported that reaper loss-of-function mutants are also viable, despite the accepted role of reaper as a key player in Drosophila apoptosis through caspase activation and inactivation of inhibitor of apoptosis proteins. In the context of the studies of immune defenses, the crucial question pertains to the potential links between the control of expression of the antibacterial peptides and the induction of apoptosis. Here, two lines of evidence draw a clear distinction: for one, bacterial challenge induces antibacterial peptide genes but does not result in detectable apoptosis; further, UV irradiation leads to P35-sensitive lethality, but does not induce antibacterial peptides. In other words, the data available today do not point to a role for apoptosis in the host response to bacterial infection (Georgel, 2001).


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 Drosophila immune system detects bacteria through specific peptidoglycan recognition

The Drosophila immune system discriminates between different classes of infectious microbes and responds with pathogen-specific defense reactions through selective activation of the Toll and the immune deficiency (Imd) signaling pathways. The Toll pathway mediates most defenses against Gram-positive bacteria and fungi, whereas the Imd pathway is required to resist infection by Gram-negative bacteria. The bacterial components recognized by these pathways remain to be defined. This study report that Gram-negative diaminopimelic acid-type peptidoglycan is the most potent inducer of the Imd pathway and that the Toll pathway is predominantly activated by Gram-positive lysine-type peptidoglycan. Thus, the ability of Drosophila to discriminate between Gram-positive and Gram-negative bacteria relies on the recognition of specific forms of peptidoglycan (Leulier, 2003).

By using highly purified products, this study has identified the bacterial compounds recognized by the Toll and Imd pathways. In contrast to vertebrates and the invertebrate horseshoe crab, this study suggests that LPS is not the main determinant for Gram-negative bacterial recognition in flies. Enterobacterial and nonenterobacterial purified LPS samples showed no stimulatory effect on expression of the Dpt gene by the fat body in Drosophila adults (Leulier, 2003).

Purified LPS did induce a weak immune response in the mbn-2 cell line. The LPS response observed in mbn-2 cells was modest, however, in comparison with the stimulatory effect of Gram-negative peptidoglycans. Although the possibility cannot be excluded that the LPS also has a weak stimulatory effect in vivo that was not detected in the assay that was used, the results indicate that peptidoglycan is the most active determinant of Gram-negative bacteria. This finding extends the results of previous studies showing that peptidoglycan but not LPS activates the prophenoloxidase cascade in the silkworm Bombyx mori (Leulier, 2003).

This study shows that the Imd pathway is activated by specific recognition of Gram-negative and Bacillus peptidoglycans, whereas the Toll pathway is more responsive to lysine-type peptidoglycans found in most Gram-positive bacteria. Purified peptidoglycans recapitulate all of the induction properties of live Gram-negative and Gram-positive bacteria, indicating that peptidoglycan is the main bacterial product recognized by the Imd and Toll pathways. Taken together, these results show that the capacity of the Drosophila immune system to discriminate between distinct classes of bacteria by the Toll and Imd pathways is mediated through specific peptidoglycan recognition (Leulier, 2003).

The data are in keeping with the identification of PGRP-LC and PGRP-SA as putative receptors of the Imd and Toll pathway. They suggest that PGRP-LC senses a specific structure that is present in Gram-negative and Bacillus peptidoglycans but absent in other Gram-positive peptidoglycans, whereas PGRP-SA binds with higher affinity to lysine-type peptidoglycans than to DAP-type peptidoglycans. These differences between PGRP-LC and PGRP-SA provide an explanation of why the Toll pathway is more activated by infections of Gram-positive bacteria than by infections of Gram-negative bacteria (Leulier, 2003).

Peptidoglycans from Bacillus and Gram-negative bacteria are crosslinked with a peptide containing a meso-DAP residue, whereas a lysine is found in the same position on other Gram-positive bacteria. This variation probably results in distinct conformational differences, allowing discriminatory recognition. The observation that Bacillus peptidoglycan is a less potent inducer of the Imd pathway than Gram-negative peptidoglycan might be explained by the fact that Bacillus peptidoglycan contains a high proportion of amidated-DAP and only 3% of meso-DAP. The idea of specific peptidoglycan recognition through PGRP is supported by the observation that another Drosophila PGRP, PGRP-LE, binds to DAP-type peptidoglycans but not to lysine-type peptidoglycans in vitro (Leulier, 2003).

The stronger activation of the Toll pathway by Gram-positive bacteria with lysine-type peptidoglycan than by Gram-negative bacteria is probably accentuated during bacterial infection, because activity of the Toll pathway is proportional to peptidoglycan concentration and Gram-positive bacterial cell walls contain much more peptidoglycan than do Gram-negative bacterial cell walls. It is also interesting to note that, within the range of peptidoglycan concentrations that were tested, maximum activation of the Imd pathway was reached by using at least 100 times less peptidoglycan than the dose required to strongly activate the Toll pathway. This finding may reflect the ability of the insect immune system to recognize Gram-negative bacteria efficiently even though these bacteria contain less peptidoglycan (Leulier, 2003).

It is surprising that flies can detect Gram-negative bacteria on the basis of a microbial component that is present at the surface of the inner membrane and is therefore hidden by the LPS-containing outer membrane. It is possible that the Imd pathway receptor, PGRP-LC, recognizes small amounts of peptidoglycan that are continuously released by Gram-negative bacteria. Alternatively, Gram-negative bacteria may be degraded by humoral or cellular mechanisms that release peptidoglycan and elicit the antimicrobial response. This latter possibility is supported by observations that P. aeruginosa cannot initiate the activation of prophenoloxidase in Galleria mellonela hemolymph unless the bacterial cells are damaged. The identification of the bacterial molecules that specifically trigger these two pathways will aid more detailed analyses of how bacterial elicitors are released and recognized during the infectious process and how Drosophila mounts specific immune responses adapted to the type of aggressor (Leulier, 2003).

Two studies have shown that mammalian Nod2 functions as a general sensor of peptidoglycans through recognition of MDP, the minimal bioactive peptidoglycan motif that is common to all bacteria. In support of these studies, peptidoglycan treated with muramidase, an enzyme that generates small peptidoglycan fragments, induces the expression of Nod2 in cell culture. The observations that after digestion with muramidase neither Gram-negative nor Gram-positive peptidoglycan activated an immune response in flies indicate that the bacterial sensing that regulates the synthesis of antimicrobial peptides by the Drosophila fat body is not mediated through a small peptidoglycan motif, as it is in the Nod2 system. The current study suggests that polymer chain size, and possibly the three-dimensional organization of the molecule, has a crucial role in bacterial sensing, in agreement with a study showing that the minimum structure of peptidoglycan required for inducing antibacterial peptides in the silkworm B. mori is two repeating GlcNAc-MurNAc units with peptide chains. It cannot, however, the possiblity cannot be definitively excluded that other immune-responsive tissues (such as hemocytes) may respond to small peptidoglycan fragments (Leulier, 2003).

This study and the work on mammalian Nod2 indicate that peptidoglycan is a complex bacterial elicitor and that the innate host defense has developed several ways to detect peptidoglycan. In vertebrates, TLR2 has been also implicated in peptidoglycan sensing, but the precise nature of the peptidoglycan fragment recognized by TLR2 is not known. It would be worthwhile determining whether vertebrates, like the fruit fly, use distinct receptors to recognize Gram-negative and Gram-positive peptidoglycans (Leulier, 2003).

Inhibitor of apoptosis 2 and TAK1-binding protein are components of the Drosophila Imd pathway

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).

An RNA interference screen identifies Inhibitor of Apoptosis Protein 2 as a regulator of innate immune signalling in Drosophila

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).

The role of ubiquitination in Drosophila innate immunity; The Drosophila homologs of Ubc13 are required in the Imd pathway for the activation of dTAK1 and the DmIKK complex

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).

An essential complementary role of NF-kappaB pathway to microbicidal oxidants in Drosophila gut immunity

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).

IAP DIAP2, acting downstream of Imd, functions in innate immunity, in a caspase independent role, and is essential to resist gram-negative bacterial infection

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).

Pirk is a negative regulator of the Drosophila Imd pathway

NF-kappaB transcription factors are involved in evolutionarily conserved signaling pathways controlling multiple cellular processes including apoptosis and immune and inflammatory responses. Immune response of the fruit fly Drosophila melanogaster to Gram-negative bacteria is primarily mediated via the Imd (immune deficiency) pathway, which closely resembles the mammalian TNFR signaling pathway. Instead of cytokines, the main outcome of Imd signaling is the production of antimicrobial peptides. The pathway activity is delicately regulated. Although many of the Imd pathway components are known, the mechanisms of negative regulation are more elusive. This study reports that a previously uncharacterized gene, pirk, is highly induced upon Gram-negative bacterial infection in Drosophila in vitro and in vivo. pirk encodes a cytoplasmic protein that coimmunoprecipitates with Imd and the cytoplasmic tail of peptidoglycan recognition protein LC (PGRP-LC). RNA interference-mediated down-regulation of Pirk caused Imd pathway hyperactivation upon infection with Gram-negative bacteria, while overexpression of pirk reduced the Imd pathway response both in vitro and in vivo. Furthermore, pirk-overexpressing flies were more susceptible to Gram-negative bacterial infection than wild-type flies. It is concluded that Pirk is a negative regulator of the Imd pathway (Kleino, 2008).

Rudra interrupts receptor signaling complexes to negatively regulate the IMD pathway

Insects rely primarily on innate immune responses to fight pathogens. In Drosophila, antimicrobial peptides are key contributors to host defense. Antimicrobial peptide gene expression is regulated by the IMD and Toll pathways. Bacterial peptidoglycans trigger these pathways, through recognition by peptidoglycan recognition proteins (PGRPs). DAP-type peptidoglycan triggers the IMD pathway via PGRP-LC and PGRP-LE, while lysine-type peptidoglycan is an agonist for the Toll pathway through PGRP-SA and PGRP-SD. Recent work has shown that the intensity and duration of the immune responses initiating with these receptors is tightly regulated at multiple levels, by a series of negative regulators. Through two-hybrid screening with PGRP-LC, this study identified Rudra (also termed Pirk), a new regulator of the IMD pathway, and demonstrates that it is a critical feedback inhibitor of peptidoglycan receptor signaling. Following stimulation of the IMD pathway, rudra expression is rapidly induced. In cells, RNAi targeting of rudra causes a marked up-regulation of antimicrobial peptide gene expression. rudra mutant flies also hyper-activated antimicrobial peptide genes and are more resistant to infection with the insect pathogen Erwinia carotovora carotovora. Molecularly, Rudra was found to bind and interfere with both PGRP-LC and PGRP-LE, disrupting their signaling complex. These results show that Rudra is a critical component in a negative feedback loop, whereby immune-induced gene expression rapidly produces a potent inhibitor that binds and inhibits pattern recognition receptors (Aggarwal, 2008).

PIMS modulates immune tolerance by negatively regulating Drosophila innate immune signaling

Metazoans tolerate commensal-gut microbiota by suppressing immune activation while maintaining the ability to launch rapid and balanced immune reactions to pathogenic bacteria. Little is known about the mechanisms underlying the establishment of this threshold. This study reports that a recently identified Drosophila immune regulator, which is termed PGRP-LC-interacting inhibitor of Imd signaling (PIMS, also termed Pirk), is required to suppress the Imd innate immune signaling pathway in response to commensal bacteria. pims expression is Imd (immune deficiency) dependent, and its basal expression relies on the presence of commensal flora. In the absence of PIMS, resident bacteria trigger constitutive expression of antimicrobial peptide genes (AMPs). Moreover, pims mutants hyperactivate AMPs upon infection with Gram-negative bacteria. PIMS interacts with the peptidoglycan recognition protein (PGRP-LC), causing its depletion from the plasma membrane and shutdown of Imd signaling. Therefore, PIMS is required to establish immune tolerance to commensal bacteria and to maintain a balanced Imd response following exposure to bacterial infections (Lhocine, 2008).

Caspase-Mediated Cleavage, IAP binding, and ubiquitination: linking three mechanisms crucial for Drosophila NF-kappaB signaling

Innate immune responses are critical for the immediate protection against microbial infection. In Drosophila, infection leads to the rapid and robust production of antimicrobial peptides through two NF-kappaB signaling pathways - IMD and Toll. The IMD pathway is triggered by diaminopimelic (DAP)-type peptidoglycan, common to most Gram-negative bacteria. Signaling downstream from the peptidoglycan (PGN) receptors is thought to involve K63 ubiquitination and caspase-mediated cleavage, but the molecular mechanisms remain obscure. This study shows that PGN stimulation causes caspase-mediated cleavage of the Imd protein, exposing a highly conserved IAP-binding motif (IBM) at its neo-N terminus. A functional IBM is required for the association of cleaved IMD with the ubiquitin E3-ligase DIAP2. Through its association with DIAP2, IMD is rapidly conjugated with K63-linked polyubiquitin chains. These results mechanistically connect caspase-mediated cleavage and K63 ubiquitination in immune-induced NF-kappaB signaling (Paquette, 2010).

Activation of the Drosophila IMD pathway by DAP-type peptidoglycan (PGN) leads to the robust and rapid production of a battery of antimicrobial peptides (AMPs) and other immune-responsive genes. Two peptidoglycan recognition protein (PGRP) receptors are responsible for the recognition of DAP-type PGN, the cell surface receptor PGRP-LC and the cytosolic receptor PGRP-LE. DAP-type PGN binding causes these receptors to multimerize or cluster, triggering signal transduction. IMD signaling culminates in activation of the NF-κB precursor Relish and transcriptional induction of AMP genes (Paquette, 2010 and references therein).

Currently, the molecular mechanisms linking these PGN-binding receptors and activation of Relish remain unclear. Genetic experiments suggest that the most receptor-proximal component of the pathway is the imd protein, while the MAP3 kinase TAK1 appears to function downstream. In turn, TAK1 is required for activation of the Drosophila IKK complex, which is essential for the immune-induced cleavage and activation of the NF-κB precursor Relish, the key transcription factor required for immune-responsive AMP gene expression. In addition to NF-κB signaling, TAK1 also mediates immune-induced JNK signaling (Paquette, 2010 and references therein).

Other major components in the IMD pathway include the caspase-8-like DREDD and its adaptor FADD. RNAi-based studies suggest that these proteins have two distinct roles in IMD pathway signaling, one relatively early in the cascade and the second further downstream. Using RNAi, DREDD and FADD have been shown to be required for immune-induced activation of the IKK complex. These data suggested that DREDD and FADD function downstream of IMD but upstream of TAK1; however, it was not established if this upstream role for DREDD involves its protease activity. In its second role, DREDD is thought to proteolytically cleave Relish (Paquette, 2010).

In addition to the components outlined above, several studies have suggested that ubiquitination plays a critical role in the IMD signaling cascade. Recently, Drosophila inhibitor of apoptosis 2 (DIAP2) was shown to be a crucial component of the IMD pathway. Typical of IAP proteins, DIAP2 has three N-terminal BIR domains, which are involved in interactions with proteins carrying conserved IAP-binding motifs (IBMs). In addition, some IAPs, including DIAP2, carry a C-terminal RING finger domain that provides these proteins with ubiquitin E3-ligase activity. Although it is unclear where in the pathway DIAP2 functions, one study showed that the RING finger is indispensable for its role in the immune response, suggesting it operates as an E3-ubiquitin ligase. Also it has been shown, using RNAi-based approaches, that the E2-ubiquitin-conjugating enzymes Uev1a and Ubc13 (bendless) are critical components of the IMD pathway. Notably, Ubc13 and Uev1a function together in a complex to generate K63-linked polyubiquitin chains. K63-polyubiquitin chains are not linked to proteasomal degradation but instead are thought to play regulatory roles. However, no K63-ubiquitinated target protein(s) has been identified in the IMD pathway. Although no connection between DIAP2 and the Bend/Uev1a E2 complex has been established, one attractive scenario is that DIAP2 functions as an E3 together with the Bend-Uev1a E2 complex (Paquette, 2010 and references therein).

The imd1 allele is a strong hypomorphic mutation that impairs innate immune responses. Surprisingly, this allele encodes a conservative amino acid substitution, alanine (A) to valine (V) at position 31, and is positioned in a region with no obvious structural motifs. The reason for the strong hypomorphic phenotype associated with the A31V substitution remains unclear. This work, demonstrates that imd protein is rapidly cleaved following PGN stimulation. Cleavage requires the caspase DREDD and occurs at caspase recognition motif 27LEKD/A31, creating a neo-N terminus at A31 that is critical for the immune-induced association of IMD with DIAP2. Substitution of the neo-N terminus with valine, as in imd1, disrupts the IMD-DIAP2 interaction. Moreover, once associated with DIAP2, cleaved IMD is rapidly K63-polyubiquitinated. Together, these data resolve a number of outstanding questions in IMD signal transduction and present a clear molecular mechanism linking caspase-mediated cleavage to NF-κB activation (Paquette, 2010).

Previous work has demonstrated that the caspase-8-like protease DREDD and its binding partner FADD are required upstream in the IMD pathway, at a position similar to Ubc13 and Uev1a (Zhou, 2005). However, it was not clear from these studies if the protease activity of DREDD is also required in this role upstream in the IMD pathway. This study shows that upon immune stimulation the imd protein is rapidly cleaved in a DREDD- and FADD-dependent manner. In fact, expression of DREDD, without immune stimulation, is sufficient to cause IMD cleavage. A caspase recognition site was identified in IMD, with cleavage predicted to occur after aspartate 30. Substitution of this residue with alanine prevents signal-induced cleavage and creates a dominant-negative allele of imd. This putative cleavage site in IMD (27LEKD/A31) is similar to the Relish cleavage site (542LQHD/G546), consistent with the notion that both proteins are cleaved by the same protease. Likewise, when IMD cleavage was blocked by caspase inhibitors, IMD was no longer ubiquitinated. Alignment of imd protein sequences from 12 Drosophila species and the Anopheles mosquito showed that the cleavage site is highly conserved (LEKD or LETD in all cases). These findings strongly argue that IMD cleavage after position 30 is mediated by DREDD and that this cleavage is critical for further downstream signaling events (Paquette, 2010).

Cleavage of IMD exposes a highly conserved IBM, which then binds the BIR2/3 domains of DIAP2. In the context of programmed cell-death regulation, these IBM motifs are best defined by their neo-N terminal alanine as well as the proline at position 3, both of which are also present in cleaved IMD, supporting the notion that IMD includes an IBM starting at position 31. The notion that IMD carries an IBM also provides a molecular explanation for the hypomorphic phenotype observed in the imd1 mutant, which carries a valine substitution for this alanine at position 1 of cleaved IMD. Although several IAP proteins have been implicated in mammalian innate immune/NF-κB signaling, the significance of their associated BIR domains, as well as their possible binding to proteins with exposed IBMs, has remained largely unexplored. This study shows that the BIR/IBM association plays a crucial role in innate immune NF-κB signaling in Drosophila. These findings present a unique role for the BIR-IBM interaction module outside of the cell-death arena (Paquette, 2010).

Furthermore, characterization of signaling in the imd1, diap2, dredd, and PGRP-LC/LE mutant flies provides critical in vivo verification of the cell-culture data and leads to a proposed model. In particular, the molecular mechanism suggests that immune stimulation leads to the DREDD-dependent cleavage of IMD, perhaps by recruiting IMD, FADD, and DREDD to a receptor complex. Consistent with this aspect of the model, dredd mutants and receptor mutants failed to cleave (or ubiquitinate IMD) following infection. Once cleaved, the exposed IBM of IMD interacts with BIR2 and BIR3 of DIAP2. Currently, it is not known precisely where in the cell the IMD/DIAP2 association occurs. Once associated with DIAP2, cleaved IMD is rapidly K63 ubiquitinated. As the RING-mutated version of diap2 failed to support IMD ubiquitination in flies, DIAP2 likely functions as the E3 for this reaction. Furthermore, the imd1 allele, which fails to interact with DIAP2 because of a mutation in the IBM, demonstrates the critical nature of the IMD-DIAP2 interaction for innate immune signaling. Consistent with the notion that cleavage precedes ubiquitination, mutants that fail to generate ubiquitinated IMD (i.e., diap2 and imd1) actually accumulate more cleaved IMD than is observed in wild-type flies. Presumably, in wild-type animals, cleaved IMD is efficiently ubiquitinated and thus is difficult to detect in assays. In contrast, dredd mutants or mutants lacking the key immunoreceptors (PGRP-LC/LE) failed to cleave and ubiquitinate IMD, consistent the cell-culture data (Paquette, 2010).

Previous work has suggested that ubiquitination plays a critical role in IMD signaling in the Drosophila immune response. However, the molecular target(s) of ubiquitination and the mechanisms of its activation have remained elusive. The data presented in this study indicate that DIAP2 functions as the E3-ligase in the IMD pathway, a function usually attributed to the TRAF or, more recently, cIAP proteins in mammalian NF-κB signaling pathways (Bertrand, 2009). The E2 complex of Bend and Uev1a also appears to be involved in IMD ubiquitination. RNAi targeting of these K63-ubiquitinating enzymes reproducibly decreases IMD ubiquitination and the induction of target genes; however, the degree of inhibition is variable and never complete (Zhou, 2005). This study show that a third E2 enzyme, Effete, the Drosophila Ubc5 homolog, also plays a vital role in ubiquitination of IMD. RNAi treatment targeting Effete, in concert with Uev1a and/or Bendless reproducibly eliminated IMD ubiquitination and the induction of Diptericin (Paquette, 2010).

Several lines of evidence argue that IMD is the critical target for K63 ubiqutination in this pathway. First, IMD is by far the most robustly modified component that identified, and the only one in which modifications can be detected in whole animals. Second, the protein produced as a result of the imd1 mutation, which does not signal, is also not ubiquitinated. Third, a deletion mutant, IMDΔ5, is present that is not ubiquitinated and fails to signal. Finally, Thevenon (2009) recently identified the Drosophila ubiquitin-specific protease, USP36, as a negative regulator of IMD ubiquitination. Functionally, USP36 is able to remove K63-polyubiquitin chains from IMD, promoting K48-mediated polyubiquitination and degradation of IMD. Consistent with the current model, animals which overexpress USP36 show decreased levels of IMD ubiquitination and reduced IMD pathway activation as monitored by Diptericin RNA expression, and are susceptible to bacterial infection. Together, these data strongly argue that IMD is the critical substrate for K63-polyubiqutination in IMD pathway signaling, although other proteins may also be conjugated to lesser degree (as shown in this study for DIAP2) and could potentially substitute for IMD as the platform for ubiquitin conjugation. Interestingly, Xia (2009) recently showed that unanchored K63-polyubiquitin chains (i.e., ubiquitin chains that are not conjugated to a target substrate) are sufficient to activate the mammalian TAK1 and IKK kinase complexes. Furthermore, unanchored polyubiquitin chains are produced after stimulation of HEK cells with IL-1β (Xia, 2009). Thus, the presence (or absence) of K63-polyubiquitin chains may be more important than their conjugation substrate (Paquette, 2010).

K63-polyubiquitin chains are likely to serve as scaffolds to recruit the key kinases TAK1 and IKK in the IMD pathway. Both of these kinases include regulatory subunits with highly conserved K63-polyubiquitin binding domains. Drosophila TAB2, which complexes with TAK1, and the IKKγ subunit are predicted to contain conserved K63-polyubiquitin-binding domains. Thus, it is hypothesized that K63-polyubiquitin chains will recruit both the TAB2/TAK1 complex and the IKK complex, creating a local environment for optimal kinase activation and signal transduction; however, this aspect of the model is still speculative (Paquette, 2010).

Although mammalian caspase-8 and FADD are best known for their role in apoptosis, a growing body of literature indicates that these factors, along with RIP1 (which has some homology to IMD), also function in RIG-I signaling to NF-κB. In addition, caspase-8 has been implicated in NF-κB signaling in B cell, T cell, and LPS signaling. Cells, from mice or humans, lacking caspase-8 have defects in immune activation, cytokine production, and nuclear translocation of NF-κB p50/p65. Furthermore, recent evidence also shows that during mammalian NOD signaling the RIP2 protein is ubiquitinated in a cIAP1/2-dependent manner. Given that Drosophila homologs of RIP1, FADD cIAP1/2, and caspase-8 also function in the IMD pathway, the results presented in this study may help elucidate the mechanism by which these factors function in these mammalian immune signaling pathways (Paquette, 2010).

Cleavage of PGRP-LC receptor in the Drosophila IMD pathway in response to live bacterial infection in S2 cells

Drosophila responds to Gram-negative bacterial infection by activating the immune deficiency (IMD) pathway, leading to production of antimicrobial peptides (AMPs). As a receptor for the IMD pathway, peptidoglycan-recognition protein (PGRP), PGRP-LC is known to recognize and bind monomeric peptidoglycan (DAP-type PGN) through its PGRP ectodomain and in turn activate the IMD pathway. The questions remain how PGRP-LC is activated in response to pathogen infection to initiate the IMD signal transduction in Drosophila. This study presents evidence to show that proteases such as elastase and Mmp2 can also activate the IMD pathway but not the TOLL pathway. The elastase-dependent IMD activation requires the receptor PGRP-LC. Importantly, it was found that live Salmonella/E. coli infection modulates PGRP-LC expression/receptor integrity and activates the IMD pathway while dead Salmonella/E. coli or protease-deficient E. coli do neither. These results suggest an interesting possibility that Gram-negative pathogen infection may be partially monitored through the structural integrity of the receptor PGRP-LC via an infection-induced enzyme-based cleavage-mediated activation mechanism (Schmidt, 2011).

Drosophila growth-blocking peptide-like factor mediates acute immune reactions during infectious and non-infectious stress

Antimicrobial peptides (AMPs), major innate immune effectors, are induced to protect hosts against invading microorganisms. AMPs are also induced under non-infectious stress; however, the signaling pathways of non-infectious stress-induced AMP expression are yet unclear. This study demonstrates that Growth-blocking peptide (GBP) is a potent cytokine that regulates stressor-induced AMP expression in insects. GBP overexpression in Drosophila elevates expression of AMPs. GBP-induced AMP expression does not require Toll and immune deficiency (Imd) pathway-related genes, but imd and basket are essential, indicating that GBP signaling in Drosophila does not use the orthodox Toll or Imd pathway but uses the JNK pathway after association with the adaptor protein Imd. The enhancement of AMP expression by non-infectious physical or environmental stressors is apparent in controls but not in GBP-knockdown larvae. These results indicate that the Drosophila GBP signaling pathway mediates acute innate immune reactions under various stresses, regardless of whether they are infectious or non-infectious (Tsuzuki, 2012).

The innate immune system of animals provides the first and most primitive line of defense against invading microorganisms. Antimicrobial peptides (AMPs) are produced as immune effector molecules to fight pathogenic infection, and the induction of AMPs is regulated through activation of the Toll and immune deficiency (Imd) pathways in Drosophila. Although the activation of both signaling pathways in response to infection has been extensively investigated, it is also known that changes in innate immune activities are sometimes unrelated to microbial infection. Various physical and physiological factors such as temperature, starvation, and diapause also elevate AMP expression levels. It is also known that AMP expression is highly sensitive to developmental stage in mammals as well as insects. Further, it has been recently reported that AMP expression in starved Drosophila is enhanced in response to the transcription factor FOXO, a key regulator of stress resistance, metabolism, and ageing, independently of the immunoregulatory pathways (Becker, 2010). Insulin signaling is currently the only known pathway for the induction of AMP expression by non-infectious stress. However, it is unlikely that animals cope with various non-infectious stressors by using the same signaling pathway that manages the regulation of innate immunity (Tsuzuki, 2012).

To investigate extracellular signaling in the innate immune regulation under non-infectious stresses, focus was placed on insect cytokines because cytokines in general regulate many physiological events including stress resistance through transmission of signals from outside the cell to the inside. While a large number of cytokines have been identified and their roles in mammals studied extensively, the number of known insect cytokines is quite limited. In Drosophila, spätzle is known as the cytokine that activates Toll signaling after microbial infection, which leads to expression of the target AMPs. In lepidopteran insects, structurally similar bioactive peptides have been reported in the last 20 years and are now recognized as the insect cytokine family referred to as the ENF peptides on the basis of their common N-terminal sequence, Glu-Asn-Phe- (Strand, 2000). These peptides are typically 23-25 amino acids long, and growth-blocking peptide (GBP) was the first member of this peptide family discovered (Hayakawa, 1990). Other known insect-specific cytokines include Unpaired-3, which was first identified in Drosophila, and hemocyte chemotactic peptide (HCP), identified in the armyworm Pseudaletia separata. Stress-responsive peptide (SRP) has also recently been identified in the common cutworm Spodoptera litura. Drosophila eda-like cell death trigger (eiger) HAS been reported to modify AMP expression levels. Among these insect cytokines, this study focused on characterizing the functional role of GBP in innate immunity because GBP was initially identified as the factor responsible for the reduced growth exhibited by armyworm P. separata larvae under stress conditions such as parasitization by the parasitoid wasp Cotesia kariyai and exposure to low temperature (Ohnishi, 1995). NMR analysis of GBP showed that it consists of flexible N- and C-termini, and a structured core stabilized by a disulfide bridge and a short antiparallel β-sheet (β-hairpin) (Aizawa, 1999). Structural comparisons indicated that the core β-hairpin region adopts the C-terminal subdomain structure of human epidermal growth factor. Consistent with this structural similarity, GBP at concentrations of 10-1 to 102 pmol/ml induced proliferation of human keratinocytes as well as insect Sf 9 cells (Hayakawa, 1998; Tsuzuki, 2012 and references therein).

At least 16 members of the insect ENF cytokine family have been identified. They have diverse functions such as growth retardation, paralysis induction, cardioacceleration, embryogenic morphogenesis, and immune cell stimulation. Characterization of some of these peptide cDNAs demonstrated that the ENF peptides are synthesized as a precursor form in which the active peptide is located at the C-terminal region. Because it has been demonstrated that ENF family peptides stimulate insect immune cells like plasmatocytes to spread on foreign surfaces, this study first examined whether GBP affects humoral immune activity in a lepidopteran insect, the silkworm Bombyx mori. As expected, injection of B. mori GBP into B. mori larvae elevated the expression of AMPs. Further, GBP-induced elevation of AMP expression was demonstrated in silkworm larvae exposed to heat stress. Although this result demonstrated GBP-dependent induction of AMP expression in non-infected stressed silkworm larvae, elucidating in detail the pathway of GBP signaling in the immune system required analysis in Drosophila because little is known about the signaling pathways that activate AMP gene expression in non-Drosophila insects like B. mori. However, none of the ENF family cytokines have been identified in insect orders outside the Lepidoptera, making it necessary to identify the Drosophila GBP homolog. Database searches did not reveal any obvious homologs in the fly genome, which suggested either that the Diptera lack ENF genes or that members of this gene family might have diverged too much to be identified on a sequence level in Diptera. Therefore, a peptidergic factor with GBP-like activity was purified from the bluebottle fly Lucilia cuprina. Using the sequence of the bluebottle fly GBP homolog for motif and FASTA searches, five Drosophila melanogaster homologs were identified, among which CG15917 was most similar to lepidopteran GBPs in terms of the primary structure of its ORF. Overexpression and RNAi knockdown of GBP in the Drosophila larvae indicated that GBP regulates the expression of AMPs through a novel pathway associated with Imd and JNK. The Drosophila GBP signaling pathway stimulated AMP expression in Drosophila larvae in response to external stressors, whether they were infectious or non-infectious (Tsuzuki, 2012).

This study has revealed the innate immune activity of GBP in insects by demonstrating that it elevates expression levels of some AMPs in Drosophila larvae as well as in Bombyx larvae. In order to clarify the role and mechanism of GBP in inducing AMP expression in insects, its signaling pathway was analyzed in Drosophila because no detailed signaling pathway of an innate immune system had been recognized in non-Drosophila species. To perform the analyses, a GBP-like cytokine was isolated from bluebottle fly larvae, and then 46 Drosophila genes were identified encoding its homologous peptide by (a cysteine-based motif search) using the C-(X except for C)6-8-C-(X except for C)1-4 pattern. Among these genes, CG14069 and CG17244 were selected as candidate Drosophila GBP genes whose ORFs consist of 100-200 amino acid residues. Subsequent FASTA searches for the C-terminal 25-amino acid peptide sequence of CG14069 identified CG11395, CG15917, and CG12517. These five genes were found to encode GBP-like peptide sequences at the C-terminal ends. Among the five genes, the peptide gene most homologous to the bluebottle fly GBP and lepidopteran GBP genes were further selected based on the characteristics common to those GBP genes: the size (100-200 amino acids) of the ORF and the presence of an arginine residue at an appropriate position (7-9 residues upstream of the first cysteine). Further, after confirming the gene expression in the larval fat body, CG15917 remained as the most plausible candidate. The functional GBP peptide located in the C-terminal region of the CG15917 ORF stimulated aggregation of hemocytes from Drosophila y w larvae. Further, overexpression of CG15917 retarded larval development, creating a phenotype similar to that seen following injection of GBP into lepidopteran larvae. These results were interpreted as an indication that the CG15917 product has a physiological role identical to that of lepidopteran GBPs (Tsuzuki, 2012).

Overexpression of proGBP as well as GBP significantly elevated the expression of the antimicrobial peptides Mtk and Dpt with the aid of the adaptor protein Imd in Drosophila larvae. The results are partially consistent with previously published data on the Drosophila gene CG15917: the gene was detected, along with many other genes, by microarray analysis as being transcriptionally activated three hours after septic injury of Drosophila adults. It was further revealed that the GBP-dependent induction of Mtk expression was abolished in larvae whose bsk expression was reduced by RNAi, indicating that the GBP pathway stimulated AMP expression through JNK signaling after recruitment of Imd to the activated GBP receptor (GBPR). This interpretation was partially confirmed by showing that GBP induced Mtk expression independently of PGRP-LC, PGRP-LE, and Relish. The biological importance of GBP was demonstrated by the finding that RNAi targeting of GBP significantly repressed AMP expression during the initial phase of bacterial infection and consequently made the larvae more susceptible to the bacteria than control larvae. Moreover, GBP RNAi repression of AMP expression was found to occur when test larvae were subcutaneously damaged without bleeding by pinching. The GBP RNAi-induced repression of AMP expression was also observed in Drosophila larvae exposed to temperature stresses: AMP expression was significantly elevated in control larvae by transferring them from 4oC to 25oC or exposing to 4oC, but it was not seen in GBP RNAi larvae. The results clearly show that induction of GBP-dependent AMP expression does not require a pathogen-associated molecular pattern, and that such non-infectious or non-injurious stimuli-dependent AMP expression is probably mediated by the GBP signaling pathway (Tsuzuki, 2012).

In lepidopteran larvae, GBP is abundantly present in a precursor form (proGBP) in hemolymph (Kamimura, 2001; Oda, 2010). The present experiments showed that this is also true for Drosophila. Non-injurious stimuli instantly trigger activation of GBP-processing enzyme(s) in hemolymph, by which the proGBP is proteolytically activated to GBP. The active GBP that results from the proteolytic processing under various stresses should trigger GBP-dependent AMP expression. It is reasonable to expect that such processing of proGBP enables the production of AMPs for the swift supply of active GBP under stress conditions. This interpretation is consistent with the fact that AMP expression was enhanced more by overexpression of active GBP than that of proGBP. Therefore, it is reasonable to propose that GBP serves as a key cytokine in the enhancement of AMP gene expression through the Imd/JNK pathway in Drosophila larvae exposed to various stressors, regardless of whether they are infectious or non-infectious (Tsuzuki, 2012).

Although there have been reports of the cross-regulation or cross-modification of two signaling pathways, Toll and Imd or JNK (or JAK/STAT) and NF-kappaB, the current knowledge is not still adequate to fully understand all mechanisms controlling the regulatory system. Further, recent studies suggest the existence of an evolutionarily conserved mechanism of cross-regulation of metabolism and innate immunity. It might be worth emphasizing that GBP was initially identified as a growth inhibitory factor in armyworm larvae. Although this study confirmed that GBP overexpression retards normal larval development in Drosophila, it was confirmed that this GBP-induced growth retardation did not affect AMP expression levels in test larvae under the present experimental conditions. Further, GBP-induced AMP expression was observed in Drosophila adults. Therefore, it is reasonable to propose that the insect cytokine GBP contributes to regulation of both growth and innate immunity. It may be possible to solve important problems concerning the adaptation of organismal defense to environmental stresses if the contributions of the novel signaling pathway through GBP-JNK are taken into account there (Tsuzuki, 2012).

Age-associated loss of lamin-B leads to systemic inflammation and gut hyperplasia

Aging of immune organs, termed as immunosenescence, is suspected to promote systemic inflammation and age-associated disease. The cause of immunosenescence and how it promotes disease, however, has remained unclear. This study reports that the Drosophila fat body, a major immune organ, undergoes immunosenescence and mounts strong systemic inflammation that leads to deregulation of immune deficiency (IMD) signaling in the midgut of old animals. Inflamed old fat bodies secrete circulating peptidoglycan recognition proteins that repress IMD activity in the midgut, thereby promoting gut hyperplasia. Further, fat body immunosenecence is caused by age-associated lamin-B reduction specifically in fat body cells, which then contributes to heterochromatin loss and derepression of genes involved in immune responses. As lamin-associated heterochromatin domains are enriched for genes involved in immune response in both Drosophila and mammalian cells, these findings may provide insights into the cause and consequence of immunosenescence during mammalian aging (Chen, 2014).

By analyzing gene expression changes upon aging in fat bodies and midguts, it was shown that an increase of immune response in the fat body is accompanied by a striking reduction in the midgut. Specifically, it was demonstrate that the age-associated increase in Immune deficiency (IMD) signaling in fat bodies leads to reduction of IMD activity in the midgut, which in turn contributes to midgut hyperplasia. This fat body to midgut effect requires peptidoglycan recognition proteins (PGRPs) secreted from fat body cells and is mediated by both bacteria dependent and independent pathways. Therefore, fat body aging contributes to systemic inflammation, which contributes to the disruption of gut homeostasis. Importantly, it was shown that the age-associated lamin-B loss in fat body cells causes the derepression of a large number of immune responsive genes, thereby resulting in fat body-based systemic inflammation (Chen, 2014).

B-type lamins have long been suggested to have a role in maintaining heterochromatin and gene repression. Consistently, this study's global analyses of fat body depleted of lamin-B revealed a loss of heterochromatin and derepression of a large number of immune responsive genes. This is further supported by ChIP-qPCR analyses of H3K9me3 on specific IMD regulators. Recent studies in different cell types show that tethering genes to nuclear lamins do not always lead to their repression. Deleting B-type lamins or all lamins in mouse ES cells or trophectdoderm cells does not result in derepression of all genes in LADs. In light of these studies, it is suggested that the transcriptional repression function of lamin-B could be gene and cell type dependent. Interestingly, GO analyses revealed a significant enrichment of immune responsive genes in Lamin-associated domains (LADs) in four different mammalian cell types and Drosophila Kc cells. Since the large-scale pattern of LADs is conserved in different cell types in mammals, it is possible that the immune-responsive genes are also enriched in LADs in the fly fat body cells. Supporting this notion, the IKKγ, key, which is one of the two derepressed IMD regulators and was found to exhibit H3K9me3 reduction and gene activation, is localized to LADs in Kc cells. It is speculated that lamin-B might play an evolutionarily conserved role in repressing a subset of inflammatory genes in certain tissues, such as the immune organs, in the absence of infection or injury. Consistently, senescence-associated lamin-B1 loss in mammalian fibroblasts is correlated with senescence-associated secretory phenotype senescence-associated secretory phenotype (SASP). Although the in vivo relevance of fibroblast SASP in chronic inflammation and aging-associated diseases in mammals remains to be established, the findings in Drosophila provide insights and impetus to investigate the role of lamins in immunosenescence and systemic inflammation in mammals (Chen, 2014).

Lamin-B gradually decreases in fat body cells of aging flies, whereas lamin-C amount remains the same. Since it has been recently shown that the assembly of an even and dense nuclear lamina is dependent on the total lamin concentration, the age-associated appearance of lamin-B and lamin-C gaps around the nuclear periphery of fat body cells is likely caused by the drop of the lamin-B level. How aging triggers lamin-B loss is unknown, but it appears to be posttranscriptional, because lamin-B transcripts in fat bodies remain unchanged upon aging. Interestingly, among the tissues examined, no changes of lamin-B and lamin-C proteins were found in cells in the heart tube, oenocytes, or gut epithelia in old flies. Therefore, the age-associated lamin-B loss does not occur in all cell types in vivo. A systematic survey to establish the cell/tissue types that undergo age-associated reduction of lamins in both flies and mammals should provide clues to the cause of loss. Deciphering how advanced age leads to lamin loss should open the door to further investigate the cellular mechanism that contributes to chronic systemic inflammation and how it in turn promotes age-associated diseases in humans (Chen, 2014).

Old Drosophila gut is known to exhibit increased microbial load, which would cause increased stress response and activation of tissue repair, thereby leading to midgut hyperplasia. Systemic inflammation caused by lamin-B loss in fat body leads to repression of local midgut IMD signaling. The upregulation of targets of IMD in the aged whole gut has been recently reported, while a downregulation of target genes was observed in the current analyses of the midgut. However, the previous study found a similar upregulation of the genes when performing RNA-seq of the whole gut (Chen, 2014).

These studies reveal an involvement of bacteria in the repression of midgut IMD signaling by the PGRPs secreted from the fat body. How PGRPs from the fat body repress midgut IMD is still unknown. One possibility is that the body cavity bacteria contribute to the maintenance of midgut IMD activity, and the increased circulating PGRPs limit these bacteria. The circulating PGRPs may also reduce midgut IMD activity indirectly by affecting other tissues. The evidence suggests that lamin-B loss could also contribute to midgut hyperplasia independent of the IMD pathway. While it will be important to further address these possibilities, the findings have revealed a fat body mediated inflammatory pathway that can lead to reduced migut IMD, increased gut microbial accumulation, and midgut hyperplasia upon aging (Chen, 2014).

Interestingly, microbiota changes also occur in aging human intestine and have been linked to altered intestinal inflammatory states and diseases. Although, much effort has been devoted to understand how local changes in aging mammalian intestines affect gut microbial community, the cause remains unclear. The findings in Drosophila reveal the importance of understanding the impact of immunosenescence and systemic inflammation on gut microbial homeostasis. Indeed, if increased circulating inflammatory cytokines perturb the ability of local intestine epithelium and the gut-associated lymphoid tissue to maintain a balanced microbial community, the unfavorable microbiota in the old intestine would cause chronic stress response and tissue repair, thereby leading to uncontrolled cell growth as observed in age-associated cancers (Chen, 2014).

Identifying USPs regulating immune signals in Drosophila: USP2 deubiquitinates Imd and promotes its degradation by interacting with the proteasome

Rapid activation of innate immune defenses upon microbial infection depends on the evolutionary conserved NFκB-dependent signals, which deregulation is frequently associated with chronic inflammation and oncogenesis. These signals are tightly regulated by the linkage of different kinds of ubiquitin moieties on proteins that modify either their activity or their stability. To investigate how ubiquitin specific proteases (USPs) orchestrate immune signal regulation, a focused RNA interference library was created and screened on Drosophila NFκB-like pathways Toll and Imd in cultured S2 cells, and the function of selected genes were further analysed in vivo. USP2 and USP34/Puf, in addition to the previously described USP36/Scny, prevent inappropriate activation of Imd-dependent immune signal in unchallenged conditions. Moreover, USP34 is also necessary to prevent constitutive activation of the Toll pathway. However, while USP2 also prevents excessive Imd-dependent signalling in vivo, USP34 shows differential requirement depending on NFκB target genes, in response to fly infection by either Gram-positive or Gram-negative bacteria. It was further shown that USP2 prevents the constitutive activation of signalling by promoting Imd proteasomal degradation. Indeed, the homeostasis of the Imd scaffolding molecule is tightly regulated by the linkage of lysine 48-linked ubiquitin chains (K48) acting as a tag for its proteasomal degradation. This process is necessary to prevent constitutive activation of Imd pathway in vivo and is inhibited in response to infection. The control of Imd homeostasis by USP2 is associated with the hydrolysis of Imd linked K48-ubiquitin chains and the synergistic binding of USP2 and Imd to the proteasome, as evidenced by both mass-spectrometry analysis of USP2 partners and by co-immunoprecipitation experiments. This work identified one known (USP36) and two new (USP2, USP34) ubiquitin specific proteases regulating Imd or Toll dependent immune signalling in Drosophila. It further highlights the ubiquitin dependent control of Imd homeostasis and shows a new activity for USP2 at the proteasome allowing for Imd degradation. This study provides original information for the better understanding of the strong implication of USP2 in pathological processes in humans, including cancerogenesis (Engel, 2014).


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 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 the antimicrobial peptides have a half-life of 2-3 weeks in Drosophila adults, suggesting that a low level of expression may also suffice to load the epidermis layer with substantial amounts of antimicrobial peptides. In addition, Drosophila cecropins are not only active against Gram-negative and Gram-positive bacteria, but also possess strong antifungal activity making them powerful weapons against most classes of microorganisms (Tingvall, 2001).

Genetic evidence has provided a model in which at least two different pathways are involved in the activation of antimicrobial peptides, the Toll pathway and the imd pathway, both suggested to activate members of the Drosophila Rel family. Although the systemic expression of the peptide drosomycin is dependent on the Toll pathway and the Rel protein Dif, the local expression of drosomycin in tracheal epithelium is independent of the same pathway. The present study demonstrates that the imd gene seems to play an important role in the local expression of cecropin. In imd mutants, the expression of CecA1 is impaired dramatically in the epidermis of embryos, and is undetectable in the epidermis underlying the cuticle of wounded larvae. Interestingly, the pro-phenoloxidase cascade is activated by injury in both wild-type and imd larvae, showing that the processes of antimicrobial defence and melanization can be uncoupled. In addition, the inducible, local expression of CecA1 in infected wounds indicates that the insect epidermis is not only a passive barrier against infection, but plays an active role in the innate defence against microorganisms (Tingvall, 2001).

In conclusion, antimicrobial cecropins are expressed locally in infected wounds in the epidermis of Drosophila larvae, and this response is amenable to genetic dissection in mutants of the fly's immune response. This has a strong potential to become valuable for the understanding of innate and epithelial immunity in higher organisms, including mammals (Tingvall, 2001).

Drosophila Sex-peptide stimulates female innate immune system after mating via the Toll and Imd pathways

Insect immune defense is mainly based on humoral factors like antimicrobial peptides (AMPs) that kill the pathogens directly or is based on cellular processes involving phagocytosis and encapsulation by hemocytes. In Drosophila, the Toll pathway (activated by fungi and gram-positive bacteria) and the Imd pathway (activated by gram-negative bacteria) leads to the synthesis of AMPs. But AMP genes are also regulated without pathogenic challenge, e.g., by aging, circadian rhythms, and mating. This study shows that AMP genes are differentially expressed in mated females. Metchnikowin (Mtk) expression is strongly stimulated in the first 6 hr after mating. Sex-peptide (SP), a male seminal peptide transferred during copulation, is the major agent eliciting transcription of Mtk and of other AMP genes. Both pathways are needed for Mtk induction by SP. Furthermore, SP induces additional AMP genes via the Toll (Drosomycin) and the Imd (Diptericin) pathways. SP affects the Toll pathway at or upstream of the gene spätzle, and the Imd pathway at or upstream of the gene imd. Mating may physically damage females and pathogens may be transferred. Thus, endogenous stimulation of AMP transcription by SP at mating might be considered as a preventive step to encounter putative immunogenic attacks (Peng, 2005).

The Toll and Imd signaling cascades are the major and best-characterized pathways involved in the activation of AMPs after pathogenic challenges. The effect of SP on AMP expression was studied by comparing the expression of Mtk, Drs, and Dipt in wt females or in females mutant in the Toll and Imd pathways, respectively, before and after mating with wt males. RNA was extracted from virgin and mated females and analyzed by quantitative PCR (Peng, 2005).

With the exception of dorsal (dl), all loss-of-function mutants of the Toll and Imd pathways abolish or strongly reduce Mtk expression after mating. Thus, Mtk expression induced by SP is dependent on both pathways. Furthermore, since spz and imd females fail to induce Mtk transcription after mating, SP must act on or upstream of spz and imd. dl and its functional homolog dif have been reported to be involved in AMP gene transcription under pathogenic challenge in the larval stage, but not functional in the adult immune defense. A partial response is observed in dl females, indicating that dl may be partially involved in the innate immune response elicited by SP in adult females (Peng, 2005).

Drs expression, controlled by the Toll pathway, is completely abolished in spz and Tl mutants. Correspondingly, Dipt expression, which is controlled by the Imd pathway, is completely abolished in the Imd pathway loss-of-function mutants imd, Tak1, and rel. It is concluded that SP can activate the Toll and the Imd pathways. The Toll pathway is essential for Drs expression, whereas the Imd pathway is essential for Dipt expression (Peng, 2005).

The SP-induced immune response activates the transcription of all three AMP genes studied. After pathogenic infections, Drs is induced by the Toll pathway and Dipt by the Imd pathway, whereas both pathways induce Mtk expression. The results obtained with the loss-of-function mutants follow this scheme. Whereas loss-of-function mutants of both pathways reduce or abolish Mtk expression after mating, induction of Drs expression is only abolished by loss-of-function mutants of the Toll pathway, whereas induction of Dipt expression is only lost in mutants of the Imd pathway. In sum, the classical pathways are activated to induce the transcription of AMP genes after mating as after microbial or fungal infections (Peng, 2005).

Detection of microorganisms and triggering the appropriate pathway is achieved by pattern recognition receptors (PRRs), immune proteins recognizing general microbial components. Two families of PRRs have been identified in Drosophila: the peptidoglycan recognition proteins (PGRPs) and the gram-negative binding proteins (GNBPs). Some of the 13 PGRPs encoded in the D. melanogaster genome have been implicated in the activation of specific immune responses. However, the signaling cascades between the PRRs and the Toll and the Imd pathways are not well characterized. Since in spz and imd null mutants AMP induction by SP is specifically abolished, the inducing signals must affect the signaling cascades at or upstream of those genes. At this stage, it cannot be determined whether SP enters the pathways at the PRR level or at an intermediate level between the PRRs and spz or imd, respectively. Furthermore, the induction of AMPs may occur systemically (e.g., in the fat body) or locally in the reproductive tract. Microarray analysis of AMP expression after mating of wt females with either wt or SP0 males, respectively, suggests that AMPs are mainly induced in the abdomen, but it does not discriminate between a systematic response in the abdomen and a specific response in the genital tract (Peng, 2005).

Drosophila females undergo dramatic physiological changes after mating, predominantly induced by SP. Mating may also physically damage females and may expose the female to pathogens transferred by the male as shown for the milkweed leaf beetle. Thus, the activation of the innate immune system to encounter putative immunogenic attacks during this sensitive phase of the life history of females makes biological sense. The signal is plausibly coupled to copulation in the form of SP transferred in the seminal fluid. Such a mechanism might allow the female to respond preventively to potential threats. In sum, these findings may describe the result of an optimal economical balance between spending costly energy for the innate immune response and preventive measures to fight a putative pathogenic attack (Peng, 2005).

Genetic rescue of functional senescence in synaptic and behavioral plasticity

Aging has been linked with decreased neural plasticity and memory formation in humans and in laboratory model species such as the fruit fly, Drosophila melanogaster. This study examined plastic responses following social experience in Drosophila as a high-throughput method to identify interventions that prevent these impairments. Young (5-day old) or aged (20-day old) adult female Drosophila were housed in socially enriched or isolated environments, then assayed for changes in sleep and for structural markers of synaptic terminal growth in the ventral lateral neurons (LNVs) of the circadian clock. When young flies are housed in a socially enriched environment, they exhibit synaptic elaboration within a component of the circadian circuitry, the LNVs, which is followed by increased sleep. Aged flies, however, no longer exhibit either of these plastic changes. Because of the tight correlation between neural plasticity and ensuing increases in sleep, sleep after enrichment was used as a high-throughput marker for neural plasticity to identify interventions that prolong youthful plasticity in aged flies. To validate this strategy, three independent genetic manipulations were used that delay age-related losses in plasticity: (1) elevation of dopaminergic signaling, (2) over-expression of the serum response factor transcription factor blistered (bs) in the LNVs, and (3) reduction of the Imd immune signaling pathway. These findings provide proof-of-principle evidence that measuring changes in sleep in flies after social enrichment may provide a highly scalable assay for the study of age-related deficits in synaptic plasticity. These studies demonstrate that Drosophila provides a promising model for the study of age-related loss of neural plasticity and begin to identify genes that might be manipulated to delay the onset of functional senescence (Donlea, 2014).


Ligation of the extracellular domain of the cell surface receptor Fas/APO-1 (CD95) elicits a characteristic programmed death response in susceptible cells. Using a genetic selection based on protein-protein interaction in yeast, two gene products have been identified that associate with the intracellular domain of Fas: Fas itself, and a novel 74 kDa protein termed RIP (receptor interacting protein). RIP also interacts weakly with the p55 tumor necrosis factor receptor (TNFR1) intracellular domain, but not with a mutant version of Fas corresponding to the murine lprcg mutation. RIP contains an N-terminal region with homology to protein kinases and a C-terminal region containing a cytoplasmic motif (death domain) present in the Fas and TNFR1 intracellular domains. Transient overexpression of RIP causes transfected cells to undergo the morphological changes characteristic of apoptosis. Taken together, these properties indicate that RIP is a novel form of apoptosis-inducing protein (Stanger, 1995).

With use of the yeast two-hybrid system, the proteins RIP and FADD/MORT1 have been shown to interact with the 'death domain' of the Fas receptor. Both of these proteins induce apoptosis in mammalian cells. Using receptor fusion constructs, evidence is provided that the self-association of the death domain of RIP by itself is sufficient to elicit apoptosis. However, both the death domain and the adjacent alpha-helical region of RIP are required for the optimal cell killing induced by the overexpression of this gene. By contrast, FADD's ability to induce cell death does not depend on crosslinking. Furthermore, RIP and FADD appear to activate different apoptotic pathways since RIP is able to induce cell death in a cell line that is resistant to the apoptotic effects of Fas, tumor necrosis factor, and FADD. Consistent with this, a dominant negative mutant of FADD, lacking its N-terminal domain, blocks apoptosis induced by RIP but not by FADD. Since both pathways are blocked by CrmA, the interleukin 1 beta converting enzyme family protease inhibitor, these results suggest that FADD and RIP can act along separable pathways that nonetheless converge on a member of the interleukin 1 beta converting enzyme family of cysteine proteases (Grimm, 1996).

The death domain of tumor necrosis factor (TNF) receptor-1 (TNFR1) triggers distinct signaling pathways leading to apoptosis and NF-kappa B activation through its interaction with the death domain protein TRADD. TRADD interacts strongly with RIP, another death domain protein that was shown previously to associate with Fas antigen. RIP is a serine-threonine kinase that is recruited by TRADD to TNFR1 in a TNF-dependent process. Overexpression of the intact RIP protein induces both NF-kappa B activation and apoptosis. However, expression of the death domain of RIP induces apoptosis, but potently inhibits NF-kappa B activation by TNF. These results suggest that distinct domains of RIP participate in the TNF signaling cascades leading to apoptosis and NF-kappa B activation (Hsu, 1996).

The CD95 (Fas/APO-1) and tumor necrosis factor (TNF) receptor pathways share many similarities, including a common reliance on proteins containing 'death domains' for elements of the membrane-proximal signal relay. Mutant cell lines have been created that are unable to activate NF-kappaB in response to TNF. One of the mutant lines lacks RIP, a 74 kDa Ser/Thr kinase originally identified by its ability to associate with Fas/APO-1 and induce cell death. Reconstitution of the line with RIP restores responsiveness to TNF. The RIP-deficient cell line is susceptible to apoptosis initiated by anti-CD95 antibodies. An analysis of cells reconstituted with mutant forms of RIP reveals similarities between the action of RIP and FADD/MORT-1, a Fas-associated death domain protein (Ting, 1996).

Although the molecular mechanisms of TNF signaling have been largely elucidated, the principle that regulates the balance of life and death is still unknown. The death domain kinase RIP, a key component of the TNF signaling complex, is cleaved by Caspase-8 in TNF-induced apoptosis. The cleavage site was mapped to the aspartic acid at position 324 of RIP. The cleavage of RIP results in the blockage of TNF-induced NF-kappaB activation. RIPc, one of the cleavage products, enhances interaction between TRADD and FADD/MORT1 and increases cells' sensitivity to TNF. Most importantly, the Caspase-8 resistant RIP mutants protect cells against TNF-induced apopotosis. These results suggest that cleavage of RIP is an important process in TNF-induced apoptosis. Furthermore, RIP cleavage is also detected in other death receptor-mediated apoptosis. Therefore, this study provides a potential mechanism to convert cells from life to death in death receptor-mediated apoptosis (Lin, 1999).

Cell death is achieved by two fundamentally different mechanisms: apoptosis and necrosis. Apoptosis is dependent on caspase activation, whereas the caspase-independent necrotic signaling pathway remains largely uncharacterized. Fas kills activated primary T cells efficiently in the absence of active caspases: this results in necrotic morphological changes and late mitochondrial damage but no cytochrome c release. This Fas ligand-induced caspase-independent death is absent in T cells that are deficient in either Fas-associated death domain (FADD) or receptor-interacting protein (RIP). RIP is also required for necrotic death induced by tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL). In contrast to its role in nuclear factor kappa B activation, RIP requires its own kinase activity for death signaling. Thus, Fas, TRAIL and TNF receptors can initiate cell death by two alternative pathways, one relying on caspase-8 and the other dependent on the kinase RIP (Holler, 2000).

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (Apo2 ligand [Apo2L]) is a member of the TNF superfamily and has been shown to have selective antitumor activity. Although it is known that TRAIL (Apo2L) induces apoptosis and activates NF-kappaB and Jun N-terminal kinase (JNK) through receptors such as TRAIL-R1 (DR4) and TRAIL-R2 (DR5), the components of its signaling cascade have not been well defined. The death domain kinase RIP is essential for TRAIL-induced IkappaB kinase (IKK) and JNK activation. Ectopic expression of the dominant negative mutant RIP, RIP(559-671), blocks TRAIL-induced IKK and JNK activation. In the RIP null fibroblasts, TRAIL failed to activate IKK and only partially activated JNK. The endogenous RIP protein was detected by immunoprecipitation in the TRAIL-R1 complex after TRAIL treatment. More importantly, RIP is not involved in TRAIL-induced apoptosis. In addition, the TNF receptor-associated factor 2 (TRAF2) plays little role in TRAIL-induced IKK activation although it is required for TRAIL-mediated JNK activation. These results indicated that the death domain kinase RIP, a key factor in TNF signaling, also plays a pivotal role in TRAIL-induced IKK and JNK activation (Lin, 2000).

To understand the mechanism of activation of the IkappaB kinase (IKK) complex in the tumor necrosis factor (TNF) receptor 1 pathway, the possibility was examined that oligomerization of the IKK complex triggered by ligand-induced trimerization of the TNF receptor 1 complex is responsible for activation of the IKKs. Gel filtration analysis of the IKK complex revealed that TNFalpha stimulation induces a large increase in the size of this complex, suggesting oligomerization. Substitution of the C-terminal region of IKKgamma, which interacts with RIP, with a truncated DR4 lacking its cytoplasmic death domain, produces a molecule that could induce IKK and NF-kappaB activation in cells in response to TRAIL. Enforced oligomerization of the N terminus of IKKgamma or truncated IKKalpha or IKKbeta lacking their serine-cluster domains can also induce IKK and NF-kappaB activation. These data suggest that (1) IKKgamma functions as a signaling adaptor between the upstream regulators such as RIP and the IKKs and that (2) oligomerization of the IKK complex by upstream regulators is a critical step in activation of this complex (Poyet, 2000).

The death domain kinase RIP and the TNF receptor-associated factor 2 (TRAF2) are essential effectors in TNF signaling. To understand the mechanism by which RIP and TRAF2 regulate TNF-induced activation of the transcription factor NF-kappaB, their respective roles in TNF-R1-mediated IKK activation was investigated using both RIP-/- and TRAF2-/- fibroblasts. It was found that TNF-R1-mediated IKK activation requires both RIP and TRAF2 proteins. Although TRAF2 or RIP can be independently recruited to the TNF-R1 complex, neither one of them alone is capable of transducing the TNF signal that leads to IKK activation. Moreover, IKK is recruited to the TNF-R1 complex through TRAF2 upon TNF treatment and IKK activation requires the presence of RIP in the same complex (Devin, 2000).

The activation of IkappaB kinase (IKK) is a key step in the nuclear translocation of the transcription factor NF-kappaB. IKK is a complex composed of three subunits: IKKalpha, IKKbeta, and IKKgamma (also called NEMO). In response to the proinflammatory cytokine tumor necrosis factor (TNF), IKK is activated after being recruited to the TNF receptor 1 (TNF-R1) complex via TNF receptor-associated factor 2 (TRAF2). The IKKalpha and IKKbeta catalytic subunits are required for IKK-TRAF2 interaction. This interaction occurs through the leucine zipper motif common to IKKalpha, IKKbeta, and the RING finger domain of TRAF2, and either IKKalpha or IKKbeta alone is sufficient for the recruitment of IKK to TNF-R1. Importantly, IKKgamma is not essential for TNF-induced IKK recruitment to TNF-R1, as this occurs efficiently in IKKgamma-deficient cells. Using TRAF2(-/-) cells, it has been demonstrated that the TNF-induced interaction between IKKgamma and the death domain kinase RIP is TRAF2 dependent and that one possible function of this interaction is to stabilize the IKK complex when it interacts with TRAF2 (Devin, 2001).

The adapter protein RIP plays a crucial role in NF-kappaB activation by TNF. Here it has been shown that triggering of the p55 TNF receptor induces binding of RIP to NEMO (IKKgamma), a component of the I-kappa-B-kinase (IKK) 'signalosome' complex, as well as recruitment of RIP to the receptor, together with the three major signalosome components, NEMO, IKK1 and IKK2, and some kind of covalent modification of the recruited RIP molecules. It also induces binding of NEMO to the signaling inhibitor A20, and recruitment of A20 to the receptor. Enforced expression of NEMO in cells reveals that NEMO can both promote and block NF-kappaB activation and dramatically augments the phosphorylation of c-Jun. The findings suggest that the signaling activities of the IKK signalosome are regulated through binding of NEMO to RIP and A20 within the p55 TNF receptor complex (Zhang, 2000).

The death domain kinase, receptor interacting protein (RIP), is one of the major components of the tumor necrosis factor receptor 1 (TNFR1) complex and plays an essential role in tumor necrosis factor (TNF)-mediated nuclear factor kappaB (NF-kappaB) activation. The activation of NF-kappaB protects cells against TNF-induced apoptosis. Heat-shock proteins (Hsps) are chaperone molecules that confer protein stability and help to restore protein native folding following heat shock and other stresses. The most abundant Hsp, Hsp90, is also involved in regulating the stability and function of a number of cell-signaling molecules. RIP is a novel Hsp90-associated kinase and disruption of Hsp90 function by its specific inhibitor, geldanamycin (GA), selectively causes RIP degradation and the subsequent inhibition of TNF-mediated IkappaB kinase and NF-kappaB activation. MG-132, a specific proteasome inhibitor, abrogates GA-induced degradation of RIP but fails to restore the activation of IkappaB kinase by TNF, perhaps because, in the presence of GA and MG-132, RIP accumulates in a detergent-insoluble subcellular fraction. Most importantly, the degradation of RIP sensitizes cells to TNF-induced apoptosis. These data indicate that Hsp90 plays an important role in TNF-mediated NF-kappaB activation by modulating the stability and solubility of RIP. Thus, inhibition of NF-kappaB activation by GA may be a critical component of the anti-tumor activity of this drug (Lewis, 2000).

The two opposite signaling pathways that stimulate NF-kappaB activation and apoptosis are both mediated by tumor necrosis factor receptor 1 (TNFR1) and its cytosolic associated proteins. The proteolytic cleavage of receptor interacting protein (RIP) by caspase-8 during TNF-induced apoptosis abrogates the stimulatory role of RIP on TNF-induced NF-kappaB activation. The uncleavable RIPD324A mutant is less apoptotic, but its ability to activate NF-kappaB activation is greater than the wild type counterpart. Ectopic expression of the pro-apoptotic C-terminal fragment of RIP inhibits TNF-induced NF-kappaB activation by suppressing the activity of I-kappaB kinasebeta (IKKbeta) which phosphorylates I-kappaB, an inhibitor of NF-kappaB, and triggers its ubiquitin-mediated degradation. The C-terminal fragment of RIP also enhances the association between TNFR1 and death domain proteins including TNFR1 associated death domain (TRADD) and Fas associated death domain (FADD), resulting in the activation of caspase-8 and stimulation of apoptosis. The present study suggests that the C-terminal fragment of RIP produced by caspase-8 activates death-inducing signaling complex (DISC), attenuates NF-kappaB activation, and thereby amplifies the activation of caspase-8, which initiates the downstream apoptotic events (Kim, 2000).

Fas-associated death domain protein (FADD), caspase-8-related protein (Casper), and caspase-8 are components of the tumor necrosis factor receptor type 1 (TNF-R1) and Fas signaling complexes that are involved in TNF-R1- and Fas-induced apoptosis. Overexpression of FADD and Casper potently activates NF-kappaB. In the presence of caspase inhibitors, overexpression of caspase-8 also activates NF-kappaB. A caspase-inactive point mutant, caspase-8(C360S), activates NF-kappaB as potently as wild-type caspase-8, suggesting that caspase-8-induced apoptosis and NF-kappaB activation are uncoupled. NF-kappaB activation by FADD and Casper is inhibited by the caspase-specific inhibitors crmA and BD-fmk, suggesting that FADD- and Casper-induced NF-kappaB activation is mediated by caspase-8. FADD, Casper, and caspase-8-induced NF-kappaB activation are inhibited by dominant negative mutants of TRAF2, NIK, IkappaB kinase alpha, and IkappaB kinase beta. A dominant negative mutant of RIP inhibits FADD- and caspase-8-induced but not Casper-induced NF-kappaB activation. A mutant of Casper and the caspase-specific inhibitors crmA and BD-fmk partially inhibit TNF-R1-, TRADD, and TNF-induced NF-kappaB activation, suggesting that FADD, Casper, and caspase-8 function downstream of TRADD and contribute to TNF-R1-induced NF-kappaB activation. Moreover, activation of caspase-8 results in proteolytic processing of NIK, which is inhibited by crmA. When overexpressed, the processed fragments of NIK do not activate NF-kappaB, and the processed C-terminal fragment inhibits TNF-R1-induced NF-kappaB activation. These data indicate that FADD, Casper, and pro-caspase-8 are parts of the TNF-R1-induced NF-kappaB activation pathways, whereas activated caspase-8 can negatively regulate TNF-R1-induced NF-kappaB activation by proteolytically inactivating NIK (Hu, 2000).


Search PubMed for articles about Drosophila immune deficiency

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Chen, H., Zheng, X. and Zheng, Y. (2014). Age-associated loss of lamin-B leads to systemic inflammation and gut hyperplasia. Cell 159: 829-843. PubMed ID: 25417159

Choe, K. M., Lee, H. and Anderson, K. V. (2005). Drosophila peptidoglycan recognition protein LC (PGRP-LC) acts as a signal-transducing innate immune receptor. Proc. Natl. Acad. Sci. 102: 1122-1126. 15657141

Choe, K. M., et al. (2002). Requirement for a peptidoglycan recognition protein (PGRP) in Relish activation and antibacterial immune responses in Drosophila. Science 296: 359-362. 11872802

Corbo, J. C. and Levine, M. (1996). Characterization of an immunodeficiency mutant in Drosophila. Mech. Dev. 55(2): 211-20. 8861100

De Gregorio, E., et al. (2002). The Toll and Imd pathways are the major regulators of the immune response in Drosophila. EMBO J. 21: 2568-2579. 12032070

Devin, A., et al. (2000). The distinct roles of TRAF2 and RIP in IKK activation by TNF-R1: TRAF2 recruits IKK to TNF-R1 while RIP mediates IKK activation. Immunity 12: 419-429. 10795740

Devin, A., et al. (2001). The alpha and beta subunits of IkappaB kinase (IKK) mediate TRAF2-dependent IKK recruitment to tumor necrosis factor (TNF) receptor 1 in response to TNF. Mol. Cell. Biol. 21(12): 3986-94. 11359906

Donlea, J. M., Ramanan, N., Silverman, N. and Shaw, P. J. (2014). Genetic rescue of functional senescence in synaptic and behavioral plasticity. Sleep 37(9): 1427-37. PubMed ID: 25142573

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