InteractiveFly: GeneBrief

Drosomycin: Biological Overview | Regulation | Developmental Biology | Evolutionary Homologs | References


Gene name - Drosomycin

Synonyms -

Cytological map position-63D2

Function - immune induced antifungal protein

Keywords - antifungal humoral response

Symbol - Drs

FlyBase ID: FBgn0283461

Genetic map position - 3L

Classification - cysteine-rich peptide

Cellular location - secreted



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Li, Y., Li, S., Li, R., Xu, J., Jin, P., Chen, L. and Ma, F. (2016). Genome-wide miRNA screening reveals miR-310 family members negatively regulate the immune response in Drosophila melanogaster via co-targeting Drosomycin. Dev Comp Immunol 68: 34-45. PubMed ID: 27871832
Summary:
Although innate immunity mediated by Toll signaling has been extensively studied in Drosophila melanogaster, the role of miRNAs in regulating the Toll-mediated immune response remains largely unknown. Following Gram-positive bacterial challenge, this study identified 93 differentially expressed miRNAs via genome-wide miRNA screening. These miRNAs were regarded as immune response related (IRR). Eight miRNAs were confirmed to be involved in the Toll-mediated immune response upon Gram-positive bacterial infection through genetic screening of 41 UAS-miRNA lines covering 60 miRNAs of the 93 IRR miRNAs. Interestingly, four out of these eight miRNAs, miR-310, miR-311, miR-312 and miR-313, are clustered miRNAs and belong to the miR-310 family. These miR-310 family members were shown to target and regulate the expression of Drosomycin, an antimicrobial peptide produced by Toll signaling. Taken together, this study implies important regulatory roles of miRNAs in the Toll-mediated innate immune response of Drosophila upon Gram-positive bacterial infection.
Li, S., Li, Y., Shen, L., Jin, P., Chen, L. and Ma, F. (2016). miR-958 inhibits Toll signaling and Drosomycin expression via directly targeting Toll and Dif in Drosophila melanogaster. Am J Physiol Cell Physiol [Epub ahead of print]. PubMed ID: 27974298
Summary:
Drosophila melanogaster is widely used as a model system to study innate immunity and signaling pathways related to innate immunity, including the Toll signaling pathway. Although this pathway is well-studied, the precise mechanisms of post-transcriptional regulation of key components of the Toll signaling pathway by microRNAs (miRNAs) remain obscure. This study used an in silico strategy in combination with the Gal80ts-Gal4 driver system and identified microRNA-958 (miR-958) as a candidate Toll pathway regulating miRNA in Drosophila. Overexpression of miR-958 significantly reduces the expression of Drosomycin, a key antimicrobial peptide involved in Toll signaling and the innate immune response. It was demonstrated in vitro and in vivo that miR-958 targets the Toll and Dif genes, key components of the Toll signaling pathway, to negatively regulate Drosomycin expression. In addition, a miR-958-sponge rescues the expression of Toll and Dif, resulting in increased expression of Drosomycin. These results not only reveal a novel function and modulation pattern of miR-958, but also provide a new insight into the underlying molecular mechanisms of Toll signaling in regulation of innate immunity.


BIOLOGICAL OVERVIEW

The initial description of the anti-fungal peptide Drosomycin is found in a study by Fehlbaum (1994). In response to a septic injury (pricking with a bacteria-soaked needle) larvae and adult Drosophila produce considerable amounts of a 44-residue peptide containing 8 cysteines engaged in intramolecular disulfide bridges. The peptide is synthesized in the fat body, a functional homologue of the mammalian liver, and secreted into the blood of the insect. It exhibits potent antifungal activity but is inactive against bacteria. This novel inducible peptide, Drosomycin, shows a significant homology with a family of 5-kDa cysteine-rich plant antifungal peptides isolated from seeds of Brassicaceae. This finding underlines that plants and insects can rely on similar molecules in their innate host defense (Fehlbaum, 1994).

There are a number of different controls on the expression of the antifungal polypeptide gene drosomycin in adults: the receptor Toll, intracellular components of the dorsoventral signaling pathway (Tube, Pelle, and Cactus), and the extracellular Toll ligand, Spätzle, but not the NF-kappaB related transcription factor Dorsal. Mutations in the Toll signaling pathway dramatically reduce survival after fungal infection. In Tl-deficient adults, the cecropin A and, to a lesser extent, attacin, drosomycin and defensin genes are only minimally inducible, in contrast with the diptericin and drosocin genes, which remain fully inducible in this context. The drosomycin gene induction is not affected in mutants deficient in gastrulation defective, snake and easter, all upstream of spätzle in the dorsoventral pathway. The involvement of Spätzle in the drosomycin induction pathway is unexpected, since, in contrast with cat, pll, tub, and Tl, the spz mutant shows no striking zygotic phenotype. The partner of Cact in the drosomycin induction pathway has not yet been identified, but it is probably a member of the Rel family, possibly Dorsal-related immunity factor (Lemaitre, 1996).

There are two distinct regulatory pathways controlling the expression of antimicrobial genes, the dorsoventral pathway and the immune deficiency (imd) gene. In contrast to the results with drosomycin, antibacterial genes, cecropin A1, diptericin, drosocin, attacin, and defensin do not give strong constitutive expression in dorsoventral pathway mutants. However, the level of constitutive expression of anti-bacterial genes in dorsoventral pathway mutants is higher than the basal level, and induction of Cecropin A genes is 4-fold lower in dorsoventral pathway mutants. The transcription of cact, dorsal, dif, pll, tub, Tl and spz genes, but not tub, are clearly up-regulated in response to immune challenge. Even though the same components of the dorsoventral pathway that are involved in antifungal response are also involved in antibacterial response, there is an additional requirement for the imd gene product (Lemaitre, 1996).

The Rel protein DIF mediates the antifungal but not the antibacterial host defense in Drosophila

Two Drosophila lines were isolated that carry point mutations in the gene coding for the NF-kappaB-like factor Dif. Like mutants of the Toll pathway, Dif mutant flies are susceptible to fungal but not to bacterial infections. Genetic epistasis experiments demonstrate that Dif mediates the Toll-dependent control of the inducibility of the antifungal peptide gene Drosomycin. Strikingly, Dif alone is required for the antifungal response in adults, but is redundant in larvae with Dorsal, another Rel family member. In Drosophila, Dif appears to be dedicated to the antifungal defense elicited by fungi and gram-positive bacteria. The possibility is discussed that NF-kappaB1/p50 might be required more specifically in the innate immune response against gram-positive bacteria in mammals (Rutschmann, 2000).

Transcription factors of the Rel family have long been suspected to play an important role in the control of the expression of insect antimicrobial peptides. To date, three members of this family have been reported in Drosophila, namely, Dorsal, DIF, and Relish. In this study, two mutations in the gene encoding Dif were generated and show that this Rel protein plays a critical role in the control of the antifungal response in Drosophila (Rutschmann, 2000).

The Dif1 mutation is a strong mutation, since no significant Drosomycin induction is observed in flies subjected to a natural infection by the fungus B. bassiana in contrast to wild-type flies, where this infection triggers a strong and sustained expression of the antifungal gene. Similar results have been obtained upon injury with either gram-positive bacteria or fungi. Furthermore, in response to a fungal or gram-positive bacterial infection, Dif1 homozygous and hemizygous flies transcribe the Drosomycin gene at the same low, residual level and show similar survival curves to fungal infections. These data indicate that the Dif1 mutation is genetically close to a null mutation. It is likely that the Dif2 mutation is also a strong mutation, since the induction of Drosomycin by immune challenge and the survival to fungal infections were similar in Dif1 and Dif2 homozygous, hemizygous, or transheterozygous Dif1/Dif2 flies (Rutschmann, 2000).

The two mutations isolated in this study were induced in the Rel homology domain (RHD). Rel proteins bind as dimers to DNA with an unusually high affinity through the conserved RHD. The structure of this domain has been determined by X-ray crystallography in several NF-κB members over the last years. Its main features have been remarkably conserved throughout evolution. The dimer wraps around the DNA, giving the appearance of a butterfly to the complex. Each monomer consists of two immunoglobulin (Ig)-like domains connected by a short linker. The N-terminal Ig-like domain consists of a nine-stranded β barrel and contains a recognition loop (L1) that makes specific contacts with the DNA binding site. This domain also contacts the binding site through a second loop (L2) that clamps the DNA at the central minor groove via basic residues. The C-terminal Ig-like domain consists of a seven-stranded β barrel, which contacts the DNA backbone through two loops (L4 and L5) and mediates homo- or heterodimerization of NF-κB subunits. Together with the nuclear localization signal located right at the C-terminal end of the RHD, the C-terminal Ig-like domain also binds to the six ankyrin repeats of the I-κB inhibitor. Finally, a basic amino acid located in the linker (loop L3) that joins the N-terminal to the C-terminal Ig-like domains makes a specific contact with a nucleotide of the DNA binding site at position ± 2 (the dyad axis of symmetry passes usually through the nucleotide in position 0) (Rutschmann, 2000).

The positions of the two mutations in the Dif structure were visualized by modeling a putative Dif based on the crystallographic data from the mosquito Rel protein Gambif, which is 37% homologous to Dif over the RHD. Glycine 181 is located in the middle of β strand E′ on the inside of the N-terminal domain, close to the DNA clamping loop L2. This glycine has been conserved in all RHDs known to date and presumably plays an important structural role. Its replacement by a bulky, negatively charged amino acid, in close vicinity to the DNA helix, certainly perturbs the local structure and prevents loop L2 from binding to DNA. Thus, it is anticipated that the Dif1 mutation severely affects the high-affinity DNA binding of Dif. The C-terminal dimerization domain of the RHD that contacts I-κBs is not affected by the mutation and nuclear import of the protein is not altered, as illustrated by nuclear localization of Dif1 following an immune challenge (Rutschmann, 2000).

The Dif2 mutation replaces a serine by a phenylalanine residue. This serine is unlikely to be the target of a kinase, since its alcoholic function is not accessible to solvents. The same holds true for serines at the same position in the other Rel structures that have been determined. It is buried on the inside of the protein where the oxygen of the alcoholic function makes a hydrogen bond with the amide function of leucine 225 in the modeled Dif structure. The minimal hypothesis is favored that the Dif2 mutation induces a strong local perturbation of the structure of the N-terminal Ig-like domain. Indeed, serine 245 is part of β strand G that connects β strand F, in particular through its alcoholic function. It is likely that this contact stabilizes the orientation of loop L3, which is highly structured when bound to DNA. It usually makes a specific contact with a central DNA base, probably through lysine 251 in Dif. As for Dif1, it is expected that the Dif2 mutation affects DNA binding, although the overall stability of the protein might also be affected. It is therefore likely that both mutations prevent the recognition of κB binding sites by Dif and that they do not impair protein–protein interactions with putative cofactors (Rutschmann, 2000).

Two lines of evidence indicate that Dif plays a pivotal role in the antifungal response in adult Drosophila. First, Dif flies are more susceptible to fungal infections than wild-type flies. Second, immune induction of Drosomycin, the predominant antifungal peptide gene of Drosophila, is severely reduced, if not abolished in those flies. Furthermore, Defensin induction is significantly decreased in Dif mutants. As regards Cecropin, Dif and dl appear to participate, at least partially, in the regulation of its immune-induced expression. Defensin and Cecropin display some antifungal properties in addition to their antibacterial activities. Even though the decreased expression of antifungal peptides certainly contributes to susceptibility to fungal infection, the possibility that Dif also activates other antifungal defense mechanisms and, namely, cellular responses, cannot be excluded (Rutschmann, 2000).

The Dif phenotype reported in this study is strikingly similar to that of mutants that affect the spz/Tl/tub/pll/cac gene cassette (Lemaitre, 1996). The epistatic relationship between Tl and Dif on one hand and cact and Dif on the other hand provides a clear demonstration that the control of Drosomycin inducibility is mediated through Dif in response to activation of the Tl pathway. Since fungal infections seem to have similar lethal effects on Dif and spz mutants, it is proposed that Dif controls the various aspects of the Tl-mediated antifungal response in adult Drosophila (Rutschmann, 2000).

As regards the induction of Cecropin, Dif and dorsal appear to be functionally redundant in adults. Indeed, immune induction of Cecropin is unaffected in Dif mutants and is significantly reduced in Dif/TW119 hemizygous flies. Since the Dif mutations are strong and are likely to affect DNA binding of Dif rather than putative interactions with cofactors, this effect cannot be ascribed to a hypomorphic effect of the Dif mutations on the Cecropin promoter. Rather, it is likely that the removal of a copy of dl in TW119 combined with the total lack of Dif is responsible for this phenotype in the Dif hemizygous background. It thus appears that the single dose of dl left in this genetic context is not sufficient to mediate the contribution of the Tl pathway to the regulation of Cecropin, and that this reduced but significant expression is regulated via the imd pathway. This inference in the case of Cecropin is further substantiated by the following observations for Drosomycin. In larvae, have shown that Dif and dl are functionally redundant in controlling the inducibility of Drosomycin (Manfruelli, 1999). This inducibility is not markedly affected in a Dif background but is significantly reduced in Dif1 and Dif2 hemizygous larvae where one wild-type copy of dl is left (Rutschmann, 2000).

In contrast to that of Defensin or Cecropin, the immune induction of Attacin does not depend on either Dif or dl, whereas it is strongly decreased in spz, Tl, and pll mutants. These results suggest that a branch point in the Tl pathway exists downstream of pll to regulate an undefined transcription factor, possibly Relish, that controls the inducibility of Attacin in adults (Rutschmann, 2000).

The Dif mutations generated in this study do not fully abolish Drosomycin induction by immune challenge with a mix of gram-positive and gram-negative bacteria. Indeed, some 25% of wild-type levels of induction were consistently observed in these conditions. In contrast, fungi or gram-positive bacteria failed to induce any induction of Drosomycin in Dif1 mutants. The residual Drosomycin expression is abolished in Dif-kenny double mutants challenged with the mix (kenny is a mutant that shows a phenotype similar to that of Relish). It is proposed that the low level of Drosomycin inducibility triggered by gram-negative bacteria is partially controlled by a Dif-independent pathway, namely, the imd-kenny-Relish pathway. This hypothesis is supported by the result that an infection with E. coli triggers a short-lived induction of Drosomycin, similar to that of fast reactants such as Cecropin and Attacin, that are predominantly controlled by imd-kenny-Relish. Similar observations have been reported in the case of the other genes of the Toll-signaling cassette, where some 25% of wild-type Drosomycin induction was observed after challenge with the mix, whereas no induction was detected when Tl mutants were coated with fungal spores. Furthermore, the low level of Drosomycin expression observed in Tl pathway mutants was totally abolished in Tl-imd double mutants. In conclusion, it is proposed that natural infections with B. bassiana spores trigger Drosomycin expression exclusively by the Tl-Dif-dependent pathway, whereas another pathway, most likely the Relish-kenny-imd pathway, induces, at least partially, Drosomycin expression in response to gram-negative bacteria (Rutschmann, 2000).

It has been shown that Relish is required for the induction of most antimicrobial peptides in response to a gram-negative immune challenge. Specifically as regards Drosomycin, only 20% of wild-type levels were induced in Relish null mutants 6 hr after a challenge with the gram-negative Enterobacter cloacae. The data presented above are compatible with the hypothesis that expression of the Drosomycin gene is controlled by a Relish-Dif heterodimer, which is also in keeping with tissue culture experiments performed with transfected S2 cells. However, the possibility that Relish mediates the gram-negative-induced, Dif-independent component of Drosomycin induction cannot be excluded (Rutschmann, 2000).

It has been demonstrated (Manfruelli, 1999) by a clonal analysis of the TW119 deficiency that Dif and dl are functionally redundant in the control of Drosomycin inducibility in larvae. In contrast, another study (Meng, 1999) has observed that Dif but not dl regulates the expression of Drosomycin in adults. This latter result was obtained by complementation with either a Dif or a dorsal transgene of the small J4 deficiency that removes both genes. The analysis of the Dif point mutants presented in this study resolves this paradox and shows that the regulation of Drosomycin expression in the adult fat body differs from that in the larval fat body. The reasons for this difference are at present unclear. It has to be keep in mind that larval and fat body cells are not fully equivalent and have distinct developmental origins. One explanation for the difference in regulation could be that larval and adult fat body cells produce different levels of either Dif or DL. Another explanation could relate to the presence or absence in larvae versus adults of various cofactors required for full activation of response genes by DL or Dif (Rutschmann, 2000).

Five distinct Rel family members are present in mammals where they play a major role in the host defense by controlling the expression of such diverse immune-response molecules as immunoreceptors, cytokines, adhesion molecules, and acute phase proteins. In addition, they can provide protection against TNFα-mediated apoptosis. The analysis of the respective roles of the five Rel proteins is hampered by partial redundancies and by the complexities or lethality of the mutant phenotypes. In particular, the identity of the Rel proteins that mediate the TLR2-dependent response to peptidoglycans and the TLR4-dependent response to LPS has not been established, since in both cases only the activation of a 'generic' NF-κB binding activity was investigated. Yet, distinct responses are likely to be elicited by different pathogens. In this respect, it is striking that p50 knockout mice are highly susceptible to infections by the pathogenic gram-positive Streptococcus pneumoniae and not by the gram-negative Haemophilus influenzae or E. coli K1, which raises the possibility that p50 is the Rel protein mediating the response to gram-positive bacteria (Rutschmann, 2000).

In Drosophila, mutants for all three individual Rel proteins are now available. Remarkably, the viability of these mutants is not impaired under normal conditions. Dif plays a critical role in mediating the antifungal response that is activated through the Tl pathway in the adult. Strikingly, Dif does not seem to play a role in the humoral immune response against gram-negative bacteria, as can be judged from the lack of effect of Dif mutations on the inducibility of Drosocin, Diptericin, and Attacin, all of which are essentially active on such bacteria. Studies underway should reveal whether the three Rel proteins are indeed complementary in mediating a humoral immune response against diverse pathogens or whether there is some degree of functional redundancy like that observed between Dif and dl in larvae (Rutschmann, 2000).

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

Jun N-terminal kinase (JNK) signaling is a highly conserved pathway that controls both cytoskeletal remodeling and transcriptional regulation in response to a wide variety of signals. Despite the importance of JNK in the mammalian immune response, and various suggestions of its importance in Drosophila immunity, the actual contribution of JNK signaling in the Drosophila immune response has been unclear. Drosophila TAK1 has been implicated in the NF-kappaB/Relish-mediated activation of antimicrobial peptide genes. However, this study demonstrates that Relish activation is intact in dTAK1 mutant animals, and that the immune response in these mutant animals is rescued by overexpression of a downstream JNKK. The expression of a JNK inhibitor and induction of JNK loss-of-function clones in immune responsive tissue revealed a general requirement for JNK signaling in the expression of antimicrobial peptides. The data indicate that dTAK1 is not required for Relish activation, but instead is required in JNK signaling for antimicrobial peptide gene expression (Delaney, 2006).

Innate immune responses are critical for a rapid host defense against pathogens. The signaling pathways that control these responses are present in all multicellular organisms, ranging from humans to flies, and are remarkably well conserved. Although the innate response lacks the antigen recognition capacity of vertebrate adaptive immunity, it is nevertheless complex and crucial for host survival. Drosophila is a proven genetic model organism for the study of innate immunity and has provided invaluable insights into the control of responses to infection (Delaney, 2006).

Toll and Imd are the founding members of two principal innate immune response signaling pathways in Drosophila. Toll signals through two NF-kappaB/Rel family transcription factors, Dif and Dorsal, and is required for responses to fungal and Gram+ bacterial infections (Rutschmann, 2000; De Gregorio, 2002). Imd signaling controls primarily Gram- bacteria-specific responses through the cleavage and activation of a third Rel family transcription factor, Relish, by the Drosophila caspase Dredd. Relish activation also requires an IkappaB kinase (IKK) complex that is itself activated by Imd signaling. The transcriptional targets of Dif and Relish are not entirely distinct. For example, cecropinA expression requires either Relish or Dif, or both, depending on the type and strain of infecting microorganism. More than 20 Drosophila genes have been implicated in these signaling pathways and nearly all of them have mammalian homologues with conserved immune functions (Delaney, 2006 and references therein).

Jun N-terminal kinase (JNK) signaling has been linked to stress responses, cell migration, apoptosis, and immune responses in both insects and mammals. JNK activity can be induced by infection, lipopolysaccharide, and inflammatory cytokines such as tumor necrosis factor (TNF) in flies and mammals. Null mutations in JNK signaling components are typically embryonic lethal in flies and thus unlikely to appear as targets of mutagenesis screens designed to detect immune response genes in living animals. An exception to this rule is dTAK1. Overexpression and dominant-negative studies indicated that dTAK1 can act as a JNK kinase kinase (Delaney, 2006 and references therein).

Previously characterized dTAK1 mutations, however, showed no apparent JNK-like phenotype, but failed to express Relish-dependent antimicrobial peptides, suggesting a role in the Imd pathway (Vidal, 2001). Previous epistasis analysis using the UAS/GAL4 overexpression system to ectopically express dTAK1 placed dTAK1 downstream of imd and upstream of the IKK complex in the Relish signaling pathway (Vidal, 2001). In vitro experiments implicated dTAK1 in the IKK-dependent phosphorylation of Relish in S2 cells (Delaney, 2006).

Evidence has been uncovered for a Relish-independent function of dTAK1 in the control of antimicrobial peptide gene expression. Several aspects of Relish activation appeared normal in infected dTAK1 mutant animals, including cleavage, nuclear localization, and promoter binding. Therefore whether JNK pathway components mediate dTAK1 function in the immune response was examined. Several lines of evidence are reported for dTAK1 acting through the JNK cascade in the innate immune response. First, overexpression of Hemipterous, a JNKK, rescued attacin and diptericin expression in dTAK1 mutant animals, whereas overexpression of the downstream Imd component Dredd did not. Second, it was found that expression of the Puckered (Puc) phosphatase, an inhibitor of JNK activity, suppressed the expression of antimicrobial peptide genes. To directly test for a JNK requirement in immune signaling, JNK mutant clones were induced in the fat body of larvae. Strikingly, diptericin, attacin, Metchnikowin, and drosomycin expression was lost in the mutant tissue (Delaney, 2006).

It is concluded that the JNK pathway is required to mediate dTAK1 signaling during the Drosophila immune response. Furthermore, a model is proposed where the JNK and NF-kappaB signaling are both required to activate antimicrobial peptide gene expression during the immune response in the Drosophila fat body (Delaney, 2006).

The function of TAK1 in vertebrates has remained enigmatic. It was originally identified as a TGFβ-activated kinase, hence the name, in mammalian cell culture assays. However, follow-up work in multicellular contexts and in vivo analyses in vertebrates, C. elegans, and Drosophila have shown no clear link to TGFβ signaling, but rather suggest a role for TAK family kinases in JNK activation or as upstream activators of Nemo-like kinases. In mammalian systems, TAK1 is one of a number of kinases that can activate IKK complexes and, consequently, NF-kappaB signaling in vitro. In vitro studies of human cells have shown that targeting of TAK1 by RNAi reduces NF-kappaB activation by TNFalpha and IL-1 stimulation. Recent studies using fibroblasts derived from TAK1 mutant mouse embryos and mice with a B-cell-specific deletion of TAK1 showed that JNK activation was impaired in response to all stimuli tested in TAK1 mutant cells. Although NF-kappaB activation was impaired in response to stimulation by IL-1β, TNF, and TLR3 and TLR4 ligands, NF-kappaB activation by B-cell receptor or LT-β stimulation remained intact, suggesting a specific role for TAK1 upstream of IKKβ and JNK, but not IKKalpha. Interestingly, IKKalpha activation leads to the phosphorylation and processing of NF-kappaB2 from the p100 to the active p52 form, reminiscent of Relish activation in Drosophila (Delaney, 2006 and references therein).

Biochemical analyses in mammalian systems have demonstrated that TAK1 functions in multimeric protein complexes that can include TAB1, TAB2, and different TRAF proteins. The exact composition of these complexes seems to determine TAK1 responsiveness and downstream effects. In the fly, genetic studies found an interaction between dTRAF1 and dTAK1 in the activation of JNK signaling and apoptosis. Gain- and loss-of-function analyses indicate that dTRAF2, but not dTRAF1, is necessary for the activation of Relish-dependent gene expression; however, no interaction between dTRAF2 and dTAK1 in the activation of antimicrobial peptides has been reported (Delaney, 2006).

Genome-wide analyses that examined in vivo responses in Drosophila identified dJun and puc as genes potentially regulated by Toll and Imd signaling, suggesting a cross-regulation between these pathways and the JNK signaling pathway. A study recently reported that RNAi knockdown of kayak, msn, hep, or aop blocked E. coli-induced attacin and drosomycin expression in S2 cells. Furthermore, in related studies, it was also observed that, although dTAK1 RNAi-treated S2 cells failed to express an attacin reporter gene, Relish cleavage and nuclear localization remain intact in these cells. Other RNAi analyses in S2 cells have concluded that JNK signaling does not have a significant role in antimicrobial peptide gene expression. However, RNAi against hep or bsk seemed to partially block antimicrobial peptide induction, especially of attacin and cecropinA and, accordingly, attacinD levels were lower in microarrays when the JNK pathway was blocked. The current results confirm a positive role for JNK signaling in the antimicrobial peptide response in vivo (Delaney, 2006).

The placement of dTAK1 function upstream of JNK, rather than IKK, requires a remodeling of the signaling pathways that activate the antimicrobial peptide genes. Earlier models were based on studies that showed that dTAK1 mutations blocked the constitutive activation of diptericin by Imd overexpression (Vidal, 2001). In turn, IKK mutations blocked dTAK1-induced diptericin expression. One interpretation of these data places IKK directly downstream of dTAK1. However, if the activation of both JNK and IKK signaling pathways is required, then a disruption in either branch would be sufficient to suppress any upstream activation (Delaney, 2006).

Overexpression of dTAK1 is sufficient to induce antimicrobial peptide expression (Vidal, 2001). However, dTAK1 is an extremely potent activator of JNK signaling and apoptosis, and overexpression of dTAK1 could activate proteins that are not normal phosphorylation targets. Based on RNAi studies in S2 cells, dTAK1 is required for dIKK complex-dependent phosphorylation of Relish in vitro. This could reflect a stringent requirement for dTAK1 in blood cell-derived S2 cells that is different in fat body tissue (Delaney, 2006).

The new model would predict that overexpression of the Dredd caspase would be insufficient to activate fully the antimicrobial peptides in dTAK1 mutant animals and this is indeed the case. Overexpression of Dredd may be sufficient to induce antimicrobial peptide gene expression in a wild-type background because of inadvertent JNK pathway activation by ectopic caspase activity or by the heat-shock protocol itself. Alternatively, an additional role for Dredd has been proposed in the ubiquitin-mediated activation of dTAK1 and the dIKK complex (Zhou, 2005). The suppression by dTAK1 mutants of ectopic Dredd expression is consistent with this model as well, and does not distinguish between the two potential functions of Dredd. The current data are consistent with a model that places dTAK1 activity in a pathway parallel to the functions of IKK and Relish and in which both these pathways are required for the activation of antibacterial peptide genes such as diptericin and attacin (Delaney, 2006).

Promoter analyses of most antimicrobial peptide genes have not revealed any obvious binding sites for activator protein-1 (AP-1) complexes, the Jun/Fos heterodimer, and transcriptional mediator of JNK signaling. However, AP-1 binding sites can be quite diverse and are not always predictable directly from DNA sequence. Nevertheless, a recent study identified a functional AP-1 binding site in the attacinA promoter. These data suggest that AP-1 binding represses attacinA transcription by recruiting histone deacetylase 1 (dHDAC1) to the promoter. In contrast, in mammalian studies, c-Jun function is itself repressed by association with HDAC3. This repression is relieved upon JNK signaling. A similar mechanism may be employed in the Drosophila fat body. Accordingly, the sustained expression of attacin and other antimicrobial peptide genes in vivo would require an activation (or de-repression) of AP-1 function at the onset of the immune response. Such positive cooperation between AP-1 and NF-kappaB transcription factors was also seen in molecular studies of the human β-defensin-2 promoter (Delaney, 2006).

AP-1-dependent gene expression is normally rapid. Thus, if AP-1 activity is not directly required for diptericin expression, it could act indirectly through the activation of other genes. Alternatively, JNK could phosphorylate some targets other than the AP-1 complex proteins Jun and Fos. In mammalian studies, it has been shown that JNK can phosphorylate, and thereby inhibit, Insulin Receptor Substrate-1. However, the recent finding (Kallio, 2005) that RNAi against kayak/dFos can block antimicrobial peptide expression and the current dJun loss-of-function studies in vivo suggest that JNK does indeed signal through AP-1 to control expression of these genes (Delaney, 2006).

It is intriguing that overexpressed Puc not only blocks Relish-dependent antimicrobial peptide gene expression, but it also strongly blocks drosomycin expression, which is not true in dTAK1 mutants (Vidal, 2001). This suggests that JNK or JNK-related proteins, for example, p38a, p38b, and MPK2, may also be important for other aspects of the immune response, for example, the Toll/Dif-dependent antimicrobial genes (Sluss, 1996: Han, 1999). The clonal analysis of JNK mutant tissue confirms that JNK is required not only for the expression of Gram--specific peptides diptericin and attacin, but also for Metchnikowin (Gram+/fungal specific) and drosomycin (fungal specific). Mutations in dTAK1 had less of an impact on Metchnikowin or drosomycin expression than on attacin, for example. Furthermore, reduced dJun activation occurred in dTAK1 mutant animals, indicating that other upstream kinases may be involved in the control of these genes. JNK is a member of a large family of mitogen-activated protein kinases (MAPKs). In the fly, there are at least five MAPKKKs, four MAPKKs, and five MAPKs, and so the potential redundancies are many. If these other proteins contribute to the immune response, how they do so has yet to be tested in genetic loss-of-function in vivo studies in the fat body (Delaney, 2006).

How JNK and NF-kappaB signals integrate to positively control gene expression is a critical question. This study has demonstrated that both are required for the expression of a particular set of immune responsive genes in vivo. Through the use of Drosophila genetics, it should be possible to identify novel immune response genes that are controlled cooperatively by JNK and NF-kappaB signaling. From promoter analysis of these genes, it may be possible to predict additional genes that are important for other biological processes. Both the JNK and NF-kappaB signaling pathways have been implicated many times in many different contexts. Continued analysis in Drosophila may lead to a general understanding of their roles in normal biological processes and developmental malignancies (Delaney, 2006).


REGULATION

Transcriptional Regulation

Interaction and specificity of Rel-related proteins in regulating Drosophila immunity gene expression

Since co-expression of Relish with Dif or Dorsal can enhance the expression of drosomycin and defensin, it has been speculated that the proteins can interact directly with the target genes to regulate their expression. Among the immunity genes, the cecropin and diptericin promoters have been the most thoroughly analyzed. Previously, it has been shown that Dif can bind to the kappaB site in the cecropin promoter, whereas Dorsal can bind to a similar site in the diptericin promoter. The binding probably leads to activated transcription of cecropin and diptericin. Furthermore, Dif and Dorsal may preferentially activate cecropin and diptericin, respectively, based on DNA binding and transfection studies. Meanwhile, the results suggest the possibility that the heterodimers formed by these Rel proteins bind to kappaB sites in the promoters of drosomycin and defensin. Based on the conserved sequence of insect kappaB motif, similar kappaB sites have been identified in the promoters of both drosomycin and defensin. It was then tested whether Rel proteins expressed in the stable cell lines can interact directly with these kappaB sites using EMSA. The parental S2* cell extract exhibits very little DNA binding activity. The cell extract that expresses FLAG-tagged Relish has strong binding activity toward the oligonucleotide containing the drosomycin kappaB site. This activity is supershifted by anti-FLAG antibody, showing Relish is the protein factor that binds directly to the kappaB site. Compared with Relish, Dif and Dorsal have relatively lower binding affinity. When Relish is co-expressed with Dif or Dorsal, new complexes are formed that have faster mobility, probably due to the formation of Dif/Relish or Dorsal/Relish heterodimer, which has lower molecular weight. The result indicates that Relish enhances the binding of Dif and Dorsal to the kappaB site. Moreover, the corresponding bands are supershifted by anti-FLAG antibody, demonstrating that FLAG-tagged Relish complexes with Dif and Dorsal in binding to DNA (Han, 1999).

To further confirm that Rel proteins bind to the kappaB sites within the oligonucleotides, competition EMSA was carried out. Whereas 20-fold excess amount of the same wild type oligonucleotide effectively blocks the binding of the Rel proteins to the 32P-labeled wild type oligonucleotide, the oligonucleotide with mutated kappaB motif does not affect the binding, suggesting that the Rel proteins bind specifically to the kappaB motif of drosomycin (Han, 1999).

The binding activity of Rel proteins to the kappaB site of defensin promoter shows similar pattern to that of drosomycin promoter, albeit with lower affinity. Interestingly, this is coincident with lower level of defensin expression in S2* cells and in adult fly. Whether or not the affinity of the Rel proteins toward the target promoter determines the inducibility requires further investigation. When the cellular extracts that contain combinations of Rel proteins were examined, Relish was found to enhance the binding activity of both Dif and Dorsal. The corresponding bands could also be supershifted using the anti-FLAG antibody, showing that the complex indeed contains Relish. The result therefore demonstrates that Rel proteins can interact directly with the defensin promoter (Han, 1999).

Toll receptor-mediated Drosophila immune response requires Dif, an NF-kappaB factor

The induction of immunity genes in Drosophila has been proposed to be dependent on Dorsal, Dif, and Relish, the NF-kappaB-related factors. This study provides genetic evidence that Dif is required for the induction of only a subset of antimicrobial peptide genes. The results show that the presence of Dif without Dorsal is sufficient to mediate the induction of drosomycin and defensin. downstream component of the Toll signaling pathway in activating the drosomycin expression. These results reveal that individual members of the NF-kappaB family in Drosophila have distinct roles in immunity and development (Meng, 1999).

The induction of immunity genes in Drosophila has been proposed to be dependent on Dorsal, Dif, and Relish, the NF-kappaB-related factors. Genetic evidence is provided that Dif is required for the induction of only a subset of antimicrobial peptide genes. The results show that the presence of Dif without Dorsal is sufficient to mediate the induction of drosomycin and defensin. Dif is a downstream component of the Toll signaling pathway in the activation of drosomycin expression. These results reveal that individual members of the NF-kappaB family in Drosophila have distinct roles in immunity and development (Meng, 1999).

A genetic experiment tested whether Dif acts downstream of Toll in regulating drosomycin gene expression. J4 (a null mutation of both Dif and dorsal) and dorsal loss-of-function mutants were crossed with the Toll10b gain-of-function mutant. The flies that contained different combinations of marker chromosomes were collected and analyzed for the expression of drosomycin. In wild-type flies, drosomycin is expressed at a basal level, and the expression is much elevated in the Toll10b flies. This Toll10b activated expression of drosomycin is clearly suppressed by the homozygous J4 chromosome. Because the dorsal mutation itself cannot suppress the Toll10b effect, the results demonstrate that Dif is an essential component of the Toll signaling pathway in the induction of drosomycin. The possibility that dorsal can replace the function of Dif in Toll signaling has not been ruled out because of the double deletion in the J4 chromosome. Nevertheless, there is no indication that Dorsal performs essential function downstream of Toll during the immune response (Meng, 1999).

A mosaic analysis in Drosophila fat body cells of the control of antimicrobial peptide genes by the Rel proteins Dorsal and DIF

Expression of the gene encoding the antifungal peptide Drosomycin in Drosophila adults is controlled by the Toll signaling pathway. The Rel proteins Dorsal and DIF (Dorsal-related immunity factor) are possible candidates for the transactivating protein in the Toll pathway that directly regulates the drosomycin gene. An examination was carried out of the requirement of Dorsal and DIF for drosomycin expression in larval fat body cells, the predominant immune-responsive tissue. The yeast site-specific flp/FRT recombination system was used to generate cell clones homozygous for a deficiency uncovering both the dorsal and the dif genes. In the absence of both genes, the immune-inducibility of drosomycin is lost but can be rescued by overexpression of either dorsal or dif under the control of a heat-shock promoter. This result suggests a functional redundancy between both Rel proteins in the control of drosomycin gene expression in the larvae of Drosophila. Interestingly, the gene encoding the antibacterial peptide Diptericin remains fully inducible in the absence of the dorsal and dif genes. Fat body cell clones homozygous for various mutations were used to show that a linear activation cascade Spaetzle->Toll->Cactus->Dorsal/DIF leads to the induction of the drosomycin gene in larval fat body cells (Manfruelli, 1999).

In contrast to the drosomycin gene, the genes encoding the antibacterial peptides Diptericin, Cecropin and Attacin are not constitutively expressed in TollD gain-of-function mutant larvae. The diptericin gene is also fully inducible in larvae deficient for the spz and Tl genes. These data indicate that diptericin induction in larvae is not dependent on the Tl pathway. Diptericin induction, however, is clearly dependent on immune deficiency (imd), a recessive mutation that impairs the inducibility of the genes encoding antibacterial peptides in both larvae and adults, while only marginally affecting the inducibility of the antifungal peptide gene drosomycin. The imd gene, which has not yet been cloned, therefore encodes a component required for the antibacterial response. The expression patterns observed for cecropin and attacinare somewhat different from those of diptericin, since the full induction of these two genes is affected in both spz and imd mutant larvae, indicating that they are regulated both by the Tl pathway and the imd gene product (Manfruelli, 1999).

The larval polyploid fat body cells differentiate from embryonic mesodermal cells whereas the adult fat body cells are derived from larval histoblasts -- presumably from adepithelial cells, associated with the imaginal discs. It was of interest therefore to compare the regulation of antimicrobial genes during an immune response in these relatively different cell types. The results point to an overall similar mode of regulation in larval and adult fat body cells. In essence, the Tl pathway controls drosomycin gene expression whereas the genes encoding the antibacterial peptides require the product of the imd gene (diptericin) or a combination of the imd and Tl pathways (cecropin and attacin). These results with respect to the larval immune response are in keeping with a correlation between the impairment of antifungal gene induction and reduced resistance to fungal infection and, conversely, between the impairment of antibacterial gene induction and reduced resistance to bacterial infection. Northern blot analysis, furthermore, indicates that the inducibility of the drosomycin gene in Tl pathway mutants is less dramatically affected in larvae than in adults. This suggests that another regulatory cascade might partially substitute for the Tl pathway in controlling drosomycin in larval fat body. Drosophila contains several Tl-like receptors, including 18-Wheeler, which is reportedly involved in the control of attacin and, to a lesser extent, cecropin induction in larvae. However, 18-wheeler mutations do not seem to affect drosomycin expression (E. Eldon, personal communication to Manfruelli, 1999). The possible contribution of these receptors to the humoral immune response, and namely to the regulation of the drosomycin gene, awaits further investigation (Manfruelli, 1999).

In larvae, as in adults, the inducibility of the drosomycin gene is slightly reduced in imd mutants. This result, in conjunction with studies on metchnikowin gene expression, leads to the proposition that each antimicrobial peptide gene is regulated by the relative dosage of inputs from several signaling cascades, which are each triggered by distinct stimuli. Current programs of mutagenesis will contribute to the identification of new components of these cascades and help understand the cross-talk between distinct pathways (Manfruelli, 1999).

Inducible expression of double-stranded RNA reveals a role for dFADD in the regulation of the antibacterial response in Drosophila adults

Isolation of Drosomycin engendered more than a decade of research to discover the pathways involved in its regulation. These studies showed that the Toll pathway, involved in Drosophila in establishment of dorsoventral polarity, is also required for induction of drosomycin. In mice, the Toll-like receptor 4 is primarily involved in the recognition of lipopolysaccharide, a component of Gram-negative bacteria, while Toll-like receptor 2 mediates Gram-positive bacterial recognition. These two receptors utilize similar signaling cascades to activate NF-κB, a central transactivator of many immune and inflammatory genes. In Drosophila innate immune system discriminates between pathogens and responds by inducing the expression of specific antimicrobial peptide-encoding genes. Genetic analysis demonstrates that the Toll signaling cascade controls an antifungal response (Lemaitre, 1996). This pathway is triggered by the proteolytic cleavage of the Toll ligand, Spätzle (Spz), and leads to activation of the rel proteins DIF and Dorsal. Mutations that block this cascade reduce the expression of the antifungal peptide gene drosomycin and increase susceptibility to fungal infection (Lemaitre, 1996). In addition, a subset of the genes encoding peptides with antibacterial activity are induced to lower levels in flies deficient for Toll signaling, indicating that this pathway also plays a role in antibacterial immune responses (Lemaitre, 1996; Leulier, 2000 and references therein).

immune deficiency (imd) gene, encoding a death domain protein that acts in the antibacterial defense upstream of the caspase Dredd and the rel protein Relish (Georgel, 2001), only affect the induction of genes with antibacterial activity and increase susceptibility to bacterial infections (Lemaitre, 1995). imd;Toll double mutants fail to express any antimicrobial genes, suggesting that imd and Toll define two essential pathways that regulate antimicrobial gene expression (Lemaitre, 1996). Hedengren (1999) determined that mutations in relish, a gene encoding a P105-like rel protein, reduce the expression of all antimicrobial genes after bacterial infection and concluded that relish may function downstream of imd. Published studies also reveal that Relish binds directly to the kappaB site of the drosomycin promoter (Han, 1999). Based on the respective activites of Dredd as a caspase and Relish as a transcriptional transactivator, it is 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 (Leulier, 2000 and references therein).

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 induce the expression of only 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 (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).

MyD88 is an adaptor protein that interacts physically with Toll and with the kinase Pelle and functions upstream of Tube and Pelle

Toll-like receptors comprise a family of cell surface receptors that play a crucial role in the innate immune recognition of both Drosophila and mammals. Previous studies have shown that Drosophila Toll-1 mediates the induction of antifungal peptides during fungal infection of adult flies. Through genetic studies, Tube, Pelle, Cactus, and Dif have been identified as downstream components of the Toll-1 signaling pathway. A Drosophila homologue of human MyD88 is an adapter in the Toll signaling pathway that associates with both the Toll receptor and the downstream kinase Pelle. Expression of Drosophila Myd88 in S2 cells strongly induces activity of a Drosomycin reporter gene, whereas a dominant-negative version of Drosophila MyD88 potently inhibits Toll-mediated signaling. Drosophila MyD88 associates with the death domain-containing adapter Drosophila Fas-associated death domain-containing protein (dFADD), which in turn interacts with the apical caspase Dredd. This pathway links a cell surface receptor to an apical caspase in invertebrate cells and therefore suggests that the Toll-mediated pathway of caspase activation may be the evolutionary ancestor of the death receptor-mediated pathway for apoptosis induction in mammals (Horng, 2001).

Drosophila MyD88 is an adapter in the Toll signaling pathway that associates with both the Toll receptor and the downstream kinase Pelle. Expression of MyD88 in S2 cells strongly induces activity of a Drosomycin reporter gene, whereas a dominant-negative version of MyD88 potently inhibits Toll-mediated signaling. MyD88 associates with the death domain-containing adapter Drosophila Fas-associated death domain-containing protein (FADD), which in turn interacts with the apical caspase Dredd. This pathway links a cell surface receptor to an apical caspase in invertebrate cells and therefore suggests that the Toll-mediated pathway of caspase activation may be the evolutionary ancestor of the death receptor-mediated pathway for apoptosis induction in mammals (Horng, 2001).

A BLAST search of the Drosophila genome identified the sequence encoding MyD88, a Drosophila homolog of human MyD88. Similar to its human homolog, Drosophila MyD88 contains an N-terminal death domain, an intermediate domain, and a TIR domain. However, unlike human MyD88, Drosophila MyD88 contains an additional 81 amino acids preceding the death domain and a 162-aa-long C-terminal region following the TIR domain (Horng, 2001).

Transfection of MyD88 into Drosophila S2 cells potently induces a Drosomycin reporter gene but not an Attacin reporter gene. This preferential ability to induce an antifungal gene is similar to that of Toll 10b, a constitutively active form of Toll, and suggests that MyD88 may be a component of the Toll-Tube-Pelle-Cactus-Dif signaling pathway. Previous studies have demonstrated that Toll-mediated Drosomycin induction requires the nuclear translocation of Dif. Dif is normally retained in the cytoplasm by the IkappaB inhibitor Cactus and is released only in response to signal-dependent degradation of Cactus. To test whether MyD88-mediated Drosomycin induction also depends on Cactus degradation, a Cactus mutant was constructed that contains mutations of the conserved serine residues that, in mammalian IkappaB, are the targets of signal-dependent phosphorylation. A Cactus mutant inhibits Drosomycin induction by MyD88 and, as expected, by Toll. This result indicates that, similar to Toll, MyD88 regulates Drosomycin induction through the Cactus-dependent pathway (Horng, 2001).

For further analyses, various deletion mutants of MyD88 were generated. Two of the deletion mutants, one containing the TIR domain and the C-terminal domain (amino acids 237-537) and another containing the intermediate, TIR, and C-terminal domains (amino acids 176-537), activate the Drosomycin reporter weakly (10-fold) in comparison to full length MyD88, indicating that the intact protein is required for optimal activity. However, the fact that these truncation mutants can still induce signaling is surprising, since they lack the death domain that mediates interactions with downstream signaling components. Moreover, similar analyses of human MyD88 have shown that a combination of the death domain and the intermediate domain is sufficient to induce signaling activity comparable to that of the wild-type protein. An equivalent truncation of dMyD88 (amino acids 1-237) retains no residual activity despite being well expressed, suggesting that there are some differences in domain function between human and Drosophila MyD88 proteins (Horng, 2001).

To determine whether MyD88 is a component of the Toll signaling pathway, attempts were made to identify a deletion mutant that would have dominant-negative activity. Therefore, three MyD88 deletion mutants that do not activate the Drosomycin reporter were tested for their ability to inhibit Toll-mediated Drosomycin induction. The strongest inhibitor was the death domain- and middle domain-containing construct (amino acids 1-237), which at low concentrations potently inhibits Toll-mediated Drosomycin induction in a dose-dependent manner (Horng, 2001).

To order MyD88 in the pathway with respect to Pelle, MyD88 was tested for its ability to be inhibited by PelleN, a dominant-negative form of Pelle that consists of the N-terminal death domain-containing region of Pelle. MyD88, like Toll, is strongly inhibited by PelleN. MyD88, however, does not inhibit Pelle, demonstrating that, similar to the mammalian pathway, MyD88 functions upstream of Pelle (Horng, 2001).

To further establish MyD88 as a component of the Toll pathway, whether MyD88 interacts with Toll was tested by coimmunoprecipitation assays. The TIR domain-containing MyD88 construct is detected in anti-Toll immunoprecipitates. Interestingly, when cotransfected with Toll 10b, MyD88 reproducibly appears as two distinct bands -- a slower migrating upper band that may correspond to phosphorylated MyD88 construct and a faster migrating lower band. The predominant form of MyD88 detected in immunoprecipitates is the faster migrating species. MyD88 therefore associates with Toll, presumably through TIR domains, and is a component of the active receptor complex (Horng, 2001).

Because human MyD88 associates with IRAK through death domains, a likely immediate downstream target of MyD88 is the IRAK homolog Pelle. Interaction between the death domain-containing dMyD88 construct (amino acids 1-237) and Pelle was examined. MyD88 is detected in Pelle immunoprecipitates, indicating that MyD88 interacts with Pelle, presumably through their death domains (Horng, 2001).

These results therefore demonstrate that MyD88 is an adaptor in the Toll signaling pathway downstream of the receptor and upstream of Pelle. From genetic analyses, the adaptor protein Tube has also been implicated to be downstream of Toll and upstream of Pelle in the Toll signaling pathway. The death domain of Tube also interacts with Pelle. Because Tube and MyD88 also contain death domains that could potentially mediate their interaction, tests were performed for association between these two proteins in immunoprecipitation assays; Tube and MyD88 do indeed interact. Therefore, MyD88 and Tube both function as adaptors downstream of Toll, exist in the same active complex along with Pelle, and are probably both involved in the recruitment and/or activation of Pelle. Understanding functional differences between these two adapters will require further analysis (Horng, 2001).

To identify other potential downstream targets of MyD88, a search of the Drosophila genome was performed for other sequences that encode death domain-containing proteins that may interact with MyD88. One such sequence encodes a protein with a death domain as well as a death effector domain and appears to be a homolog of mammalian FADD. This cDNA has been identified and named FADD (Hu, 2000). Whether FADD can interact with MyD88 was tested. Lysates from S2 cells transfected with MyD88 were incubated with anti-Flag beads to immunoprecipitate FADD, and immunoprecipitates were blotted with anti-V5 antibody to look for associated MyD88. A strong band corresponding to MyD88 was observed, indicating that MyD88 can interact with FADD through death domains. Overexpression of FADD in S2 cells, however, does not lead to activation of either the Drosomycin or Attacin reporters (Horng, 2001).

Mammalian FADD is recruited to the tumor necrosis factor receptor complex through homophilic death domain interactions with the adapter TNFR-associated death domain-containing protein (TRADD). In turn, FADD recruits procaspase-8 through homophilic death effector domain associations. It is speculated that Drosophila FADD may likewise recruit a Drosophila caspase to the Toll receptor complex. A potential candidate caspase is Dredd, an apical caspase with a long prodomain shown to be essential for induction of antibacterial genes. Indeed, analysis of immunoprecipitated lysates from cells cotransfected with Drosophila FADD, and either full length Dredd or the death effector domain of Dredd showed strong association of Dredd with FADD. A second study (Hu, 2000) has also shown interaction of dFADD with Dredd (Horng, 2001).

Thus Drosophila MyD88 is an adapter in the Toll signaling pathway. MyD88 associates with both Toll and Pelle and functions upstream of Pelle. Tube is known from genetic studies to be an adapter in the Toll pathway that functions upstream of Pelle. Why Toll should signal through MyD88 and Tube, two receptor-proximal adapters with seemingly similar functions, is not yet clear. MyD88 associates with the receptor Toll as well as the downstream adapter FADD, which in turn interacts with the apical caspase Dredd. Because caspases are essential executioners of the apoptotic machinery in organisms from nematodes to mammals, and because Dredd has been shown to be involved in apoptosis during Drosophila development, it is possible that Toll-1 or some of the other eight Tolls that exist in Drosophila may induce apoptosis (or another Dredd-dependent pathway) through the MyD88/dFADD/Dredd pathway in a cell-type specific and/or developmental stage-specific manner. The pathway comprised of Toll, MyD88, dFADD, and Dredd would be the first description of a pathway in invertebrates that links a cell surface receptor to an apical caspase. Such a pathway, if it exists, would enable extracellular stimuli, perhaps ligands secreted by other cells during development or pathogen-derived products during infection, to instruct invertebrate cells to undergo cell death. In addition, the Toll/MyD88/dFADD/Dredd pathway is remarkably similar to that activated by the receptors of the tumor necrosis factor receptor (TNFR) superfamily in mammals, in which FADD-mediated recruitment of caspase-8 leads to induction of apoptosis. Since the Drosophila genome does not encode any cell surface receptors homologous to TNFRs, it appears that the Toll/MyD88/dFADD/Dredd pathway is the evolutionary ancestor of the mammalian death receptor pathways. This possibility is further supported by the recent finding that human TLR2 can induce apoptosis through the MyD88/FADD/Caspase-8 pathway (Horng, 2001).

MyD88 encodes the Drosophila homolog of mammalian MyD88. DmMyD88 combines a Toll-IL-1R homology (TIR) domain and a death domain. Overexpression of DmMyD88 is sufficient to induce expression of the antifungal peptide Drosomycin, and induction of Drosomycin is markedly reduced in DmMyD88-mutant flies. DmMyD88 interacts with Toll through its TIR domain and requires the death domain proteins Tube and Pelle to activate expression of Drs, which encodes Drosomycin. DmMyD88-mutant flies are highly susceptible to infection by fungi and Gram-positive bacteria, but resist Gram-negative bacterial infection much as do wild-type flies. Phenotypic comparison of DmMyD88-mutant flies and MyD88-deficient mice shows essential differences in the control of Gram-negative infection in insects and mammals (Tauszig-Delamasure, 2002).

Mutations in the Drosophila Tak1 gene reveal a conserved function for MAPKKKs in the control of rel/NF-kappaB-dependent innate immune responses

In mammals, TAK1, a MAPKKK kinase, is implicated in multiple signaling processes, including the regulation of NF-kappaB activity via the IL1-R/TLR pathways. TAK1 function has been studied primarily in cultured cells, and its in vivo function is not fully understood. Null mutations have been isolated in the Drosophila Tak1 gene that encodes Tak1, a homolog of TAK1. Tak1 mutant flies are viable and fertile, but they do not produce antibacterial peptides and are highly susceptible to Gram-negative bacterial infection. This phenotype is similar to the phenotypes generated by mutations in components of the Drosophila Imd pathway. Genetic studies also indicate that Tak1 functions downstream of the Imd protein and upstream of the IKK complex in the Imd pathway that controls the Rel/NF-kappaB like transactivator Relish. In addition, epistatic analysis places the caspase, Dredd, downstream of the IKK complex, which supports the idea that Relish is processed and activated by a caspase activity. This genetic demonstration of Tak1's role in the regulation of Drosophila antimicrobial peptide gene expression suggests an evolutionary conserved role for TAK1 in the activation of Rel/NF-kappaB-mediated host defense reactions (Vidal, 2001).

The Toll signaling pathway, which was first identified as a regulator of embryonic dorsal-ventral patterning, is one regulator of antimicrobial peptide gene expression in Drosophila. Upon infection, the Spaetzle (Spz) protein is cleaved to generate a ligand for the Toll transmembrane receptor protein; Toll binding by Spz stimulates the degradation of the IkappaB homolog, Cactus, and the nuclear translocation of the Rel proteins Dorsal and Dorsal-like immunity factor (Dif). A second pathway regulating antimicrobial peptide gene expression in flies was initially identified by a mutation in the immune deficiency (imd) gene that results in susceptibility to Gram-negative bacterial infection and an impairment of antibacterial peptide gene expression (Lemaitre, 1995). imd encodes a homolog of the mammalian Receptor Interacting Protein (RIP) (P. Georgel, in preparation). The imd gene encodes a death domain-containing protein. In mammals, RIP appears to function in an adaptor complex associated with the tumor necrosis factor (TNF) receptor, and genetic placement of imd suggests that IMD has a conserved function in flies as part of a receptor-adaptor complex that responds to Gram-negative bacterial infection. Molecular studies have isolated four additional factors that appear to define the Imd pathway: Relish, a third Drosophila Rel protein; two members of a Drosophila IkappaB kinase (IKK) complex, that is, the kinase DmIKKß and a structural component DmIKKgamma and Dredd, a caspase. Relish is a homolog of the mammalian P100 and P105 compound Rel proteins that contain both Rel domains and inhibitory ankyrin domains. A receptor for the Imd pathway has not been identified, but infection triggers Relish cleavage and nuclear translocation of the Rel domain (Stöven et al. 2000). Relish cleavage requires DmIKKß activity, indicating that, like the mammalian IKK complex that functions in the IL1-R and TNF-R pathways, the Drosophila IKK complex regulates Rel protein activity. However, and in contrast to P100 and P105 processing, Relish cleavage is not blocked by proteasome inhibitors but does require a functional Dredd gene, suggesting that Dredd may cleave Relish directly after infection. 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 identify Drosophila genes that mediate defense reactions to bacterial infection, ~2500 lines carrying ethyl methanesulfonate (EMS)-induced mutations on the X chromosome were tested for susceptibility to bacterial infection: male adult flies were pricked with a needle dipped into a pellet of the Gram-negative bacterial species Erwinia carotovora carotovora 15 (E. carotovora 15) and screened for mutants that failed to survive infection. Using this assay, nine recessive, homozygous viable mutations were isolated that render flies highly susceptible to E. carotovora 15 infection: Less than 10% of the mutated flies survived 48 h postinfection, whereas more than 90% of the wild-type flies survived. These nine mutations fall into two complementation groups: B118, which represents five of the mutations, and D10, corresponding to Tak1, which represents the other four mutations. The B118 group corresponds to the caspase encoding gene Dredd (Vidal, 2001),

To compare the D10 phenotype with the phenotypes generated by mutations in other genes that regulate Drosophila immune responses, the susceptibility of D10 and other mutant lines to infection by four microorganisms was assayed: flies were pricked with the Gram-negative bacteria Escherichia coli (E. coli), the Gram-positive bacteria Micrococcus luteus (M. luteus), or the fungus Aspergillus fumigatus, and flies were naturally infected with the entomopathogenic fungus Beauveria bassiana (B. bassiana). The D10 phenotype is similar to the imd and Relish phenotypes; flies carrying the D10, imd, and Relish mutations are susceptible to Gram-negative bacterial infection and resistant to Gram-positive bacterial and fungal infections, although D10 flies, like imd flies, exhibit slightly lower susceptibility to Gram-negative bacterial infection compared to Relish mutants. In contrast, mutations in the spz gene render flies susceptible to fungal infections, and only flies carrying mutations in both spz and imd are susceptible to Gram-positive bacterial infection. This survival analysis demonstrates that the D10 gene product, like Imd and Relish, is required to resist Gram-negative bacterial infection (Vidal, 2001).

The Toll pathway is required for the full induction of the antifungal peptide genes and a subset of the antibacterial peptide genes. Mutations that block the Toll pathway reduce the expression of these genes; conversely, mutations that block the Imd pathway reduce the expression of genes with antibacterial activity. To determine how the D10 mutation affects antimicrobial peptide gene expression, the levels of Diptericin, Cecropin A, Defensin, and Attacin, which encode antibacterial peptides, Drosomycin, which encodes an antifungal peptide, and Metchnikowin, which encodes a peptide with both antibacterial and antifungal activity, were monitored in flies homozygous for two D10 alleles. In addition, the D10 phenotype was compared with all of the previously identified mutations affecting the Imd pathway and with a spz mutation that blocks the Toll pathway (Vidal, 2001).

Pricking adult flies with a mixture of Gram-positive and Gram-negative bacteria activates the expression of all the antimicrobial peptide genes; in the D10 mutants, however, mixed Gram-negative/Gram-positive infections induce significant levels of only Drosomycin and Metchnikowin. Quantitative measurements of three independent RNA blot experiments show that in D10 flies, Drosomycin is induced to wild-type levels; Metchnikowin is induced to 70% of wild-type levels; Cecropin A, Defensin, and Attacin are induced to <25% of wild-type levels, and Diptericin is induced to <5% of wild-type levels. This pattern of antimicrobial peptide gene expression in the D10 mutants is similar to the patterns displayed in mutants of the Imd pathway, although the D10 mutations, like imd, have slightly weaker effects on antimicrobial peptide gene expression compared to the Dredd, DmIKKß, DmIKKgamma, and Relish mutations. This weaker phenotype in D10 and imd flies correlates well with their lower susceptibility to E. coli infection (Vidal, 2001).

Because of its chromosomal location and the functions of its mammalian homologs, Tak1 was chosen as a candidate gene mutated in D10 flies. Several different experiments to determine whether the D10 alleles correspond to mutations in Tak1. Tested first was the ability of a 15 Kb genomic fragment that contains Tak1 to rescue the immune response deficiency in D10 flies. D10 adults carrying the Tak1 transgene (P[Tak1+]) both express Diptericin and resist Gram-negative bacterial infection at levels comparable to wild-type flies, demonstrating that this genomic fragment rescues the D10 phenotypes. Overexpression of the Tak1 cDNA using the UAS/GAL4 system partially rescues Diptericin expression in the D101 mutant. The Tak1 genomic coding sequence was tested. The four D10 alleles all contain mutations within the Tak1 kinase domain. This led to the renaming of the D10 alleles Tak11 to Tak14: Tak11 and Tak14 were generated by missense mutations in conserved residues, Tak12 was generated by a point mutation that creates a stop codon, and Tak13 contains a deletion of 31 base pairs that also results in a premature stop codon. All four Tak1 alleles inhibit Diptericin induction by Gram-negative bacterial infection to the same degree, and this inhibition is not enhanced in flies heterozygous for each allele and a deficiency spanning Tak1. In addition, flies homozygous for the four alleles are equally susceptible to Gram-negative bacterial infection. The apparent null phenotype manifested by the four Tak1 alleles indicates that the Tak1 kinase domain is essential for Tak1 function in the Imd pathway. This observation is supported by results from experiments with a kinase dead form of Tak1, Tak1-K46R, which acts as a dominant negative inhibitor of Tak1. Tak1-K46R expression driven by the UAS/GAL4 system blocks Diptericin expression after mixed bacterial infection, confirming that Tak1 is required for the Drosophila antibacterial immune response. The results of the rescue experiments, sequencing data, and the dominant negative Tak1 mutant phenotypes together demonstrate that D10 encodes the MAPKKK Tak1 (Vidal, 2001).

The loss-of-function Tak1 mutations display immune response phenotypes that are very similar to the phenotypes generated by mutations in imd, DmIKKß, DmIKKgamma, Dredd, and Relish, suggesting that these genes function together in the Imd pathway. Previous studies indicated that DmIKKgamma and DmIKKß directly regulate Relish activity (Silverman, 2000); however, the positions of Imd, Tak1, and Dredd in the Imd pathway were not determined. To avail of a genetic approach for ordering the Imd pathway, both Dredd and Tak1 were overexpressed via the UAS/GAL4 system. In lines carrying a heat shock (hs)-GAL4 driver and either the Dredd or Tak1 cDNAs under the control of a UAS promoter, heat shock induces Diptericin expression to about 15%-20% of the level observed in adults 6 h after bacterial infection. This result indicates that the overexpression of these two genes is sufficient to activate the antibacterial pathway in the absence of infection. By using this UAS/GAL4 system to overexpress Tak1 and Dredd in various mutant backgrounds, the epistatic relationships were tested between Tak1 and Dredd, and the other genes of the Imd pathway. Mutations in DmIKKß and DmIKKgamma, but not imd, block Diptericin induction by Tak1 overexpression, indicating that Tak1 functions downstream of Imd and upstream of the IKK complex. However, the Diptericin expression induced by Dredd overexpression is not affected by mutations in imd or DmIKKgamma. For genetic reasons, Northern blot analysis could not be used to test the effect of mutations in Relish and DmIKKß, which are on the third chromosome, on the UAS-Dredd-induced Diptericin expression. Therefore, the UAS-Dredd transgene was overexpressed through a female, adult fat body driver (yolk-GAL4) and Diptericin expression was monitored with a Diptericin-LacZ reporter gene. LacZ titration assays have demonstrated that ß-galactosidase activity is induced in lines overexpressing UAS-Dredd in the absence of infection. The Dredd-mediated Diptericin-LacZ induction is strongly reduced in Relish but not in DmIKKß mutants, confirming the Northern blot results showing that Dredd functions downstream of the IKK complex and demonstrating that Dredd regulates Diptericin expression through Relish (Vidal, 2001).

Natural fungal infections highlight the ability of Drosophila to discriminate between pathogens and activate specific immune response pathways that lead to adapted immune responses. Although only the Imd pathway is required to resist Gram-negative bacterial infection, the Toll pathway is still activated to some degree in flies pricked with Gram-negative bacteria. This suggests that injury (and associated contamination) contributes to a nonspecific immune response. E. carotovora 15 naturally infects the Drosophila larval gut and triggers both the local and systemic expression of antimicrobial peptide genes. In contrast to infection by pricking, natural E. carotovora 15 infection induces Diptericin expression more strongly than Drosomycin expression. To compare the contribution of the Toll and Imd pathways to antimicrobial peptide induction after natural E. carotovora 15 infection, Diptericin and Drosomycin expression were quantified in larvae from various mutant lines after natural E. carotovora 15 infections. The Diptericin expression induced by E. carotovora 15 natural infection is entirely dependent on the genes of the Imd pathway, confirming that Diptericin is exclusively regulated by the Imd pathway. Interestingly, the level of Diptericin induction is higher in spz mutants than in wild-type larvae. In addition, Drosomycin induction after E. carotovora 15 natural infection of larvae is also dependent on the Imd pathway: mutations blocking the Imd pathway have stronger effects than the spz mutation on Drosomycin expression induced by natural infection. These data contrast somewhat with previous observations that the imd mutation does not block Drosomycin induction by E. carotovora 15 infection. However, the earlier analysis of Drosomycin induction by E. carotovora 15 was based on the qualitative analysis of a Drosomycin-lacZ reporter gene; it is thought that that the quantitative analysis of Drosomycin expression by Northern blot is a more accurate determination of Drosomycin expression patterns. In conclusion, the current results indicate that Drosomycin gene induction after natural Gram-negative bacterial infection is largely mediated by the Imd pathway (Vidal, 2001).

Natural infections by E. carotovora 15 also trigger the local expression of antimicrobial peptide genes in various epithelial tissues, and both Diptericin expression in the anterior midgut and Drosomycin expression in the trachea are dependent on the imd gene. By assaying the expression of Diptericin-lacZ and Drosomycin-GFP reporter genes in naturally infected Tak1 and Dredd mutant larvae, it has been shown that both genes are required for Diptericin and Drosomycin expression in epithelial tissues after natural E. carotovora. These data confirm the predominant role of the Imd pathway in antimicrobial peptide regulation after natural E. carotovora 15 infection and suggest that Gram-negative bacterial recognition in flies preferentially activates the Imd pathway (Vidal, 2001).

Impact of Toll and Imd pathways on Drosomycin induction

Microarray studies have shown 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).

The effect was analyzed of mutations affecting Imd and Toll pathways on the expression of antimicrobial peptide (AMP) genes after injection of E.coli, M.luteus or A.fumigatus. Northern blot analysis was performed for of two antibacterial peptide genes (attacin and diptericin) and two antifungal peptide genes (drosomycin and metchnikowin). The double mutants rel,spz and rel,Tl failed to show induction of AMP genes. In fact, the only AMP transcript detectable in these flies is the antifungal drosomycin, which is present at a level similar to that in unchallenged flies. Diptericin is regulated by the Imd pathway, while metchnikowin, attacin and drosomycin are regulated by both pathways. Interestingly, the contribution of each pathway to the expression of each AMP gene depends on the type of infection. For example, in agreement with previous studies (Leulier, 2000), drosomycin expression is affected similarly by the rel and Toll pathway mutants after E.coli infection, but is regulated predominantly by the Toll pathway during M.luteus or A.fumigatus infections (De Gregorio, 2002).

The results obtained by northern blot analysis correlate with the data from survival experiments. The contribution of the two pathways to the control of the antibacterial peptides is consistent with the augmented sensitivity to bacterial infection of double mutant flies versus single mutants. The level of the antifungal peptide Drosomycin transcript after fungal infection is very similar in the double mutant flies (rel,spz and rel,Tl) compared with Tl and spz single mutants, consistent with a similar resistance to A.fumigatus and B.bassiana displayed by these four lines. Importantly, the Tl and spz alleles alone, or in combination with rel, display the same behaviour in all survival experiments performed and have a similar pattern of AMP gene expression, suggesting that Spaetzle is the sole extracellular activator of the Toll pathway in response to microbial infection. However, it was noticed that attacin and diptericin expression after A.fumigatus infection is reduced in Tl but not in spz flies. The analysis of A.fumigatus infection was extended to pelle, tube and dif mutants, which display the same AMP expression profile as spz, suggesting that the effect observed in Tl flies is due to the genetic background of the strain used. The complete survival and northern analysis presented in this study was extended to a strong allele of pelle alone or in combination with rel, which gave similar results to spz and Tl alleles (De Gregorio, 2002),

A heterotrimeric death domain complex involving Tube, Pelle and Myd88

Tube is a scaffolding protein containing an N-terminal interaction motif belonging to the death domain family, as well as C-terminal Tube repeats that mediate binding to Dorsal. Pelle is a serine/threonine-specific protein kinase with a death domain N-terminal to its catalytic domain (Sun, 2002).

Although no Tube homolog has been found in mammals, four Pelle homologs, named IL-1 receptor-associated kinases (IRAKs), have been identified: IRAK1, -2, -M, and -4 . IRAKs function in signaling by a family of Toll-like receptors, as well as the IL-1 receptor (IL-1R), each of which contains a TIR domain, a conserved cytoplasmic signaling motif. An adaptor molecule, Myd88, associates with the C-terminal TIR domain of Toll-like receptors and the IL-1 receptor and with the N-terminal death domain (DD) of IRAKs (Sun, 2002).

During the past few years, genomic sequencing has allowed the identification of Drosophila genes with mammalian homologs functioning in Toll/IL-1 receptor-signaling pathways. These genes include IKK (a homolog of mammalian IKKalpha/ß), Kenny (a homolog of mammalian IKKgamma), IK2 (a homolog of mammalian TBK1/IKKepsilon), Myd88, TAK1, three TRAF loci, and ECSIT. These genes have been studied systematically by using RNA interference (RNAi). RNAi provides a ready means to inactivate a given gene or genes and has facilitated the dissection of Drosophila signaling pathways in cultured S2 cells. To search for essential components of the Toll pathway, an RNAi-based screen was performed among these potential Drosophila NF-kappaB regulators. This approach, coupled with genetic and biochemical analyses, has allowed the dissection of the molecular interactions among death domain-containing proteins in the Drosophila Toll pathway (Sun, 2002).

To investigate the mechanism of Toll signaling, a reporter assay was used in conjunction with RNAi in cultured Drosophila cells. A constitutively active form of Toll, TollDeltaLRR, was stably expressed in S2 cells under the control of a metallothionein promoter, such that the addition of CuSO4 to the cell culture medium initiates Toll signal transduction. To assay signal transduction downstream of Toll, these S2 cells were transiently transfected with a Drosomycin-luciferase construct. Expression of TollDeltaLRR consistently induces a significant activation (~100 fold) of the Drosomycin reporter (Sun, 2002).

To confirm the efficacy of RNAi in these cells, dsRNA was generated for several genes known to function in the Drosophila Toll pathway. RNAi against Pelle, Tube, or Dorsal significantly inhibits the activation of the Drosomycin reporter, with the effect of Dorsal RNAi relatively stronger than that of Pelle or Tube RNAi. In contrast, RNAi against Cactus dramatically enhances the activation of the Toll pathway. These observations are consistent with the fact that Pelle, Tube, and Dorsal promote Toll signaling, whereas Cactus plays an inhibitory role in the pathway. In this and all subsequent experiments, Easter RNAi serves as a negative control for any nonspecific effect of dsRNA, because Easter acts upstream, and not downstream, of Toll (Sun, 2002).

Next, RNAi-based screening was performed against fly counterparts of mammalian Toll and tumor necrosis factor pathway components, specifically Drosophila IKK, IKKgamma (Kenny), IK2, Myd88, TAK1, ECSIT, TRAF1, TRAF2, and TRAF3. Expression of each of these genes was interrupted individually by RNAi in S2 cells and the effect on Toll signaling was assayed. RNAi was also conducted against combinations of genes, in particular IKK and Pelle;IKK and IKKgamma;IKK and IK2;TRAF1, 2, and 3. To determine whether requirements are specific to the Toll pathway, the same panel of RNAi analysis was conducted in S2 cells treated with LPS. An Attacin-luciferase reporter was used to indicate LPS-mediated activation of the response pathway for Gram-negative bacteria (Sun, 2002).

When the effects of RNAi on the Toll and LPS pathways were compared, it was found that Drosophila Myd88, like Tube and Pelle, is required for activation of the Drosomycin, but not the Attacin, reporter; Drosophila Myd88 is thus essential for Toll signaling. In contrast, a requirement for TAK1 was found only in the LPS pathway and no essential role was found for fly IK2, ECSIT, or TRAF 1, 2, and 3 in either Toll or LPS signaling. Inactivating IKK and IKKgamma affected both types of signaling, with the LPS pathway being more severely inhibited than the Toll pathway. These results are consistent with the fact that inactivating IKK in flies disrupts Toll-dependent axis formation in a small fraction of embryos, although neither IKK nor IKKgamma is strictly required for Toll signal transduction (Sun, 2002).

It is known that Tube acts downstream of Toll and upstream of Pelle in signal transduction. To place Drosophila Myd88 in this pathway precisely, the epistatic relationship was examined among Myd88, Tube, and Pelle. Expression of wild-type Myd88, which has been shown to activate the Drosomycin reporter, was induced. RNAi against Pelle, Tube, or Myd88 blocks this Myd88-induced activation. These results, as well as similar findings in adult flies (Tauszig-Delamasure, 2002), indicate that Myd88 acts either upstream of or in parallel to Tube (Sun, 2002).

To dissect the signaling hierarchy further, a constitutively active form of Tube was used. This Tube-initiated activation of the Drosomycin reporter does not require Myd88, but does require Pelle. Furthermore, Pelle-induced activation of the Drosomycin reporter is diminished only by RNAi against Pelle, but not Tube or Myd88. Thus, epistasis analysis defines a linear order of action, with Tube downstream of Myd88 and upstream of Pelle (Sun, 2002).

Myd88, Tube, and Pelle each contain a death domain, a motif known to form homotypic interactions. Tube and Pelle interact directly by means of their death domains. Furthermore, Myd88 has been found to coimmunoprecipitate with Pelle in S2 cells (Tauszig-Delamasure, 2002). It was therefore interesting to discover the role of binding interactions mediated by death domains in the hierarchy defined by epistasis analysis (Sun, 2002).

To assay the interaction of Myd88 with either Pelle or Tube, full-length Myd88, as well as the death domain of Pelle (PelleDD) and a slightly larger Tube death domain peptide (TubeDD*) were epitope tagged. Also, an antiserum was generated against Drosophila Myd88. Immunoprecipitation experiments, using the alpha-Myd88 for the precipitation step and alpha-V5 to detect the tagged peptides was carried out. In pair-wise experiments, substantial interaction was detected between Myd88 and the Tube death domain. (In addition, a reduction occurred in the abundance of a fast migrating Myd88 species, perhaps reflecting a TubeDD-mediated protection from proteolysis). In contrast, only a trace amount of PelleDD coprecipitated with Myd88 (Sun, 2002).

PelleDD, TubeDD, and Myd88 were co-expressed to assay for higher-order complexes. Under such conditions, the amount of Myd88-associated PelleDD was dramatically increased. Indeed, the relative amount of TubeDD and PelleDD coimmunoprecipitated with Myd88 was indistinguishable. It is concluded that Tube forms a stable complex with Myd88 and is also strictly required for the recruitment of Pelle into a complex with Myd88 (Sun, 2002).

Two alternative models were envisioned for the role of Tube in complex formation. In one, the interaction of Pelle and Tube is essential for Pelle to join the Myd88 complex. In the alternative model, Pelle can stably associate with Myd88, provided Myd88 is bound by Tube. To discriminate between these two models, interaction surface mutations were used in characterizing a dimer between the Tube and Pelle death domains (Sun, 2002).

The crystal structure of the complex formed by the death domains of Tube and Pelle suggests that residue E50 in Tube and R35 in Pelle form a salt bridge that is critical for dimer formation. By using an RNA injection bioassays, it has been demonstrated that mutation of residue 50 in Tube to lysine (E50K mutation) abolish Tube function in Toll signaling. It was therefore surprising to find that the E50K mutation has no discernible effect on the binding of the Tube death domain to Myd88 (Sun, 2002).

Although Tube E50K has an apparently wild-type interaction with Myd88, this mutation blocks the binding of Tube to Pelle in the coimmunoprecipitation assay. Furthermore, a mutational change in Pelle (Pelle R35E) that is predicted to reconstitute the salt bridge, fully restores the Tube-Pelle interaction, just as these compensatory mutations in Tube and Pelle together allow signaling in embryos. Thus, at least two types of death domain contacts are in the Toll signaling complex: one between Tube and Pelle that involves Tube E50 and a second between Tube and Myd88 that is E50-independent (Sun, 2002).

To determine whether binding to Tube is essential for Pelle recruitment into the Myd88 complex, advantage was taken of the compensatory mutations in Tube and Pelle. In cells coexpressing TubeDD and PelleDD, the association of PelleDD with Myd88 is greatly inhibited by either individual mutation, Pelle R35E or Tube E50K, that blocks the Tube-Pelle interaction. Remarkably, the simultaneous presence of these compensatory mutations restores the recruitment of PelleDD to the Myd88 complex. It is therefore concluded that Pelle must bind directly to Tube to join the Myd88 complex (Sun, 2002).

A model is proposed that describes the three way interaction between Pelle, Tube and Myd88. The Tube-mediated complex formation involves two distinct binding surfaces on Tube death domain, which allow simultaneous association of Myd88 and Pelle. Direct binding of Myd88 to Toll and of both Tube and Pelle to Cactus-bound Dorsal would result in a complex facilitating efficient signal transduction from Toll to Dorsal. On the basis of this model of the heterotrimeric death domain complex, it is predicted that expression of the wild-type death domain of either Tube or Pelle might disrupt formation of an endogenous trimeric complex and thereby interfere with the normal function of the Toll pathway. Moreover, distinct outcomes are expected for expression of mutant forms of the Tube and Pelle death domains. The E50K mutant of TubeDD, although incapable of interacting with Pelle, nevertheless binds to Myd88 and hence might interfere with the formation of the complex of Myd88, Tube, and Pelle. By the same logic, expressing the R35E mutant of Pelle, which cannot stably interact with Tube, and hence the trimeric complex, might not interfere with signaling (Sun, 2002).

To test these hypotheses, the effect of expressing Tube and Pelle death domains was assayed in the context of an active Toll pathway. Wild-type and E50K TubeDD each significantly block TollDeltaLRR-induced activation of the Drosomycin reporter, as does wild-type PelleDD. However, the R35E mutant of PelleDD, expressed at the same level as its wild-type counterpart, has no discernible effect on Drosomycin activation. These results thus confirm the predictions of the model for heterotrimer formation and demonstrate that formation of the trimeric Myd88, Tube, and Pelle complex is a critical step in Toll signaling (Sun, 2002).

The death domain was originally identified as a protein module transducing apoptotic signals. It has been found, for example, that death domain mediated interactions between Fas and FADD or between tumor necrosis factor receptor and TRADD provide the basis for assembling the death-inducing signaling complex. The death effector domain and caspase recruitment domain also form homotypic interactions involved in apoptotic signaling and are structurally similar to a death domain. These motifs, together with the death domain, comprise the death domain superfamily (Sun, 2002 and references therein).

Experimental data demonstrate that PelleDD and Myd88 are found in the same complex when each is physically associated with TubeDD. The association of three different death domains has also been implied by studies on the tumor necrosis factor receptor complex, in which TRADD was found to facilitate the recruitment of FADD or RIP to tumor necrosis factor receptor. This study has probed the nature of such a complex and it was found that Myd88, Pelle, and Tube form a heterotrimer, with the TubeDD interacting with Myd88 and PelleDD by distinct binding surfaces (Sun, 2002).

Recently, molecular modeling based on available structural data has suggested that the homotypic interaction among death domain superfamily modules could be multivalent. Higher-order multimers, such as a heterohexamer, can be modeled by docking the death domains of Fas and FADD together. Furthermore, the structural plasticity observed in the PelleDD:TubeDD dimerhypothetically allows it to accept a third death domain into a three-fold symmetric structure. Whether the death domains of Myd88, Tube, and Pelle can form such a structure, as opposed to a linear array, awaits biophysical characterization of this trimeric complex. It is noted, however, that no evidence has been provided for any physical interaction between Pelle and Myd88. In addition, the fact that the PelleDD R35E mutant fails to dominantly interfere with Toll signaling in a functional assay argues against the possibility of such a direct contact between Pelle and Myd88 (Sun, 2002).

Because Myd88 binds to Toll through interaction between TIR domains on both proteins, it is envisioned that Myd88 connects both Tube and Pelle to Toll. Toll-initiated aggregation of these signaling molecules could trigger Pelle activation. Such a model is consistent with epistasis analyses indicating a linear order of action for Toll, Myd88, Tube, and Pelle in primary signaling (Tauszig-Delamasure, 2002). Furthermore, because Dorsal binds directly to Pelle, Tube, and Cactus, it is conceivable that the entire signaling cassette exists, at least transiently, in a single complex. As suggested by both biochemical and biological assays, Pelle-catalyzed phosphorylation may then lead to both Dorsal nuclear transport and complex dissociation (Sun, 2002).

In mammalian signaling pathways initiated by either Toll-like receptors or IL-1 receptors, Myd88 associates with IRAK. Because this study shows that a third death domain is required to mediate the interaction between Myd88 and Pelle in Drosophila, does a parallel exist in mammals? Although no known Tube ortholog exists in mammals, multiple IRAKs are present. It is speculated, therefore, that two or more IRAKs may participate in one protein complex, with the death domain of one IRAK bridging the interaction of another with Myd88. In this way, a particular IRAK isoform might act together with Myd88 to regulate the activity of a second IRAK through the oligomerization of death domains, resulting in isoform-specific biological functions (Sun, 2002).

The Drosophila Toll-9 activates a constitutive antimicrobial defense

The Toll family of transmembrane proteins participates in signaling infection during the innate immune response. The nine Drosophila Toll proteins were analyzed and it was found that wild-type Toll-9 behaves similar to gain-of-function Toll-1. Toll-9 activates strongly the expression of Drosomycin, and utilizes similar signaling components to Toll-1 in activating the antifungal gene. The predicted protein sequence of Toll-9 contains a tyrosine residue in place of a conserved cysteine, and this residue switch is critical for the high activity of Toll-9. The Toll-9 gene is expressed in adult and larval stages prior to microbial challenge, and the expression correlates with the high constitutive level of drosomycin mRNA in the animals. The results suggest that Toll-9 is a constitutively active protein, and implies its novel function in protecting the host by maintaining a substantial level of antimicrobial gene products to ward off the continuous challenge of microorganisms (Ooi, 2002).

In both dorsal–ventral development and antifungal response, activated Toll-1 recruits Tube and Pelle to initiate signaling. Both Tube and Pelle contain death domains, and Pelle is a kinase. Recruitment of Pelle somehow leads to degradation of the inhibitor Cactus and release of the transcription factors, Dorsal and Dif. Whether Toll-9 employs the same signaling components to activate drosomycin expression was examined. A construct for Pelle containing only the death domain (PelleDD), but lacking the kinase domain, was generated. This mutated Pelle protein should function as dominant negative by binding to the death domain of Tube but cannot phosphorylate downstream substrates. Transfection of wild-type Pelle activated the reporter gene efficiently, consistent with an important role of the protein in antifungal response. As expected, PelleDD did not activate the reporter. In contrast, the PelleDD construct inhibited all the Toll-1-, Toll10b- and Toll-9-mediated drosomycin reporter activities (Ooi, 2002).

Cactus uses its ankyrin repeats to bind to the Rel homology domains of Dif and Dorsal. The Cactus protein degradation is regulated both by signal dependent and signal independent mechanisms, through the N-terminal serine residues and C-terminal PEST sequence, respectively. Therefore, a construct CactusDelta125DeltaPEST was used that contained only the ankyrin repeats. This mutant Cactus should stably bind to and inhibit Dif and Dorsal, even when the signaling pathway is stimulated. Co-transfection of wild-type Cactus did not lead to significant changes in the activation of drosomycin by Toll-1, Toll10b and Toll-9. In contrast, the CactusDelta125DeltaPEST construct abolished all these Toll signaling activities. Therefore, Cactus and Pelle, and probably the binding partners Dif and Tube, are likely signaling components that mediate the activation of drosomycin by Toll-9 (Ooi, 2002).

The homeobox gene Caudal regulates constitutive local expression of antimicrobial peptide genes in Drosophila epithelia

In Drosophila, although the NF-kappaB transcription factors play a pivotal role in the inducible expression of innate immune genes, such as antimicrobial peptide genes, the exact regulatory mechanism of the tissue-specific constitutive expression of these genes in barrier epithelia is largely unknown. This study shows that the Drosophila homeobox gene product Caudal functions as the innate immune transcription modulator that is responsible for the constitutive local expression of antimicrobial peptides cecropin and drosomycin in a tissue-specific manner. These results suggest that certain epithelial tissues have evolved a unique constitutive innate immune strategy by recruiting a developmental 'master control' gene (Ryu, 2004).

In silico identification of putative genomic binding sites of AMP genes and their transcription factors was performed by using the MatInd and MatInspector systems. In this analysis, several cis elements (such as the kappaB motif, the GATA motif, and Cad binding motifs) were commonly found in the promoter regions of all known AMPs. Transcription factors resulting from this analysis were systematically tested for their capacity to induce AMP genes in the immunocompetent Schneider cell line SL2. In Schneider cells stably expressing Cad, the expression of all seven AMP genes was greatly enhanced, suggesting that Cad is a potential transcription regulator. This result prompted an in-depth investigation into the in vivo role of Cad using two representative AMP genes (IMD pathway-controlled Cec and Toll pathway-controlled Drs). Because the Cad gene product contains a homeodomain, which indicates that the protein has a DNA-binding capability, the cis elements responsible for Cad-induced Cec and Drs expression were examined. To identify the cis elements responsible for Cad-induced Cec and Drs expression, a luciferase reporter assay of various mutant constructs having deletions in the Cec promoter region was performed in Drosophila Schneider cells. Cad-induced luciferase activity in cells transfected with the plasmid with a deletion from -751 to -484 bp was found to be almost invariant compared with that in cells transfected with the wild-type construct. However, luciferase activity remained at the basal level in cells transfected with the plasmid having a deletion from bp -751 to -377. These results suggest that the region from bp -484 to -377 of the Cec promoter is a candidate region for Cad-protein DNA recognition elements (CDREs). For Drs, the region covering bp -1082 to -1008 was identified as a candidate region for CDREs of Drs. Based on these results, six putative binding sites (S1 to S6) with the consensus Cad binding motif (T>C/A>G)TTT(A>G>C)(T>G/A/C)(G>T/C/A)(A>G/T/C) were identified in the promoter region of Cec and Drs. To determine whether Cad possesses a DNA binding capability with these putative binding sites of the Cec promoter, DNA-binding experiments were performed with the recombinant Cad protein using wild-type probes and various mutant probes. The results showed that GST-Cad is able to bind to two Cad binding motifs, at the S2 and S5 sites. Luciferase reporter analysis with a plasmid carrying double mutations in the putative binding sites (S2 and S5) revealed that these sites are essential for Cad-mediated Cec promoter regulation. Similar methods were employed to identify the CDREs for Cad-mediated Drs promoter regulation. The luciferase reporter assay with plasmids carrying point mutations in the putative CDREs together with the EMSA and supershift assay revealed that Cad is capable of directly regulating the expression of Drs via four CDREs (S1 to S4) found in its promoter. These results demonstrate the involvement of Cad in the regulation of AMP genes, providing yet another function for this homeotic transcriptional regulator, well known for its key role in anteroposterior patterning of the embryo (Ryu, 2004).

This study shows that a Cad-LacZ reporter and endogenous Cad mRNA are expressed in the salivary glands and ejaculatory duct, where AMP expression is constitutive. Vertebrate Cad homologues are well known to participate in early embryogenesis, the development of the intestine, and colon tumorigenesis. However, apart from their developmental roles, the physiological functions and target genes of the Cad homeobox gene family are unknown. The observation that Cad regulates AMP gene expression in a subset of epithelia indicates a new function for this trans-activator in the local defense against microbial infection and/or maintenance of microbial flora. At present, the real in vivo function of AMP gene expression in local epithelia in Drosophila is not known. In the local-infection experiment, enhanced mortality in the Cad-RNAi-expressing flies was not obserfed following short-term (1 h) bacterial feeding. However, although local AMP expression is not directly related to the rate of survival of infection, the locally secreted AMPs may help to prevent the onset of infections (Ryu, 2004).

It is well known that the Toll/NF-kappaB signaling pathway for dorsoventral body axis formation mainly regulates the inducible expression of the Drs gene during the systemic immune response. Interestingly, this pathway has been well conserved during evolution and assists NF-kappaB activation via Toll-like receptors in the human innate immune system. The results show that, in the local epithelial immune system, NF-kappaB-independent, constitutive expression of Drs and Cec in the barrier epithelial tissues is mainly controlled by the homeobox gene Cad, a master controller of anteroposterior body axis formation. The developmental genes involved in specification of the fly body plan (dorsoventral and anteroposterior body axes) have been recruited for this evolutionally ancient first line of defense (Ryu, 2004).

The involvement of Cad in the constitutive local innate immunity illustrates the complexity of the tissue-specific regulation of AMP expression in Drosophila. To better visualize the complexity and dynamic of the innate immune response in Drosophila, a comprehensive scheme was constructed. Experimental infection such as septic injury rapidly induces various AMPs, mainly in the fat body (known as systemic immunity), via two different NF-kappaB pathways (Toll and IMD pathways), whereas natural infection, such as local bacterial infection, activates the expression of AMPs via the IMD pathway only in a subset of epithelial tissues (known as inducible local innate immunity). These two inducible innate immune systems in Drosophila are rather distinct because septic injury cannot activate the inducible local immune system. The third type of AMP regulation is the constitutive local expression of AMPs in an NF-kappaB-independent manner in several epithelia. This type of strategy is believed to be very ancient in evolution and may be very efficient in certain epithelia by avoiding chronic NF-kappaB activation where the contact with microbes is continuous (Ryu, 2004).

This study showed that Cad is capable of directly regulating Cec and Drs via CDREs found in their promoters in Drosophila Schneider cells. Furthermore, Cad binds in vitro to the CDREs found upstream of AMP genes in a gel shift assay. These results demonstrate that Cad is a direct trans-activator of AMP genes. The in vivo reporter analysis demonstrates that mutations affecting CDREs do not abolish the inducible systemic Drs expression in the fat body. These results clearly indicate that the CDREs, in contrast to kappaB sites, are not required for inducible Drs expression in the fat body during a systemic immune response. In addition to the fat body, the trachea is involved in inducible Drs expression. This tissue, in which Drs expression is normally absent but rapidly induced in response to local infection by Erwinia carotovora, is known to be involved in inducible local immunity. Surprisingly, even though there is no appreciable role for CDREs in the fat body, it was found that all 12 independent Drsmut-GFP-expressing fly lines (larvae and adults) exhibited spontaneous constitutive expression of Drs reporter activity in the trachea in the absence of local infection. One may speculate that CDREs can also act as negative cis elements in some epithelial tissues such as the trachea, where they can maintain the silencing of Drs expression, and that this depends on the specific cell type. Further studies will be needed to understand the complete tissue-specific Cad signaling pathway for AMP regulation in all epithelial tissues. In contrast to kappaB-dependent inducible AMP expression, the constitutive local innate immunity employs Cad for the expression of AMPs through CDRE motifs rather than kappaB motifs. Interestingly, for salivary glands, overexpression of the Cad-RNAi construct is sufficient to severely reduce Drs expression, indicating that constitutive local expression of Drs in salivary glands is greatly dependent on Cad. For the ejaculatory duct, although partial reduction of Cad modestly reduces Cec expression, only minor expression (~20%) of the Cec reporter was detected in flies carrying Cecmut-GFP, as well as flies overexpressing the dominant-negative form of Cad. This also indicates that Cec expression in this tissue is largely dependent on Cad. Interestingly, this study showed that not all constitutive local expression is dependent on Cad. The constitutive local expression in the female reproductive organs is completely CDRE independent, suggesting the existence of yet another unknown signaling pathway(s). Recently, Drosophila Toll-9, one of the Toll-related receptors, was found to trigger the constitutive expression of Drs in cultured cells (Ooi, 2002). It is possible that Toll-9 may control constitutive Drs expression in certain epithelia. The studies on the in vivo function of Toll-9 should elucidate this issue (Ryu, 2004).

The expression of various AMPs in analogous human epithelial tissues suggests that epithelial innate immunity is well conserved and that the careful regulation of AMP levels may be needed to maintain homeostasis in these tissues from Drosophila to humans. The presence of human Cad homologues, CDXs, raises interesting questions concerning their putative role(s) in human epithelial innate immune gene regulation (Ryu, 2004).

The moleskin gene product is essential for Caudal-mediated constitutive antifungal Drosomycin gene expression in Drosophila epithelia

The homeobox gene, Caudal, encodes the DNA-binding nuclear transcription factor that plays a crucial role during development and innate immune response. The Drosophila homologue of importin-7 (DIM-7), encoded by moleskin, was identified as a Caudal-interacting molecule during yeast two-hybrid screening. Both mutation of the minimal region of Caudal responsible for Moleskin binding and RNA interference (RNAi) of moleskin dramatically inhibited the Caudal nuclear localization. Furthermore, Caudal-mediated constitutive expression of antifungal Drosomycin gene was severely affected in the moleskin-RNAi flies, showing a local Drosomycin expression pattern indistinguishable from that of the Caudal-RNAi flies. These in vivo data suggest that DIM-7 mediates Caudal nuclear localization, which is important for the proper Caudal function necessary for regulating innate immune genes in Drosophila (Han, 2004).

dUbc9 negatively regulates the Toll-NF-kappaB pathways in larval hematopoiesis and drosomycin activation in Drosophila

Highly conserved during evolution, the enzyme Ubc9 activates the small ubiquitin-like modifier (SUMO) prior to its covalent ligation to target proteins. Mutations in the Drosophila Ubc9 (dUbc9) gene have been used to understand Ubc9 functions in vivo. Loss-of-function mutations in dUbc9 cause strong mitotic defects in larval hematopoietic tissues, an increase in the number of hematopoietic precursors in the lymph gland and of mature blood cells in circulation, and an increase in the proportion of cyclin-B-positive cells. Some blood cells are polyploid and multinucleate, exhibiting signs of genomic instability. Also, an overabundance of highly differentiated blood cells (lamellocytes), normally not found in healthy larvae, are observed. Lamellocytes in mutants are either free in circulation or recruited to form tumorous masses. Hematopoietic defects of dUbc9 mutants are strongly suppressed in the absence of the Rel/NF-κB-family transcription factors Dorsal and Dif or in the presence of a non-signaling allele of Cactus, the IκB protein in Drosophila. In the larval fat body, dUbc9 negatively regulates the expression of the antifungal peptide gene drosomycin, which is constitutively expressed in dUbc9 mutants in the absence of immune challenge. dUbc9-mediated drosomycin expression requires Dorsal and Dif. Together, these results support a role for dUbc9 in the negative regulation of the Drosophila NF-κB signaling pathways in larval hematopoiesis and humoral immunity (Chiu, 2005).

Ubc9 was discovered in Saccharomyces cerevisiae based on its sequence similarity to other known ubiquitin-conjugating enzymes. A loss of function of Ubc9 causes an increase in the S- and M (B type)-phase cyclins, resulting in an arrest of the cell cycle at the G2 or early M phase. This cessation of the cell cycle causes an accumulation of large budded cells with a single nucleus and replicated DNA. Ubc9 was also identified in a screen for DNA damage checkpoint control in S. pombe. Although not directly involved in the checkpoint control, Ubc9 (encoded by hus5) is required for the efficient recovery from DNA damage or S-phase arrest, and for chromosome segregation. Yeast mutants display severe impairment in growth and exhibit a high frequency of failed mitosis. Further studies in yeast revealed that unlike other E2 ubiquitin-conjugating enzymes, Ubc9 is unable to form a thioester bond with ubiquitin; instead it conjugates the ubiquitin-like protein SUMO/Smt3 to specific targets in a yeast extract (Chiu, 2005 and references therein).

Vertebrate homologs of Ubc9 were subsequently identified in many laboratories, showing that Ubc9 may interact with a wide variety of cellular proteins, regulating cellular processes such as cell division, protein trafficking, signal transduction, and transcriptional regulation. Studies designed to identify biochemical targets of Ubc9 highlighted a role for sumoylation in the regulation of chromatin organization, gene expression, and genome surveillance (Chiu, 2005 and references therein).

While SUMO-1 is structurally similar to ubiquitin and sumoylation and ubiquitination are enzymatically similar processes, the conjugation of SUMO-1 or ubiquitin to the same protein can have opposite effects. For example, ubiquitin or SUMO-1 can be directly conjugated to lysine residues 21 and 22 of mammalian IκBα in reactions catalyzed by activating enzymes Ubch5 and Ubc9, respectively. IκBα is a cytoplasmic inhibitor of the transcription factor NF-κB. In unstimulated cells, cytoplasmic NF-κB, complexed with IκBα, remains inactive. Activation of NF-κB is achieved by ubiquitination and proteasome-mediated degradation of IκBα, allowing NF-κB translocation to the nucleus. However, when the same lysine residues in IκBα are conjugated to SUMO-1/Smt3, ubiquitination is blocked, thereby stabilizing the cytoplasmic pool of this protein. This increase in stabilization of IκB sequesters NF-κB in the cytoplasm, leading to a downregulation of the NF-κB pathway. Thus, while ubiquitination targets proteins for degradation, SUMO-1 modification acts antagonistically to render proteins resistant to degradation. Given that SUMO modification alters the ability of proteins to interact with their partners, alters their subcellular localization, and controls their stability, understanding the role of sumoylation in different cellular processes is of fundamental importance in normal and diseased cells (Chiu, 2005 and references therein).

Drosophila has been used as a model system to understand the functions of Ubc9 in vivo. An alignment of the Drosophila Ubc9 (dUbc9) with its counterparts in yeast, C. elegans, and humans shows that dUbc9 shares a higher level of structural similarity with the human Ubc9 (84% identical) than with either the C. elegans (76% identical) or yeast (35% identity) proteins. Strikingly, expression of either human or Drosophila Ubc9 can rescue an S. cerevisiae Ubc9ts mutant. dUbc9, also called semushi and lesswright, (lwr), has many biological functions. For example, mutation in dUbc9 disrupts anterior segmentation in embryogenesis by interfering with the nuclear uptake of the homeodomain transcription factor Bicoid. Mutants in dUbc9 also suppress the nodDTW (dominant antimorphic allele of no distributive disjunction) phenotype, implying a role in chromosome segregation during meiosis in females. Biochemically, the SUMO-1/Ubc9 pathways are conserved between flies and humans: (1) dSmt3 and dUbc9 are coexpressed during development, (2) dSmt3 can be processed and conjugated in human cells, and (3) human transcription factor PML can be modified by dSmt3 in Drosophila SL2 or human HeLa cells. Like their human counterparts, dSmt3 and dUbc9 colocalize in nuclear foci (Chiu, 2005 and references therein).

Drosophila Ubc9 was also identified in a yeast two-hybrid screen for Dorsal-interacting proteins. Dorsal is one of three Drosophila Rel/NF-κB-family proteins. The nuclear localization of Dorsal is controlled by the Toll receptor. Toll activation leads to signal transduction via Tube and Pelle, as well as the phosphorylation and degradation of Cactus, the Drosophila IκB protein. Components of the Toll-Dorsal pathway were first identified as maternal-effect genes controlling the development of the embryonic dorsal-ventral axis. Although the precise mechanism underlying Cactus degradation in the embryo is still unclear, in vivo studies suggest that, like mammalian IκBα, Cactus degradation is regulated by Toll signal-dependent phosphorylation. dUbc9 conjugates a Drosophila SUMO/Smt3 to lysine 382 of Dorsal. Whether Cactus also serves as a sumoylation target is not known (Chiu, 2005 and references therein).

In Drosophila, the NF-κB pathway regulates many biological processes at different developmental stages. The Toll-Dorsal/Dif pathway activates transcription of antifungal and antibacterial (Gram-positive) peptide genes in the larval and adult fat body. Dif (Dorsal-related immunity factor) belongs to the NF-κB family . The regulation of genes encoding antibacterial peptides that kill Gram-negative bacteria (e.g., diptericin) is under the control of Relish, the third Drosophila Rel/NF-κB protein similar to the mammalian p100/p105 proteins. Relish activation is Toll-independent. Instead, the intracellular Relish phosphorylation and activation are regulated by activities of Immune deficiency (Imd) pathway including proteins of the IKK complex and the Dredd caspase. This pathway is negatively regulated by the ubiquitin proteasome system (Chiu, 2005 and refenreces therein).

The Toll-IκB pathway also contributes to proliferation of blood cells (hemocytes) during normal larval hematopoiesis and during the hematopoietic proliferation that accompanies immune challenge. Unchallenged Drosophila larvae have two hemocyte types in circulation: the plasmatocyte and the crystal cell, both of which are specified and formed during embryonic stages. More than 90% of all hemocytes in circulation are plasmatocytes. Plasmatocytes are phagocytic cells, ridding the larvae of microbial infections. The remaining hemocytes in circulation (5% or less), the crystal cells, carry prominent crystalline inclusions and, when activated, lyse and release their contents, melanizing target cells. Mutations that upregulate the Toll pathway (loss-of-function mutations in cactus, gain-of-function mutations in Toll, or overexpression of Dorsal in larval hemocytes) result in overproliferation of hemocytes, whereas mutations that downregulate the pathway (loss-of-function in Toll, tube, or pelle) lead to a reduction in the number of circulating hemocytes. Changes in hemocyte counts in mutations affecting either dorsal or Dif are unremarkable (Chiu, 2005 and references therein).

A third kind of hemocyte, the lamellocyte, appears in the larval hemolymph only in response to infections by naturally occurring endoparasitoid wasps and other foreign bodies. The parasitoids constitute a major threat to the Drosophila population in nature, as they hijack the larval body for their own development. Lamellocytes are adhesive, and rapidly aggregate around a parasite egg to form a cellular capsule. Parasite-induced lamellocyte differentiation in the lymph gland is accompanied by a modest increase in the number of plasmatocytes and crystal cells. The encapsulated wasp egg is melanized. Lamellocyte precursors are normally quiescent. However, mutations that lead to the overproliferation of hemocytes (loss-of-function cactus alleles, gain-of-function Toll alleles) also result in constitutive differentiation of lamellocytes, resulting in the encapsulation of self-tissue in the absence of wasp infection (innate autoimmune response). Because of their dark appearance, these capsules are called melanotic tumors (Chiu, 2005 and references therein).

This study reports that larvae carrying loss-of-function mutations in dUbc9 show strong hematopoietic proliferation and differentiation defects. Furthermore, the antifungal gene drosomycin is constitutively active in developmentally delayed dUbc9 mutants. Both constitutive humoral and cellular immune defects are rescued by mutations in dorsal and Dif. These results suggest that dUbc9 contributes to the regulation of both humoral immunity and hemocyte proliferation by acting as a negative regulator of the Toll pathway (Chiu, 2005).

Genetic experiments in Drosophila emphasize the central regulatory role for Ubc9 function and sumoylation in different cells and during different life cycle stages. lwr adults exhibit defects in eye, wing, and leg morphogenesis: mutant males die and the few surviving females are sterile, revealing roles for dUbc9 in cell division and differentiation, in tissue patterning, and in oogenesis. This paper describes additional defects in dUbc9 mutants, based on studies of mutant larvae. Phenotypes are described that point to the involvement of dUbc9 in larval metamorphosis, proliferation, and differentiation of hematopoietic precursors, in antimicrobial gene expression, and in NF-κB signal transduction. These observations show that diverse biological processes share a common regulatory mechanism involving sumoylation and the variety of the phenotypes observed suggest the possibility of multiple biochemical targets in vivo. Drosophila is an excellent model for the identification of cell- and tissue-specific sumoylation targets of Ubc9, and it is very likely that their conserved mammalian counterparts will be similarly modified and regulated (Chiu, 2005).

Like many larval lethals (e.g., cact mutants), mutations in dUbc9 result in a prolonged third instar period followed by larval death. Hematopoietic defects are observed as early as 4 days after egg lay, whereas the immune defects are evident 6 days after egg lay. Defects of both types become severe as mutant animals persist in larval stages even 10 days after their birth. Indeed, constitutive expression of antimicrobial genes during larval stages continues through lwr4-3/lwr5 pupae and adults. How dUbc9 affects the rate of development is currently not known. Epistasis experiments between lwr and mutants of the ecdysone genetic hierarchy may reveal a role for dUbc9 sumoylation during development and metamorphosis. Also not known is whether hemocyte survival or their apoptosis is affected in lwr mutants. One study of salivary gland apoptosis in Drosophila during pupariation provides evidence that the Rel family members are not required for salivary gland cell death during metamorphosis. The current results provide a link between lwr negative regulation of dl and Dif in the larval fat body and hematopoietic tissues. In any case, the hematopoietic and immune defects in lwr mutants may be tied to abnormalities in larval development as these defects are most pronounced in older animals. The rescue of the immune defects by expression of the dUbc9 protein in the fat body suggests that the misregulation of drom expression in lwr mutants is due to a specific reduction or an absence of the Ubc9 function. Analysis of mutant clones in the fat body or lymph gland will reveal if the requirement of this enzyme is cell autonomous or not (Chiu, 2005).

Four distinct hematopoietic defects affecting hemocyte abundance, differentiation, and morphology are observed in both the lymph gland and circulating hemocyte populations in 4-day-old lwr larvae. The mean CHC values in mutants are significantly higher than those of the pooled control class of larvae; in two of the three lwr genetic combinations studied, this increase in abundance correlates with an increase in cyclin-B-positive hemocytes. Such increase in cyclin B-positive hemocyte population is reminiscent of increase in the B type cyclins in mutant yeast lacking Ubc9. The current observations suggest that dUbc9 negatively regulates the rate at which hematopoietic cells divide. The prehemocyte classes influenced by the dUbc9 mutation are not known, however, it is interesting that while there is an increase in circulating plasmatocyte and lamellocyte percentages, there is a clear reduction in the number of crystal cells. The opposite effect of lwr on crystal cell numbers indicates a distinct role for dUbc9 function in this hemocyte lineage. Prohemocytes following the crystal cell fate require a combination of signals from the transcription factors Serpent (Srp; human GATA-2) and Lozenge (lz; human AML-1), along with permissive signals from Ser/Notch. Perhaps dUbc9 asserts a role in crystal cell development by propagating the above required signals or alleviating suppression of this cellular fate exerted by the combinatorial interaction of SrpNC (Srp isoform containing both N- and C-terminal Zinc finger domains) and the U-shaped protein. These observations suggest multiple requirements for dUbc9 in the hematopoietic tissue. Furthermore, as mitotic defects are observed in both lymph gland hemocytes and circulating hemocytes, and since these groups of hemocytes originate independently, it is likely that dUbc9 function is independently required in each hemocyte population (Chiu, 2005).

The presence of large numbers of lamellocytes accounts for a significant fraction (10%-20%) of the increase of CHC in lwr mutants. The coincident expansion of the plasmatocyte population and constitutive differentiation of lamellocytes are hallmarks of melanotic tumor mutants in which affected genes are not necessarily related by either structure or function. Yet, hemocyte proliferation and differentiation have distinct genetic requirements. For example, while the Drosophila kinase Hopscotch and transcription factor STAT92E are required for lamellocyte differentiation, Toll and Tube proteins are not, even though upregulation of either the JAK/STAT or the Toll/NF-κB pathways results in the production of melanotic tumors. One explanation for the simultaneous expansion of the plasmatocyte and lamellocyte populations in melanotic tumor mutants such as lwr, cact, and Toll10b is that these mutations affect proliferation of precursors in the lymph gland (or in circulation) that differentiate as plasmatocytes or lamellocytes (Chiu, 2005).

The aberrant nuclear morphologies observed in lwr4-3/lwr14 and lwr4-3/lwr5 hemocytes are variable in appearance and fall into four categories: aneuploidy, abnormal nuclear shapes, presence of multiple nuclei, and presence of fragmented nuclear material (additional smaller Hoechst-positive structures). Genetic evidence suggests that dUbc9 is required for the proper disjunction of homologous chromosomes in meiosis I. It is thus possible that dUbc9 is also involved in chromosome segregation in mitotic divisions. Indeed, defects in lwr hemocytes are strikingly similar to proliferation defects in cultured chicken cells conditionally depleted of Ubc9 protein, in which cells with multiple or fragmented nuclei are also observed. The defects in chicken cells arise due to chromosomal loss during chromosome segregation. In both Drosophila and chicken, the frequency of cells with multiple or fragmented nuclei increases with age. Thus, it is possible that the biochemical targets of Ubc9 in chromosome segregation in Drosophila and chicken cells (and possibly other vertebrates) are conserved and that chromosomal damage accumulates in Ubc9-depleted cells because of similar molecular processes. The proportion of multinucleate hemocytes (in lwr4-3 larvae) is reduced in lwr4-3 dl as well as lwr4-3 Dif dl mutant larvae. The identity of the extra chromosomes and extrachromosomal DNA in dUbc9 mutant hemocytes is not known nor the specificity of this phenotype to lwr allele 4-3. The presence of multinucleate hemocytes in circulation within hemocyte overproliferation mutants is unique to lwr and, to date, has not been reported in other mutants where similar overproliferation and lamellocyte differentiation defects have been documented (e.g., hopTum-l; Toll10b/+; cactus), reflecting the multiplicity of effects of dUbc9 on the cell cycle (Chiu, 2005).

The genetic interaction and immunohistochemical studies presented in this study constitute the first clear evidence of a role for Dorsal and Dif during hemocyte proliferation in Drosophila larvae, even though these functions were predicted from previous experiments. Like Cactus, the cellular function of dUbc9 is to regulate the nuclear localization of NF-κB proteins. Interestingly, Dorsal and Dif appear to have somewhat redundant functions as suppression of lwr phenotypes is stronger in triple mutants than in double mutants and suppression appears earlier in development in triple mutants than in double mutants. Functional redundancy may also explain why clear hematopoietic phenotypes (e.g., lowered hemocyte concentration or reduced encapsulation of wasp egg) have not been observed in dl or Dif single mutants. Similar redundancy in Dorsal and Dif function has been reported for antimicrobial peptide gene activation in the larval fat body (Chiu, 2005).

Genetic experiments here support a model for negative regulation by dUbc9 of antifungal peptide-encoding genes drosomycin and Cecropin, making it the second negative regulator of the Toll pathway to be identified so far. In general, the effects of lwr mutants on drosomycin expression are stronger than on that of Cecropin. These effects are evident by characterization using promoter-driven GFP reporters representative of the Toll and Imd downstream antimicrobial genes and subsequent confirmation by Northern analysis in whole animals. Perhaps the expression pattern of Cecropin A observed in lwr mutants is partially independent of the Toll pathway. cecropin A has been shown to be regulated by both Toll and Imd immunity pathways. Indeed, dynamic expression patterns of the two variant Cec A1 and A2 transcripts are detected in adult flies, after specific microbial infection regiments, further delineating their expression into the two immune pathways. A detailed analysis of Cecropin A regulation in lwr mutant larvae in combination with loss of function alleles of the Imd pathway may elucidate control of this gene further (Chiu, 2005).

The constitutive activation of drosomycin and Cecropin in lwr mutants is also dependent on Dorsal and Dif, whose roles and functional redundancy in the larval fat body are already recognized. Furthermore, genetic epistasis experiments described here place dUbc9 function upstream of Dorsal/Dif and Cactus. These observations are largely consistent with previous biochemical experiments on these proteins and provide additional support for a model in which Dorsal, Dif, Cactus and dUbc9 exist in a complex that is activated by a Toll-dependent signal. Significantly, however, the results suggest that dUbc9 blocks the nuclear localization of Dorsal and Dif and differ from observations made in Drosophila S2 cell cultures, in which dUbc9 facilitates the nuclear localization of Dorsal-GFP. This divergence in experimental results is likely to be due to different experimental models used in the two studies. The genetic results are consistent with Ubc9's role in IκB sumoylation and downregulation of the mammalian NF-κB pathway. Consistent with this model of mammalian Ubc9 function, it is likely that sumoylation of Cactus by dUbc9 protects it from phosphorylation, assisting the retention of Dorsal/Dif in the cytoplasm. This model requires biochemical support as Cactus sumoylation has not been demonstrated (Chiu, 2005 and references therein).

The intracellular Toll-Dorsal/Dif pathways in both the fat body and in hemocytes include Toll, Tube, Pelle, Ubc9, Dorsal, and Dif, and in both cases, dUbc9 appears to function upstream of Cactus and Dorsal/Dif. These observations with GFP reporter constructs suggest that the effect of dUbc9 is restricted to the Toll pathway and it is possible that the Imd signaling cascade is not regulated by sumoylation. Indeed, a ubiquitin proteasome pathway involving function of SkpA and Slimb has been identified and shown to repress the Imd pathway. Thus, sumoylation and ubiquitination of specific targets appear to have parallel but specific effects in downregulating these pathways (Chiu, 2005).

In conclusion, strong evidence is presented that dUbc9 is a negative regulator of the Toll-NF-κB pathways that control both the humoral and cellular aspects of immune responses in Drosophila. Spatzle activates the Toll pathway in the fat body; however, a role for Spatzle function in hemocyte proliferation or differentiation has not been demonstrated, and therefore the mechanism of Toll activation in the hemocytes is not known. Similarly, target genes of the NF-κB pathway in hemocytes (besides the antimicrobial peptide genes) have not yet been identified. The differences in phenotypes observed in lwr and cact mutants are likely to arise from differences in gene expression programs in the two mutants. These differences provide a unique opportunity to resolve issues of biological specificity in the regulation of NF-κB activation and gene expression in vivo (Chiu, 2005).

A Drosomycin-GFP reporter transgene reveals a local immune response in Drosophila that is not dependent on the Toll pathway

In Drosophila, a septic wound induces the rapid appearance in the hemolymph of a battery of antibacterial peptides that includes the Cecropins, Drosocin, insect Defensin, Metchnikowin, Attacin and one major antifungal peptide, Drosomycin. These peptides are synthesized mostly in the fat body, a functional equivalent of the liver, and secreted into the hemolymph. This reaction constitutes a systemic antimicrobial response. Since experimental wounds are restricted to a single point of entry and since all of the disseminated fat body is responding to the attack, it is thought that a signal is transmitted to the fat body through the hemolymph from the entry site of microorganisms, where non-self recognition presumably occurs. This study asked whether antimicrobial peptides are also expressed in barrier epithelia in Drosophila, independent of a systemic response. This question was specifically addressed regarding the expression of the antifungal peptide Drosomycin in both larvae and adults. Using a drosomycin-green fluorescent protein (GFP) reporter gene, it was shown that in addition to the fat body, a variety of epithelial tissues that are in direct contact with the external environment, including those of the respiratory, digestive and reproductive tracts, can all express the antifungal peptide, suggesting a local response to infections affecting these barrier tissues. As is the case for vertebrate epithelia, insect epithelia appear to be more than passive physical barriers and are likely to constitute an active component of innate immunity. In contrast to the systemic antifungal response, this local immune response is independent of the Toll pathway (Ferrandon, 1998).

Inhibitor kappaB-like proteins from a polydnavirus inhibit NF-kappaB activation and suppress the insect immune response

Complex signaling pathways regulate the innate immune system of insects, with NF-kappaB transcription factors playing a central role in the activation of antimicrobial peptides and other immune genes. Although numerous studies have characterized the immune responses of insects to pathogens, comparatively little is known about the counter-strategies pathogens have evolved to circumvent host defenses. Among the most potent immunosuppressive pathogens of insects are polydnaviruses that are symbiotically associated with parasitoid wasps. This study reports that the Microplitis demolitor bracovirus encodes a family of genes with homology to inhibitor kappaB (IkappaB) proteins from insects and mammals. Functional analysis of two of these genes, H4 and N5, were conducted in Drosophila S2 cells. Recombinant H4 and N5 greatly reduce the expression of drosomycin and attacin reporter constructs, which are under NF-kappaB regulation through the Toll and Imd pathways. Coimmunoprecipitation experiments indicated that H4 and N5 bind to the Rel proteins Dif and Relish, and N5 also weakly binds to Dorsal. H4 and N5 also inhibit binding of Dif and Relish to kappaB sites in the promoters of the drosomycin and cecropin A1 genes. Collectively, these results indicate that H4 and N5 function as IkappaBs and, circumstantially, suggest that other IkappaB-like gene family members are involved in the suppression of the insect immune system (Thoetkiattikul, 2005).

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).

Mating in D. melanogaster and in many other insects elicits various postmating responses (PMR) in females, e.g., enhanced ovulation and oviposition, reduced receptivity (willingness to remate) and stimulation of the innate immune system. The PMR are mainly elicited by seminal fluid transferred during copulation. One of its components, Sex-peptide (SP; a 36 amino acid long peptide synthesized in the male accessory glands (Chen, 1988), is the major agent eliciting oviposition and reduction of receptivity. This study investigates the time course of AMP induction after copulation; SP has been determined to be one of the major seminal components eliciting transcription of AMP genes after mating (Peng, 2005).

D. melanogaster produces about 20 different antimicrobial peptides. The AMPs can be classified into seven distinct families: Attacins, Cecropins, Diptericins, Drosocins (against gram-negative bacteria), Defensins, Metchnikowin (against gram-positive bacteria), and Drosomycins (against fungi). Mating induces transcription of all probed AMP genes: Attacin A and C, Cecropin B, Diptericin, Drosocin, Defensin, Metchnikowin, and Drosomycin, thus confirming the data obtained by microarray analysis (Peng, 2005).

To cover the whole spectrum of AMPs induced by different types of pathogens, focus was placed on the transcription of the Metchnikowin (Mtk; induced by both pathways), Drosomycin (Drs; induced by the Toll pathway), and Diptericin (Dipt; induced by the Imd pathway) genes, respectively. In mated females Mtk, Drs, and Dipt induction are observed as early as 1 hr after mating. Expression peaks between 2 and 4 hr and fades away after 8 hr, again reaching the virgin level. However, the degree of upregulation varies, Mtk showing the strongest response (Peng, 2005).

To identify the elements responsible for the elevated transcription of the AMP genes, males without functional SP (SP0 males) and males without germline (germline-less [GLL] males; sons of tropomyosinII mutant [TmIIgs1/TmIIgs1] females lacking the germline) were mated with virgin wt females. 2 hr after mating, RNA was extracted from the mated females and analyzed by Northern blots and quantitative PCR. SP0 males fail to induce the transcription of AMP genes, whereas GLL males induce AMP genes at about 4/5th of the level of wt males. The latter finding indicates that sperm plays a minor role in eliciting Mtk transcription (Peng, 2005).

To confirm the capability of SP to induce the transcription of the Mtk gene, transgenic females expressing SP ectopically and constitutively (driven by the promoter of the Yp1 gene; i.e., Yp-SP females; Aigaki, 1991) were analyzed for Mtk expression. In contrast to the very low level of Mtk expression in virgin control females, Mtk expression in virgin Yp-SP transgenic females is already high, even higher than in mated control females. It is not further increased by mating, i.e., Mtk in Yp-SP virgins is already transcribed at a maximal rate. It is concluded that SP is the major agent eliciting Mtk expression after mating and that constitutive expression of SP leads to permanent high levels of Mtk transcription. Furthermore, since SP concentration in the hemolymph of transgenic Yp-SP females is higher than in the hemolymph of mated females and Mtk transcription is statistically significantly higher in Yp-SP transgenic females than in wt females mated with wt males, the level of transcription of AMP genes is very likely dependent on SP concentration (Peng, 2005).

The SP-induced expression of two additional AMPs dependent on the Toll (Drosomycin) or the Imd (Diptericin) pathways, respectively, was investigated. Expression of Drs and Dipt was monitored in virgin wt females, in wt females mated with wt or SP0 males, respectively, and in virgin and mated Yp-SP transgenic females (the latter mated with wt males). Sex-peptide also induces the transcription of Drs and Dipt, but the induction of Drs and Dipt is weaker by orders of magnitude than that of Mtk. Constitutive expression of SP in Yp-SP transgenic females leads to continuous expression of Drs and Dipt and elevates the expression of Dipt statistically significantly above the level induced by mating. It is concluded that specific AMP genes respond differentially to SP induction (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).

In vivo RNA interference analysis reveals an unexpected role for GNBP1 in the defense against Gram-positive bacterial infection in Drosophila adults

The Drosophila immune system discriminates between different classes of infectious microbes and responds with pathogen-specific defense reactions via the 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 Gram-negative bacterial infection. Microbial recognition is achieved through peptidoglycan recognition proteins (PGRPs); Gram-positive bacteria activate the Toll pathway through a circulating PGRP (PGRP-SA), and Gram-negative bacteria activate the Imd pathway via PGRP-LC, a putative transmembrane receptor, and PGRP-LE. Gram-negative binding proteins (GNBPs) were originally identified in Bombyx mori for their capacity to bind various microbial compounds. Three GNBPs and two related proteins are encoded in the Drosophila genome, but their function is not known. Using inducible expression of GNBP1 double-stranded RNA, it has been demonstrated that GNBP1 is required for Toll activation in response to Gram-positive bacterial infection; GNBP1 double-stranded RNA expression renders flies susceptible to Gram-positive bacterial infection and reduces the induction of the antifungal peptide encoding gene Drosomycin after infection by Gram-positive bacteria but not after fungal infection. This phenotype induced by GNBP1 inactivation is identical to a loss-of-function mutation in PGRP-SA, and the genetic studies suggest that GNBP1 acts upstream of the Toll ligand Spatzle. Altogether, these results demonstrate that the detection of Gram-positive bacteria in Drosophila requires two putative pattern recognition receptors, PGRP-SA and GNBP1 (Pili-Floury, 2004).

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).

DEAF-1 regulates immunity gene expression in Drosophila

Immunity genes are activated in the Drosophila fat body by Rel and GATA transcription factors. Evidence that an additional regulatory factor, Deformed epidermal autoregulatory factor-1 (DEAF-1), also contributes to the immune response and is specifically important for the induction of two genes encoding antimicrobial peptides, Metchnikowin (Mtk) and Drosomycin (Drs). The systematic mutagenesis of a minimal Mtk 5' enhancer identified a sequence motif essential for both a response to LPS preparations in S2 cells and activation in the larval fat body in response to bacterial infection. Using affinity chromatography coupled to multidimensional protein identification technology (MudPIT), DEAF-1 was identified as a candidate regulator. DEAF-1 activates the expression of Mtk and Drs promoter-luciferase fusion genes in S2 cells. SELEX assays and footprinting data indicate that DEAF-1 binds to and activates Mtk and Drs regulatory DNAs via a TTCGGBT motif. The insertion of this motif into the Diptericin (Dpt) regulatory region confers DEAF-1 responsiveness to this normally DEAF-1-independent enhancer. The coexpression of DEAF-1 with Dorsal, Dif, and Relish results in the synergistic activation of transcription. It is proposed that DEAF-1 is a regulator of Drosophila immunity (Reed, 2008).

Transcriptional regulation of Drosophila antimicrobial genes depends on Rel and GATA transcription factors. Many immunity genes contain tightly linked Rel- and GATA-binding sites in promoter-proximal regions. GATA sites are important for establishing responses in distinct tissues such as the fat body and midgut. Serpent (dGATAb) is thought to regulate antimicrobial gene expression in the fat body, whereas dGATAe activates such genes in the midgut in response to ingested microbes. In contrast, Dorsal, Dif, and Relish, the NF-kappaB homologues in flies, shuttle between the cytoplasmic and nuclear compartments, acting as 'on/off switches' for induction). Additional factors, such as HOX and POU domain proteins, bind to distal enhancer elements and maintain constitutive domains of gene activity. A regulatory element (R1) also has been described within the CecA1 enhancer, although the factor that interacts with this motif is unknown (Reed, 2008).

Deformed epidermal autoregulatory factor-1 (DEAF-1) is a transcription factor that was originally shown to bind the autoregulatory enhancer of the Deformed (Dfd) Hox gene, which is activated in embryonic head segments of Drosophila (Gross, 1996). DEAF-1 recognizes several TTCG motifs within the portion of the Dfd autoregulatory region termed 'module E.' In addition, DEAF-1 binds several similar motifs within a Dfd response element (DRE) from the 1.28 gene that enhances maxillary gene expression during embryogenesis. The DEAF-1 binding elements identified in these studies are reportedly not required for enhancer activity however (Reed, 2008).

The 576-aa DEAF-1 protein possesses two conserved domains, SAND and MYND. The 113-aa SAND domain (named for SP100, AIRE-1, NucP41/75, and DEAF-1) is responsible for DNA binding via a highly conserved KDWK peptide motif. The 32-aa MYND domain (for myeloid, Nervy, and Deaf-1) contains non-DNA-binding zinc fingers that are thought to mediate protein-protein interactions. DEAF-1 is maternally expressed, and the encoded protein is broadly distributed throughout the early embryo. It exhibits augmented expression in the CNS after stage 14. Zygotic mutants develop to pupal stages, but do not eclose, whereas maternal mutants display severe defects in early embryonic patterning. Overexpression of DEAF-1 by using a maternal driver inhibits germ-band retraction and causes defects in dorsal closure, whereas overexpression at later stages causes cell death (Reed, 2008).

In vertebrates, the closest relatives of DEAF-1 are nuclear DEAF-1-related factor (NUDR) and Suppressin (SPN). Both factors are expressed in a wide variety of tissue types. NUDR functions to either activate transcription depending on its context, and it binds sequences bearing TTCGGG or TTTCCG motifs. SPN does not have a characterized role in transcription. It was originally identified as a protein secreted by the bovine pituitary gland that, when added to tissue culture media, inhibits splenocyte proliferation and stimulates IFN-gamma production in leukocytes (Reed, 2008).

Studies in Drosophila have identified a 208-bp proximal enhancer that regulates the expression of the Mtk gene. This enhancer directs high levels of transcription in the fat bodies of infected larvae and also is induced by LPS preparations in S2 cells. These regulatory activities depend on a cluster of Rel- and GATA-binding sites. This study presents evidence that an additional sequence motif (E8) contributes to Mtk activation. Enhancer DNA affinity chromatography assays and proteomic analysis identified DEAF-1 as a protein that interacts with the E8 motif. DEAF-1 binds to the consensus sequence TTCGGBT, which is contained within the E8 region of the Mtk enhancer. Additional DEAF-1 consensus motifs are found in the regulatory regions of other immunity genes, such as Drosomycin (Drs). Evidence is presented that DEAF-1 works synergistically with Dorsal, Dif, and Relish to induce gene expression in response to LPS. It is proposed that DEAF-1 is an essential component of the immune response in Drosophila (Reed, 2008).

To identify regulatory motifs within the minimal 208-bp Mtk enhancer, the nucleotide sequences of 11 regions (E1-E11) flanking the previously identified Rel- and GATA-binding sites were scrambled. Several of these scrambled elements (SEs) were found to alter the activities of Mtk-Luciferase (Mtk-Luc) promoter-reporter fusion genes in transient transfection assays with S2 cells in the presence of LPS. It should be emphasized that this assay does not measure a response to LPS, but rather to Gram-negative peptidoglycans that commonly occur in commercial preparations of LPS. Gram-negative peptidoglycans have been shown to signal through the Imd and, to a lesser degree, the Toll pathway, so this assay probably reflects both types of signaling cascades. Fusion genes bearing scrambled sequences in region 4, 5, or 6 are roughly twice as active as the native enhancer, suggesting a disruption of potential repressor elements. A 50% reduction in induction is seen for the fusion gene containing scrambled sequences in region 9, suggesting the loss of a weak activator element. Most notably, there is a severe 21-fold decrease in the induced expression of the Mtk-Luc fusion gene containing scrambled sequences in region 8 (SE8). There also is a 3-fold reduction in the constitutive activities of this fusion gene. Thus, region 8 appears to contain an essential activator element (Reed, 2008).

To test the activities of the E8 sequence in vivo, transgenic larvae carrying an Mtk SE8-LacZ transgene were examined. Upon septic injury with a mixture of Escherichia coli and Micrococcus luteus, the wild-type Mtk-lacZ transgene drives intense lacZ expression in the fat body. Mutation of E8 essentially abolishes reporter gene expression in three of four independent lines and allows only a weak response in the fourth. The wild-type Mtk-lacZ fusion gene is constitutively active in the posterior proventriculus of most larvae and in the anterior midgut of ?20% of the larvae. Upon ingestion of Erwinia carotovora, there is at least a doubling in the number of larvae that exhibit expression in the anterior midgut. In contrast, the Mtk SE8-LacZ transgene completely lacks both constitutive and induced activity throughout the midgut in three of four transgenic lines, with a weak response in the fourth line (Reed, 2008).

A proteomics approach was used to identify proteins that bind E8. The entire Mtk regulatory domain (WT and SE8) was biotinylated and coupled to magnetic Dynal beads. An EcoRI restriction site was included proximal to the biotin moiety. Dynal bead-DNA complexes were incubated with nuclear extracts from S2 cells that had been treated with LPS. The resulting nucleoprotein complexes were washed extensively and eluted with a brief EcoRI digest. Eluted proteins were then subjected to Multidimensional Protein Identification Technology (MudPIT) analysis (Reed, 2008).

MudPIT identified several candidate proteins that were uniquely present in the eluate from the native Mtk regulatory sequence and not from the SE8 mutant enhancer sequence. One of these, DEAF-1, was particularly interesting because it recognizes a sequence motif, TTCG, which resembles the E8 sequence (TCATTCGGC). This led to a pursuit of the role of DEAF-1 in regulating Mtk expression (Reed, 2008).

To test whether DEAF-1 recognizes Mtk regulatory sequences, gel shift assays were performed with increasing amounts of recombinant DEAF-1 protein and radiolabeled E8 and SE8 oligonucleotides. The DEAF-1 protein binds to E8, but not the SE8 scrambled sequence. Competition assays were done by incubating DEAF-1 with the radiolabeled E8 sequence, followed by the addition of an excess of unlabeled E8 or SE8 oligonucleotides. A 10-fold excess of unlabeled E8 removes DEAF-1 from the radioactive probe, whereas the same amount of the SE8 oligonucleotide only weakly disrupts binding (Reed, 2008).

The previously published DEAF-1-binding site, TTCG, is based on footprint assays using the Dfd autoregulatory enhancer and the 1.28 gene enhancer. This analysis was extended by performing systematic evolution of ligands by exponential (SELEX) enrichment experiments. Recombinant His-tagged DEAF-1 protein was incubated with a random library of radiolabeled oligonucleotides. Protein-DNA complexes were gel-purified and PCR-amplified, and the selected DNA was subjected to two additional rounds of binding and amplification. The DNAs were sequenced and aligned with the published footprint data to generate a position-weighted matrix. The broadest consensus sequence using this approach is TTCGGBT. The SELEX data show a weaker preference for cytosine at position 3 than the previous footprinting data and a stronger selection for guanine at position 5. This finding may reflect differences in the binding of DEAF-1 to individual sites, compared with the clustered sites seen in the Dfd enhancer (Reed, 2008).

The strongest selection by DEAF-1 occurs at positions 2-5 (TCGG). The importance of each position was verified by performing gel shift competition assays. DEAF-1-E8 complexes were incubated with unlabeled oligonucleotides bearing mutations at each position along the consensus binding sequence. Oligonucleotides bearing mutations at position 1, 6, or 7 (TTCGGBT) successfully competed with radiolabeled E8 for DEAF-1 binding, suggesting that they contain an intact core-binding sequence. In contrast, mutations at positions 2-5 (TTCGGBT) greatly impaired competitive binding of the modified oligonucleotides. Hence, strong DEAF-1-binding sites appear to contain a TCGG core sequence (Reed, 2008).

Transient transfection assays were done to investigate the ability of DEAF-1 to activate transcription in S2 cells. DEAF-1 was expressed in S2 cells by placing the DEAF-1 coding sequence under the control of the actin promoter (pMA6-DEAF-1). This DEAF-1 expression vector was cotransfected with an Mtk-Luc reporter construct in S2 cells. The fusion gene is normally induced 12-fold upon addition of LPS to the culture medium. The addition of pMA6-DEAF-1 causes a 5-fold increase in the basal expression of the Mtk-Luc reporter gene and a 28-fold increase upon addition of LPS. In contrast, an Mtk-Luc reporter construct bearing the scrambled E8 sequence (Mtk SE8-Luc) did not respond to expression of DEAF-1 (Reed, 2008).

Other immunity genes were surveyed for sequences that conformed to the DEAF-1-binding consensus. The 746-bp 5' enhancer of Drs contains five potential DEAF-1-binding sites (E8.1-E8.5). Each site binds DEAF-1 with a different affinity; sites E8.3 and E8.4 bind particularly well. The five recognition sequences were scrambled in the context of an otherwise normal Drs enhancer and analyzed in S2 cells by using a Drs-Luc reporter construct. A Drs-Luc reporter containing wild-type sequences is induced ~2-fold upon LPS addition. Cotransfection with the pMA6-DEAF-1 expression vector causes an additional 12-fold increase in Drs-Luc reporter gene expression in the absence of LPS and a 17-fold increase with LPS. Mutations in individual binding sites reduce both basal and activated transcription. Mutation of site 8.3 causes the most dramatic reduction in activity (Reed, 2008).

The 201-bp Diptericin enhancer mediates strong expression in the fat bodies of infected larvae, but is only weakly induced in S2 cells (~2-fold). The enhancer likely lacks DEAF-1-binding sites based on DNA sequence analysis, and a Dpt-Luc reporter construct responds only weakly to transfected DEAF-1 in S2 cells. To determine whether insertion of a DEAF-1 consensus binding site can confer an ability to respond to DEAF-1 expression, a single, optimal DEAF-1 site was placed 10 bp upstream of the endogenous GATA motif, either in the forward or reverse orientation. Both constructs exhibit a significant increase in luciferase expression when coexpressed with a DEAF-1 expression construct, compared with the unmodified reporter fusion gene. In contrast, insertion of the scrambled E8 (SE8) sequence led to only a modest increase in reporter gene expression (Reed, 2008).

Mutation of all three Rel sites or all three GATA sites within the Mtk enhancer causes a complete loss of induced activity in S2 cells. Thus, Rel and GATA factors function synergistically to activate immunity gene expression. The presence of DEAF-1-binding sites near the Rel and GATA sites suggests that it may cooperate with these factors during the mounting of an immune response. To test this, pMA6-DEAF-1 were cotransfected with expression vectors for Dorsal, Dif, and Relish. Both Mtk-Luc and Drs-Luc fusion genes were used as reporters to monitor the combinatorial activities of these transcription factors (Reed, 2008).

The Mtk-Luc reporter gene is induced 12-fold with LPS. Addition of pMA6-DEAF-1 raised the basal activity 5-fold and boosts the response to added LPS ~30-fold. Separate transfections with individual Dorsal, Dif, and Relish expression vectors also result in substantial activation (up to 67-fold for Dorsal, 166-fold for Dif, and 350-fold for Relish with LPS). Cotransfection of pMA6-DEAF-1 with each Rel factor results in some degree of synergy. The level of synergy is calculated by dividing the activity of two factors working together by the sum of their individual activities. The average fold synergy with DEAF-1 on the Mtk-Luc reporter is 1.6-fold with Dorsal, 2.2-fold with Dif, and 1.7-fold with Relish (Reed, 2008).

Similar results were obtained by using the Drs-Luc reporter, which is induced an average of 2.8-fold upon addition of LPS in S2 cells. The addition of pMA6-DEAF-1 raises basal activity 6-fold and LPS induction 10-fold. Separate transfections of either Dorsal or Dif dramatically activate this reporter (up to 60-fold with LPS), whereas Relish appears to be a much weaker activator (7-fold with LPS). Cotransfection of pMA6-DEAF-1 with each Rel factor results in an average of 3-fold synergy with Dif and 1.9-fold synergy with Dorsal. Only additive effects (average 1.2-fold synergy) were obtained with Relish. Altogether, these results suggest that DEAF-1 differentially augments the activities of different Rel factors during the induction of immunity genes. Particularly strong synergy is seen between DEAF-1 and Dif (Reed, 2008).

In summary, DEAF-1 is an essential component of the immune response in both Drosophila larvae and S2 cells. It appears to augment the synergistic activities of Rel and GATA transcription factors during the immune response. In so doing, it provides a signal amplification mechanism so that certain innate immunity genes, such as Mtk, can be transcribed at particularly high levels while using the same signaling pathways as other less highly expressed immunity genes. The critical E8 motif, TTCGGCT, is highly conserved among the Mtk enhancers of divergent Drosophilids and is closely linked to a paired set of Rel and GATA sites. It is therefore conceivable that DEAF-1 facilitates the binding or transcriptional efficacy of Rel and GATA factors at linked sites (Reed, 2008).

The requirement for DEAF-1 in the regulation of Drs, but not Dpt, hints at a possible function for DEAF-1 in Toll signaling. Cotransfection experiments using Mtk-Luc and Drs-Luc reporter genes demonstrate that DEAF-1 synergizes with Dorsal and especially Dif, two effectors of Toll signaling. Only weak cooperation occurs between DEAF-1 and Relish, a target of the Imd pathway. Microarray studies of flies mutant for Toll and Imd pathway components have comprehensively identified groups of genes coregulated by each pathway. Interestingly, several genes that require Toll signaling for regulation, such as Cactus, IM1, and Dif, contain one perfect and several near-perfect consensus DEAF-1-binding sites within 1 kb of the transcription start sites. Future studies should determine whether DEAF-1 is a constitutive component of immune tissues like the GATA factors or is regulated in response to infection by Toll signaling as seen for the Rel factors (Reed, 2008).

A serpin that regulates immune melanization in the respiratory system of Drosophila

Epithelial tissues facing the external environment are essential to combating microbial infection. In addition to providing a physical barrier, epithelial tissues mount chemical defenses to prevent invasion of internal tissues by pathogens. The melanization reaction implicated in host defense is activated in the respiratory system, the trachea, of Drosophila. Tracheal melanization can be activated by the presence of microorganisms but is normally blocked by Spn77Ba, a protease inhibitor in the serpin family. Spn77Ba inhibits a protease cascade involving the MP1 and MP2 proteases that activates phenol oxidase, a key enzyme in melanin biosynthesis. Unexpectedly, it was found that tracheal melanization resulting from Spn77Ba disruption induces systemic expression of the antifungal peptide Drosomycin via the Toll pathway. Such signaling between local and systemic immune responses could represent an alarm mechanism that prepares the host in case a pathogen breaches epithelial defenses to invade internal tissues (Tang, 2008).

Innate immunity is an ancient system of host defense against pathogens used by a wide range of organisms. In humans, innate immunity is the front line of host defense that acts before the adaptive immune system. Whereas the adaptive immune system can be programmed to recognize an almost infinite number of specific antigens, the innate immune system is hardwired to recognize a limited number of common determinants found in infectious agents. Nonetheless, the innate immune system involves a sophisticated repertoire of humoral and cellular responses, acting at local as well as systemic levels, which together provide an effective barrier to infection by pathogens. In recent years, many fundamental mechanisms and principles of innate immunity have been revealed from studies with the fruit fly Drosophila (Tang, 2008).

A key innate immune mechanism found in both humans and Drosophila involves signaling by receptors in the Toll family. In humans, upon microbial infection, Toll-like receptors (TLRs) activate the synthesis of cytokines as well as other molecules that stimulate induction of the adaptive immune system. In Drosophila, Toll activates the synthesis of antimicrobial peptides (AMPs) by the fat body, the functional equivalent of human liver and adipose tissue. Since AMPs synthesized by the fat body are secreted into the blood-like hemolymph which bathe all internal tissues and organs, they provide systemic protection against pathogens that may invade the body cavity. In humans, pathogens are directly sensed by TLRs, but in Drosophila, they are detected by circulating pattern recognition receptors (PRRs) that trigger protease cascades, which lead to cleavage of the Spätzle protein to generate the ligand that activates Toll signaling. In Drosophila, the Toll pathway is mainly responsive to infection by Gram-positive bacteria and fungi, whereas the Imd pathway activates AMP synthesis in response to infection by Gram-negative bacteria. Both pathways, as well as human TLRs, trigger immune gene expression by activating a transcription factor in the NF-κB family (Tang, 2008).

Another major immune response in Drosophila, and more generally in insects and other arthropods, is the melanization reaction. Melanization involves the rapid synthesis of melanin at the site of infection and injury in order to contain a microbial pathogen as well as to facilitate wound healing. A key enzyme in melanin biosynthesis is phenol oxidase (PO), which catalyzes the oxidation of phenols to quinones that then polymerize into melanin. A by-product of PO activity is reactive oxygen species (ROS), a potent antimicrobial agent. PO is synthesized as an inactive zymogen called proPO (PPO), which is released by crystal cells that circulate in the hemolymph and cleaved to generate active PO at the end of a protease cascade. The proteases in this cascade also exist as inactive zymogens in the hemolymph, analogous to components of the complement pathway found in human blood and tissues. Thus, like the human complement pathway, the melanization reaction functions as a component of the humoral immune response to contain pathogens that invade internal body spaces (Tang, 2008 and references therein).

As illustrated by the Toll pathway and the melanization reaction in Drosophila but also by the complement pathway in humans, a recurring theme in innate immunity is the use of protease cascades to activate immune responses. Such protease cascades, because of their potential for enormous signal amplification, are advantageous for detecting minute levels of pathogens. However, tight regulation must exist to prevent their activation under normal conditions and to localize their action both temporally and spatially. Indeed, C1 inhibitor, a member of the serpin family of protease inhibitors, is an essential regulator of the complement pathway. Moreover, genetic studies in Drosophila have defined important roles for serpins in inhibiting the protease cascades that activate Toll and the melanization reaction (Tang, 2008 and refereces therein).

Epithelial tissues exposed to microorganisms in the environment play an important role in innate immunity. In addition to providing a physical barrier, such epithelia secrete chemical defenses to prevent pathogen penetration of internal tissues. Recent studies have shown that epithelia in Drosophila, as in humans, mount local immune responses. In particular, the respiratory and reproductive tracts express the antifungal peptide Drosomycin (Drs) in response to contact with pathogens, but interestingly, this response depends on the Imd pathway, not the Toll pathway utilized by the fat body during systemic antimicrobial responses. Moreover, reactive oxygen species generated by a dual oxidase are essential for the antimicrobial activity of the gastrointestinal tract (Tang, 2008 and references therein).

The melanization reaction can be activated in the trachea, the respiratory system, of Drosophila. In an RNAi screen to investigate the function of serpins in Drosophila, it was discovered that the serpin Spn77Ba is a trachea-specific inhibitor of melanization. As in other tissues, melanization of the trachea is strongly enhanced by the presence of microorganisms. Surprisingly, however, tracheal melanization induces a systemic response (Drs expression in the fat body) via the Toll pathway, which may represent a mechanism for alerting and preparing the host for possible infection of internal tissues. These data suggest that melanization is an intrinsic immune response of respiratory epithelium under tight negative control by a serpin (Tang, 2008).

Transgenic flies were generated bearing an inverted repeat construct of serpin Spn77Ba under control of the UAS element recognized by the Gal4 transcriptional activator (UAS-Spn77Ba-IR). These flies were crossed to Gal4 'driver' flies in order to activate RNAi of Spn77Ba in the progeny. When the act-Gal4 driver was used to activate Spn77Ba RNAi in virtually all tissues during development, the result was a striking phenotype in larva: melanization of the tracheal system, the respiratory organ. Tracheal melanization was first visible during the second or third instar larval stage and increased in intensity until the affected larva died within a few days before reaching the pupal stage. This phenotype was observed in experiments with two different inverted repeat constructs targeting different regions of the Spn77Ba gene and with multiple independently generated transgenic lines for each construct. By quantitative RT-PCR analysis, it was estimated that RNAi was able to knock down Spn77Ba expression to 20%-30% of the wild-type level. These results suggest that Spn77Ba is required for inhibiting melanization in the tracheal system and for larval viability (Tang, 2008).

A spn77Ba null mutation, l(3)77ABi[W7], was identified that causes early lethality, which was rescued by overexpression of the Spn77Ba protein using the act-Gal4 driver. To bypass this early essential requirement for Spn77Ba function, Spn77Ba was overexpressed in the l(3)77ABi[W7] mutant background, using the hs-Gal4 driver. At 18°C, leaky expression of Spn77Ba was sufficient for some larvae to grow to the second instar stage, and these survivors had melanized trachea and in most cases died before reaching the third instar stage. At 22°C-25°C, some larvae survived to third instar and even grew to pupae. These larvae and pupae also had melanized trachea, which was similar to that induced by Spn77Ba RNAi. These results confirm that l(3)77ABi[W7] is a mutation of the spn77Ba gene and that the phenotype induced by RNAi is due to specific knockdown of the spn77Ba gene. They also suggest that low level of Spn77Ba activity, such as left by RNAi, was sufficient to fulfill Spn77Ba's essential function during early development, but not its role in inhibiting tracheal melanization during the larval stage (Tang, 2008).

Spn77Ba regulates tracheal melanization by inhibiting a protease cascade that activates phenol oxidase, a key enzyme in melanin biosynthesis. This protease cascade involves the proteases MP1 and MP2, which previously were shown to regulate the melanization reaction in the hemolymph where it is inhibited by the serpin Spn27A (Tang, 2006). While hemolymph melanization involves MP1 and MP2 made by fat body cells and/or hemocytes, tracheal melanization involves the same proteases locally synthesized by tracheal cells. Spn77Ba is required, as well as expressed, in tracheal cells to prevent tracheal melanization. A previous study showed that the cuticular PO involved in host defense and wound healing in the silkworm is transported from the hemolymph. In contrast, the current results obtained with the Bc1 mutation indicate that tracheal melanization is not mediated by hemolymph PO. In preliminary experiments, tracheal-specific RNAi of each of several PO genes (CG2952, CG5779, CG8193, CG10484) did not suppress tracheal melanization resulting from Spn77Ba RNAi, perhaps indicating that multiple PO genes are responsible for tracheal melanization. Interestingly, the RelE20 mutation in Relish, a transcription factor in the Imd pathway, partially suppressed tracheal melanization and overall Drs expression resulting from Spn77Ba RNAi, which suggests that Rel could be important for the expression of a component required for tracheal melanization (Tang, 2008).

Tracheal melanization is inducible by microorganisms. Thus, tracheal melanization may be a local immune response analogous to the local synthesis of reactive oxygen species and antimicrobial peptides by epithelia facing the external environment. Such a role is consistent with the observation that Spn77Ba is localized in the extracellular space facing the tracheal lumen. Melanization could be used to encapsulate pathogens killed by these other local immune responses, and reactive oxygen species generated during the melanization reaction may aid in killing pathogens as well. Melanization could also create a physical barrier to impede pathogen penetration of the tracheal epithelium, as in the case where melanin is deposited at the site of entry in defense against entomopathogenic fungi that infect by crossing the insect cuticle. Spn77Ba overexpression or MP1 RNAi did not make larvae more susceptible to natural B. bassiana infection. However, testing of more pathogens will be needed to determine the importance of melanization in combating infection (Tang, 2008).

An issue that remains to be addressed is the mechanism by which microorganisms activate the melanization cascade in the tracheal system. Overexpression of PGRP-LE, a pattern recognition receptor (PRR) expressed in tracheal epithelia, does not lead to constitutive tracheal melanization, suggesting that this PRR is not a tracheal-specific activator of melanization. Interestingly, under axenic conditions, Spn77Ba RNAi flies still showed tracheal melanization, although at a lower strength than seen under normal conditions or in the presence of bacteria. This low level of melanization could reflect activation of the melanization cascade by dead microorganisms present in the culture food medium. Alternatively, even in the absence of microbial infection, the target protease of Spn77Ba could exist in the tracheal system already activated rather than entirely as inactive zymogen, but is prevented from activating the whole cascade and thus melanization by the presence of Spn77Ba. In this case, microbial infection could trigger tracheal melanization by either reducing the level of Spn77Ba or increasing the amount of activated target protease. Defining the actual mechanism may require identifying a natural pathogen that strongly induces tracheal melanization (Tang, 2008).

Regardless of how it is activated, tracheal melanization appears to play a key intermediary role in inducing systemic expression of the antifungal peptide gene Drosomycin (Drs). It had previously been reported that melanotic 'tumors' resulting from melanization of aberrant host tissue are associated with systemic Drs expression, but the mechanism linking melanization and Drs expression has been obscure. The data suggest that melanization induces Drs expression, rather than the two immune responses being triggered in parallel, and that a product of the melanization reaction is involved in inducing Drs expression. However, as melanization does not necessarily activate Drs expression in all other contexts, this product may not be melanin itself but rather a melanin metabolite or a secondary signal induced when melanization is activated in the trachea. Indeed, the mechanical and ROS-related insults resulting from tracheal melanization may themselves induce a stress response that contributes to the systemic induction of Drs. In any case, it appears that hemocytes are not involved in transmitting signals that connect tracheal melanization to fat body Drs expression, since l(3)hem larvae lacking hemocytes retain the systemic response (Tang, 2008).

Whatever the molecular nature of this product, it is presumably a diffusible molecule that can pass through the tracheal system basement membrane into the hemolymph, where it activates the Toll pathway to induce Drs synthesis by the fat body. Moreover, it apparently acts upstream or at the level of the Persephone (Psh) protease involved in cleaving Spätzle to generate the Toll ligand. Interestingly, it was recently shown that Psh, which specifically functions to activate Toll in response to fungal infection, is proteolytically activated by a secreted fungal protease and thus acts as a direct sensor of this virulence factor. These data suggest that local melanization is involved in inducing Drs expression after fungal infection and that Psh can be activated by an alternative mechanism in which a host factor arising from melanization triggers Psh activation, which could be analogous to a danger signal from damaged tissue that activates the immune system. It is therefore speculated that induction of Drs synthesis by tracheal melanization represents signaling between local and systemic immune responses that alerts and prepares the host for potential invasion of internal tissues by pathogens such as entomopathogenic fungi. Such an alarm system could be advantageous for organisms in which pathogens are naturally first encountered at epithelial surfaces (Tang, 2008).

FOXO-dependent regulation of innate immune homeostasis

The innate immune system represents an ancient host defence mechanism that protects against invading microorganisms. An important class of immune effector molecules to fight pathogen infections are antimicrobial peptides (AMPs) that are produced in plants and animals. In Drosophila, the induction of AMPs in response to infection is regulated through the activation of the evolutionarily conserved Toll and immune deficiency (IMD) pathways. This study shows that AMP activation can be achieved independently of these immunoregulatory pathways by the transcription factor FOXO, a key regulator of stress resistance, metabolism and ageing. In non-infected animals, AMP genes are activated in response to nuclear FOXO activity when induced by starvation, using insulin signalling mutants, or by applying small molecule inhibitors. AMP induction is lost in foxo null mutants but enhanced when FOXO is overexpressed. Expression of AMP genes in response to FOXO activity can also be triggered in animals unable to respond to immune challenges due to defects in both the Toll and IMD pathways. Molecular experiments at the Drosomycin promoter indicate that FOXO directly binds to its regulatory region, thereby inducing its transcription. In vivo studies in Drosophila, but also studies in human lung, gut, kidney and skin cells indicate that a FOXO-dependent regulation of AMPs is evolutionarily conserved. These results indicate a new mechanism of cross-regulation of metabolism and innate immunity by which AMP genes can be activated under normal physiological conditions in response to the oscillating energy status of cells and tissues. This regulation seems to be independent of the pathogen-responsive innate immunity pathways whose activation is often associated with tissue damage and repair. The sparse production of AMPs in epithelial tissues in response to FOXO may help modulating the defence reaction without harming the host tissues, in particular when animals are suffering from energy shortage or stress (Becker, 2009).

In Drosophila, eight families of AMPs have been described. During infection, their induction depends on members of the nuclear factor-κB (NF-κB) family of inducible transactivators. They are regulated by two distinct signalling cascades, the Toll and IMD pathways, which are highly conserved in evolution and show strong parallels to the mammalian Toll-like receptors (TLR) and tumour necrosis factor receptor pathways. Under non-infected conditions, AMPs are synthesized constitutively in specific tissues, in particular in barrier epithelia in both insects and mammals where they probably confer a first line of defence against opportunistic and pathogenic bacteria. The regulatory mechanisms promoting constitutive and inducible AMP expression in barrier epithelia in combination with systemically induced gene expression are not well understood (Becker, 2009).

The cytohesin Steppke was recently identified as an essential component of insulin/insulin-like growth factor signalling (ILS) in both Drosophila (Fuss, 2006) and mice (Hafner, 2006). While studying the phenotype of steppke (step) mutant larvae, a significant upregulation of different AMP genes was observed in non-infected animals. Other mutants of the ILS cascade, including the Drosophila insulin receptor substrate homologue chico, likewise showed an upregulation of AMP genes in non-infected conditions. Because it is known that AMP transcription is highly sensitive to variations in the developmental stage, allelic combinations of step and chico mutants were carefully compared with wild-type controls at different stages and it was determined that the effects were not due to developmental delay of the ILS mutants. To exclude developmental effects further, AMP regulation in the adult stage was examined. Feeding adult wild-type flies for 8-10 days with SecinH3, which was previously shown to result in the downregulation of ILS, resulted in an induction of AMP levels (Becker, 2009).

Starvation is a stress situation known to phenocopy the growth defects of genetic ILS mutants and reflects the physiological condition of an animal searching for food. In a series of nutrient deprivation experiments, it was found that AMP expression was strongly induced in wild-type larvae after starvation. These data were further corroborated by recent gene expression studies showing that AMP genes are induced upon starvation in adult flies (Bauer, 2006). AMP gene expression could also be induced in Drosophila S2 cells after starvation in PBS or by growth factor removal. Together, these results indicate that reduction of ILS by pathway-specific mutation, feeding chemical inhibitors or by starvation results in AMP activation in tissue culture cells, larvae and adult flies (Becker, 2009).

The signal transducer of the ILS cascade, the forkhead transcription factor FOXO, has a pivotal role in adapting metabolism to nutrient conditions. When energy levels are low and ILS is reduced, FOXO enters the nucleus, resulting in enhanced target gene expression. To test whether the observed activation of AMP expression was dependent on FOXO activity, foxo21/W24 null mutants were analysed. It was found that AMP upregulation was lost in foxo mutant larvae after starvation and in adults after SecinH3 feeding. In control experiments, lipase 3 was shown to be properly regulated in starving foxo mutants, demonstrating that a FOXO-independent response to nutrient deprivation is still functional. In contrast, overexpression of FOXOTM, a dominant active form of the protein expressed in the nucleus, resulted in a strong induction of AMP expression in both normal fed larvae and adults. Together, the loss- and gain-of-function data demonstrate a prominent role of nuclear FOXO in the activation of AMP genes (Becker, 2009).

Sequence analysis of the regulatory regions identified highly conserved FOXO/forkhead consensus binding sites in most of the AMP genes, especially in the Drosomycin (Drs) promoter. Therefore, focus was placed on Drs. In situ hybridization showed that Drs is strongly upregulated in wild-type larvae after starvation, but not in foxo mutants, indicating that regulation of Drs is FOXO-specific. A fragment of the Drs promoter, which reproduces the endogenous Drs expression in vivo, contains a cluster of five putative FOXO binding sites and in addition a NF-κB site. This regulatory region was used to drive luciferase expression, and an upregulation of reporter gene activity and endogenous Drs expression was found when FOXO-GFP was overexpressed in S2 cells. In contrast, luciferase expression was lost in this assay when the cluster of FOXO binding sites, but not the NF-κB binding site, was deleted. The same response of luciferase expression was found in transgenic larvae after starvation as well as FOXOTM overexpression, further corroborating the functionality of these FOXO binding sites in vivo. Moreover, electrophoretic mobility band shift and supershift assays (EMSA) showed that FOXO binds directly to the FOXO sites in the Drs promoter. To test the in vivo significance of these FOXO sites further, each binding site or combinations of them was mutated. It was found that mutating the site at position -990 already strongly reduced FOXO-GFP-dependent luciferase expression, whereas it was entirely lost when four of five sites were mutated. Similar results were obtained by using just the isolated cluster of putative FOXO binding sites to drive luciferase expression, either containing wild-type or mutated sites. Together, these data provide strong evidence for an essential role of FOXO for Drs regulation (Becker, 2009).

These results show a direct regulation of AMP genes by FOXO that is dependent on the energy status of the cells. To exclude that this effect depends on a cross-regulation triggering the immunity pathways, double mutants for Relish (Rel) and spätzle (spz) were used in which both the IMD and Toll pathways are defective. It has been shown that NF-κB responses after bacterial challenge fail in these mutants. When nuclear FOXO activity was induced in Rel,spz mutant larvae by starvation, an upregulation of Drs expression was detectable. Similarly, feeding SecinH3 to adult Rel,spz double mutants resulted in an upregulation of different AMPs (<2">Fig. 2k). Together, these data show that FOXO can activate AMP expression independently of the NF-κB-derived innate immune pathways. To test whether the IMD and Toll pathways require FOXO to operate, adult foxo mutants were infected and it was found that NF-κB-derived AMP expression was strongly induced, enabling survival of the animals. This further indicates that the NF-κB pathways are operating independently of FOXO in activating AMP expression and that NF-κB signalling and not FOXO seems to be essential after an acute infection (Becker, 2009).

To obtain further insight into where the FOXO-dependent mechanism of AMP induction is operating, tissues of wild-type larvae were isolated that were either yeast fed or starved, and the expression of AMPs was analyzed. Focus was placed on barrier tissues, in which AMPs are known to be expressed in both insects and mammals, and on the fat body, which is a major site for the regulation of both energy homeostasis and systemic innate immunity in insects. All tissues showed a starvation-dependent upregulation of multiple AMPs. In agreement with this, enhanced Drs promoter activity was found in several tissues, most prominently in trachea and fat body after starvation of flies carrying a Drs-GFP reporter transgene. When FOXO-GFP was overexpressed in a mosaic pattern in the fat body using the FLP/GAL4 technique, it was found by using in situ hybridization that the FOXO-GFP-expressing cells showed a strong upregulation of Drs expression and a reduced cell size as compared to neighbouring cells not expressing FOXO-GFP. These data demonstrate that the FOXO-dependent mechanism of AMP gene regulation functions cell-autonomously (Becker, 2009).

Because FOXO is conserved from worms to humans, tests were performed to see whether a FOXO-dependent regulation of AMPs may also occur in mammalian cells. The regulation of defensin β-1 (also known as DEFB1), defensin β-3 (DEFB103A), defensin α-1 (DEFA1) and drosomycin-like defensin was examined in several human cell lines. In all these cell types, expression of AMPs was ILS-dependent, as already observed in Drosophila. This indicates that the FOXO-dependent regulation of AMP genes is conserved (Becker, 2009).

These results demonstrate a new mechanism of cross-regulation between metabolism and innate immunity, in which FOXO directly regulates AMP genes in non-infected animals in response to the oscillating energy and stress status of cells. This regulation is independent of the pathogen-responsive innate immunity pathways and seems to be evolutionarily conserved in mammals. In the course of an infection, high levels of AMPs are induced by the NF-κB-dependent immunity pathways to ensure bacterial clearance. However, AMP induction is normally downregulated within several hours, as continuous upregulation is detrimental to the host tissues. In the Drosophila gut epithelium, the induction of NF-κB-dependent AMP expression is repressed by the intestinal homeobox gene caudal, thereby regulating symbiotic interactions of commensal bacteria with the intestinal epithelium. Similarly, TLR signalling is downregulated in the mammalian intestine to avoid chronic inflammation in response to constant exposure of the host cells to microorganisms. The FOXO-dependent mechanism ensures the sparse production of AMPs, which may help maintaining and strengthening the defence barrier, in particular when animals are suffering from energy shortage or stress. This would be consistent with previous studies reporting that genetic mutants of the ILS pathway, which have an extended life span, such as chico mutants in Drosophila or daf-2, age-1 and akt-1 mutants in Caenorhabditis elegans, show enhanced pathogen resistance. It is also conceivable that interactions of the host with severe pathogens can be independent of FOXO-dependent immune and barrier functions. Along these lines it is therefore not surprising that foxo mutants can be more resistant to some infections, as exemplified by Mycobacterium marinum, a model organism for tuberculosis disease. Taken together, these data reveal a new evolutionarily conserved mechanism of cross-regulation of metabolism and innate immunity, which allows the adaptation of organismal defence to environmental conditions (Becker, 2009).

The sleeping beauty: How reproductive diapause affects hormone signaling, Metabolism, immune response and somatic maintenance in Drosophila melanogaster

Some organisms can adapt to seasonal and other environmental challenges by entering a state of dormancy, diapause. Thus, insects exposed to decreased temperature and short photoperiod enter a state of arrested development, lowered metabolism, and increased stress resistance. Drosophila melanogaster females can enter a shallow reproductive diapause in the adult stage, which drastically reduces organismal senescence, but little is known about the physiology and endocrinology associated with this dormancy, and the genes involved in its regulation. Diapause was induced in D. melanogaster and effects were monitored over 12 weeks on dynamics of ovary development, carbohydrate and lipid metabolism, as well as expression of genes involved in endocrine signaling, metabolism and innate immunity. During diapause food intake diminishes drastically, but circulating and stored carbohydrates and lipids are elevated. Gene transcripts of glucagon- and insulin-like peptides increase, and expression of several target genes of these peptides also change. Four key genes in innate immunity can be induced by infection in diapausing flies, and two of these, Drosomycin and Cecropin A1, are upregulated by diapause independently of infection. Diapausing flies display very low mortality, extended lifespan and decreased aging of the intestinal epithelium. Many phenotypes induced by diapause are reversed after one week of recovery from diapause conditions. Furthermore, mutant flies lacking specific insulin-like peptides (dilp5 and dilp2-3) display increased diapause incidence. This study provides a first comprehensive characterization of reproductive diapause in D. melanogaster, and evidence that glucagon- and insulin-like signaling are among the key regulators of the altered physiology during this dormancy (Kubrak, 2014: 25393614).


DEVELOPMENTAL BIOLOGY

One of the Drosomycin cDNA clones was used after nick translation to detect by Northern blotting experiments the presence of transcripts in untreated and bacteria-challenged Drosophila. A faint signal is present in naive insects and is noticeably increased in bacteria-challenged larvae, pupae, and adults. In situ hybridization was performed on paraffin-embedded naive and bacteria-challenged larvae and adults using digoxigenin- labeled cDNA probe. A marked reaction was observed in the fat body cells of challenged insects. A fainter, but distinct, reaction was also seen in the fat body in absence of challenge (Fehlbaum, 1994).


EVOLUTIONARY HOMOLOGS

The inducible antifungal peptide of Drosophila is a small sized, slightly cationic molecule (calculated PI = 7.81, which is particularly resistant to heat treatment, to the action of various proteases, and to pH variations. It has four intramolecular disulfide bridges, and is assumed to adopt a very compact tridimensional arrangement. Of major interest is the fact that the Drosophila peptide has a significant sequence homology with plant antifungal peptides recently isolated from seeds of Brassicaceae which play a role in plant defense. Indeed, apart from the already well known plant antifungal proteins (chitinases, glucanases, thionins, chitin-binding lectins, ribosome-inactivating proteins) an increasing number of new plant proteins capable of inhibiting fungal growth in vitro is emerging. In particular, several 5-kDa cationic antifungal peptides which contain disulfide bridges were isolated from seeds (AFPs, antifungal peptides) (Fehlbaum, 1994).

Drosomycin peptide was compared with Rs-AFP2 (from seeds of radish Raphanus satiuus). Both peptides have 8 cysteines which are engaged in intramolecular disulfide bridges. One of these cysteines is positioned C-terminally in both molecules. Allowing for several minor gaps in both sequences, is it apparent that the eight cysteines are arranged in a similar pattern. The overall homology between the Drosophila peptide and Rs-AFP2 is 38%, taking into account conservative replacements. It will be of great interest to work out the tridimensional structure of the Drosophila peptide and compare it to the structure of the plant antifungal peptides in the future. It is also interesting to note that the production of Rs-AFP2 and its homologue in radish is not restricted to seeds but occurs in leaves after challenge with fungal pathogens (Fehlbaum, 1994).

As is the case for Rs-AFP2, the Drosophila peptide is active against a relatively broad spectrum of filamentous fungi. It inhibits spore germination at high concentrations and, at lower concentrations, delays growth of hyphae which subsequently exhibit abnormal morphology. The IC50 values indicate an exceptionally high potency of the Drosophila peptide. Indeed, for most of the fungi tested, the IC50 values were in the low micromolar ranges and even below in the case of N. crassa. Experiments with the highly sensitive N. crassa point to a fungicidal activity (Fehlbaum, 1994).

Drosomycin is the first strictly antifungal protein isolated from an insect. The solution structure of this 44-residue protein has been reported previously. It involves a three-stranded beta-sheet and an alpha-helix, the protein global fold being maintained by four disulfide bridges. Rs-AFP2 is a plant antifungal protein exhibiting 41% sequence similarity with drosomycin. Mutational analysis of Rs-AFP2 showed the importance of some residues in the antifungal activity of the protein against the fungus target. In order to determine the structural features responsible for antifungal activity in both drosomycin and Rs-AFP2, the three-dimensional structure of Rs-AFP2, and of other antifungal proteins, was modeled using the solution structure of drosomycin as a template. Structure analysis of drosomycin and Rs-AFP2, and comparisons with the other modeled antifungal structures, revealed that the two proteins share a hydrophobic cluster located at the protein surface in which a lysine residue is embedded. Based on these close structural similarities and the experimental data available for Rs-AFP2 mutants, an antifungal active site of the insect protein is proposed (Landon, 2000).

Mutational analysis of Rs-AFP2 has shown the importance of some residues in the antifungal activity of the protein against the fungus target. In order to determine the structural features responsible for antifungal activity in both drosomycin and Rs-AFP2, the three-dimensional structure of Rs-AFP2, and of other antifungal proteins, was modeled using the solution structure of drosomycin as a template. Structure analysis of drosomycin and Rs-AFP2, and comparisons with the other modeled antifungal structures, revealed that the two proteins share a hydrophobic cluster located at the protein surface in which a lysine residue is embedded. Based on these close structural similarities and the experimental data available for Rs-AFP2 mutants, an antifungal active site of the insect protein is proposed (Landon, 2000).

The solution structure, stabilized by four disulfide bridges, involves an α-helix linked to a three-stranded β-sheet and includes the well-known cystein stabilized α-helix β-sheet (CSαβ) motif. The protein super-family containing this CSαβ motif includes the antibacterial insect defensins, scorpion toxins and plant defense proteins. Among the CSαβ proteins, antifungal properties were observed for some plant defense proteins, called 'plant defensins'. Even though the mode of action of these antifungal proteins is not yet understood, two distinct classes have been described according to the effects observed on the fungus at low concentrations. Morphogenic proteins, belonging to the first class, reduce hyphal elongation with a concomitant increase in hyphal branching. Non-morphogenic proteins, belonging to the second class, slow hyphal branching but do not induce marked morphological distorsions. The insect antifungal drosomycin causes delayed growth of hyphae with abnormal morphology and is then comparable with plant defensins of the first class (Landon, 2000).

The three-dimensional structure of a plant defensin, Rs-AFP1 extracted from the radish Raphanus sativus revealed very close structural similarities with the solution structure of drosomycin. Mutational analysis of Rs-AFP2, the primary structure of which differs only by two residues from Rs-AFP1 and which exhibits a more potent fungicidal activity, led to a proposal of possible sites for the interaction between this protein and the fungus target. In the absence of a three-dimensional structure for Rs-AFP2 and in order to establish a structure-activity relationship for drosomycin, comparative modeling was performed of the plant protein structure using the insect protein solution structures as template. Taking into account amino acid conservation between drosomycin and plant antifungal proteins and Rs-AFP2 mutation analysis results, a site of interaction between drosomycin and the putative target in fungal hyphae is proposed (Landon, 2000).

Most insects from temperate regions have evolved hibernal diapause to survive seasonal periods of adversity. Diapause-associated gene expression was studied in Drosophila triauraria using subtractive hybridization. Two genes that were shown to be upregulated in diapausing flies by Northern hybridization have similarity to genes encoding antifungal peptides of Drosophila melanogaster, members of the drosomycin family (drosomycin, CG10812, CG10813, CG10815 and CG11520). In addition, a signal peptide and Knot 1 domain are shared with them. The genes cloned from D. triauraria were tentatively named drosomycin-like. However, the similarities between drosomycin-like in D. triauraria and the members of the drosomycin family in D. melanogaster are quite lower than those between other homologous genes in these species. In addition, neighbor-joining analysis revealed that drosomycin-like in D. triauraria is not closely related to known members of the family in D. melanogaster. Thus, it is most plausible that drosomycin-like is not a D. triauraria counterpart of known members of the family, but a novel member belonging to the family. The drosomycin-like gene is expected to have a few copies, because at least two sequences having unique 3′-ends were obtained in RACE, and multiple bands were observed in Southern hybridization. However, these sequences from RACE had the same ORF. Probes for genes encoding additional antimicrobial peptides were used to evaluate expression during diapause. Like drosomycin-like, drosomycin was upregulated during diapause, but defensin and drosocin were not (Daibo, 2001).

Drosomycin (Drs) gene encodes a 44-residue inducible antifungal peptide, Drosomycin, in Drosophila. Six genes, Drs-lC, Drs-lD, Drs-lE, Drs-lF, Drs-lG and Drs-lI, show homology to the Drs form in a multigene family on the 3rd chromosome of Drosophila. This study is the first experimental demonstration that the six members in the Drs family act as functional genes. To further delineate the functional divergence of these six members, their cDNA sequences were cloned respectively into the pET-3C vector and expressed in the E. coli. The antifungal activity of the expression products was assayed. The results showed a difference among the six isoforms in antifungal activity against the tested fungal strains: Drs was most effective and showed antifungal activity to all seven fungal strains, whereas isoform Drs-lC was effective to six strains, Drs-lD was effective to five strains, Drs-lG was effective to four strains, and Drs-lE and Drs-lF were effective to only three strains. Drs-lI had no activity against any tested fungal strains. By comparing the variable residue sites of these six isoforms to that of Drosomycin in the three-dimensional structure, it is suggested that the reduction in the antifungal activity os due to the variable residues that are not in the α-helix. In addition, two inserted residues (RV) in Drs-lI may affect the dimensional structure and resulted in a functional change. These results may explain the evolution of the Drosomycin multigene family and its functional divergence (Yang, 2006).

The three-dimensional structure of Drosomycin in solution has been determined by Landon (1997). The sequence of 44-residue peptide includes eight cysteine residues engaged in the formation of four internal disulfide bridges: Cys1–Cys8, Cys2–Cys5, Cys3–Cys6 and Cys4–Cys7. The fourth disulfide bridge connects the N-terminal short strand of β-sheet to the C-terminal cysteine residue and forms the βαββ scaffold with three β-sheets and one α-helix. The six isoforms of the Drosomycin family have 12 conservative residues with the Drosomycin. Eight cysteine residues are among these conservative residues. These conservative residues maintain the similar four internal disulfide bridges as in the Drosomycin, therefore the 3-D structure of the isoforms may be also identical to that of Drosomycin. Comparing the variable residues of the isoforms with those of the Drosomycin, most of the variable residues were within the α-helix, whereas only 6 variable residues were in 3 β-sheets area. In the first β-sheet, L (Leu) of Drosomycin changed to P (Pro) of Drs-lF; in the second β-sheet, P of Drosomycin changed to G (Gly) of Drs-lE, A (Ala) of Drs-lF and Drs-lI; S (Ser) of Drosomycin changed to A of Drs-lE and R (Arg) of Drs-lI; L (Leu) of Drosomycin changed to M (Met) of Drs-lE; K (Lys) of Drosomycin changed to Q (Gln) of Drs-lI; in the third β-sheet, G (Gly) of Drosomycin changed to Q (Gln) of Drs-lF. The variable residues in the three β-sheet occur only in Drs-lE, Drs-lF and Drs-lI which showed lower antifungal activity. Drs-lE and Drs-lF were active to only 3 fungi strains, while Drs-lI couldn't inhibit any tested fungi strains. These results suggest that the β-sheet structure is very important for the antifungal activity function and the residue change in the β-sheet might reduce antifungal activity. In addition to the residues change in the β-sheet, one remarkable change in the amino acid sequence divergence of Drs-lI is that two residues, R and V, were inserted between 29th and 30th amino acid of Drosomycin. Whether or not this insertion, which is located in the outside of the β-sheet and α-helix, results in the reduction of the antifungal function is not clear. If this is the case, it might cause the 3-D structure change of Drs-lI. Other variable residues of Drs-E and Drs-F were located outside of the β-sheets, as what happened in Drs-lC, Drs-lD and Drs-lG. These residues are almost inside the α-helix. Although Drs-lC had the most variable residues (12 residues) in α-helix, its antifungal activity was the strongest among the other isoforms except the Drs of the Drs family. This reveals that the residues change in the α-helix may not affect the antifungal function (Yang, 2006).

Three alternative outcomesin the evolution of duplicate genes have been suggested: (1) one copy may simply become silenced by degenerative mutations (nonfunctionalization); (2) one copy may acquire a novel and beneficial function and become preserved by natural selection, with the other copy retaining original function (neo-functionalization); or (3) both copies may become partially compromised by mutation accumulation to the point at which their total capacity is reduced to the level of single copy ancestral gene (subfunctionalization). The function of duplicate genes may be predicted by the bioinformatics approach. The evolution of some antimicrobial peptide multigene family, such as Cecropin, Attacin, Defencin and Drosomycin multigene families, has been well studied, but these approaches lack experimental support. A change of amino acid residues could alter the conformation of a protein, resulting in a change of biological activity. Interestingly, in the Drosomycin family, only a few changes of amino acid residues in the β-sheets cause the loss of antifungal activity. However, changes of amino acid residues in the α-helix were not critical. Each gene of the Drs multigene family expresses after microbial infection, but they showed different expression patterns depending upon the microbial sources. The result of this study demonstrates a functional divergence in the antimicrobial peptides, and provides evidence for predicting crucial functional sites for engineering new antimicrobial reagents (Yang, 2006).

The biological activity of a peptide is related to its three-dimensional structure. The dimensional structure of the expressive AMP gene product might change and lost the biological activity during purification. It happened in these experiments. To refold the three-dimensional structure of a peptide, different conditions were tried to get the highest antifungal activity for each peptide. However, a possibility that there may be a difference in the extent of configurational refolding among seven Drs products could not be excluded. Future work will measure the proportion with the right configuration, and use more fungal strains to test whether or not the Drs-I has lost function or changed function to resist other fungal strains (Yang, 2006).


REFERENCES

Search PubMed for articles about Drosophila Drosomycin

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