dorsal-related immunity factor


REGULATION

Transcriptional Regulation

The Toll10b allele has been shown to encode a mutated form of the Toll receptor that is constitutively activated. This causes Dorsal protein, in both dorsal and ventral regions, to enter nuclei. In Toll10b/+ heterozygotes, only a fraction of the total DIF protein is localized in the nuclei of fat bodies. Toll10b/+ fat bodies display about a 5-fold increase in staining intensity, as compared with normal tissue, with a simular increase in DIF mRNA. This suggests that DIF might influence its own expression (Ip, 1993).

Targets of Activity

The upstream sequences of the Diptericin and Cecropin Al genes, which have been investigated in detail, contain, respectively, two and one sequence elements homologous to the binding site of the mammalian nuclear factor kappaB. These elements are mandatory for the immune-induced transcription of both genes. Functional studies have shown that these kappaB-related elements can be the target for the Drosophila Rel proteins Dorsal and Dif. A comparative analysis of the transactivating capacities of these proteins has been performed on reporter genes fused to either the Diptericin or the Cecropin kappaB-related motifs. The kappaB motifs of the Diptericin and Cecropin genes are not functionally equivalent and complexes formed with kappaB-Dipt and kappaB-Cec motifs have different protein compositions. Dorsal and Dif proteins manifest distinct DNA-binding characteristics. Dif can bind to any kappaB-related motif of either Zerknüllt, Diptericin or Cecropin. A single motif is sufficient for binding; double copies (2x kappaB-Dipt) yield a stronger signal. In contrast, Dorsal, which binds strongly to the motif present in the zen promoter, does not detectably bind to either the kappaB-related motif of the Cecropin or the Diptericin promoter. Dorsal can bind only to the kappaB-Dipt site if this motif is duplicated, as is the case in the native diptericin promoter. Even under these circumstances, the binding is not as marked as for Dif. Mutants containing no copies of Dorsal and a single copy of Dif retain their full capacity to express the Diptericin and Decropin genes in response to challenge (Gross, 1996).

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 Dif (Lemaitre, 1996).

Dif gene product trans-activates the Drosophila Cecropin A1 gene in co-transfection assays. The transactivation requires a 40 bp upstream element, including an insect kappa B-like motif. A dimer of the kappa B-like motif 5'-GGGGATTTTT inserted into a minimal promoter confers high levels of reporter gene expression by Dif; a multimer of several mutated versions of this motif is not activated, demonstrating the sequence specificity of Dif. Full trans-activation by Dif requires the C-terminal part of the protein. The morphogen Dorsal can also activate the Cecropin A1 promoter, but to a lesser extent and in a less sequence-specific manner than Dif. Simultaneous overexpression of Dif and DL in co-transfection assays reveals that DL possesses a dominant negative effect on Dif transactivation (Peterson, 1995).

NF-kappa B-like motifs have a regulatory role in the synthesis of cecropins, a set of anti-bacterial peptides, triggered by the presence of bacterial cell wall components in the insect blood. The upstream region of the Cecropin gene CecA1 contains elements responsible for inducible and tissue-specific expression. A trimer of kappa B-like motif confers high levels of inducible expression from a reporter gene, after transfection in a Drosophila blood cell line. As in the moth Hyalophora cecropia, stimulation with bacterial lipopolysaccharide induces a nuclear factor that specifically binds to the kappa B-like motif. These data suggest a functional and evolutionary relationship between these insect immune response factors and the mammalian NF-kappa B (Engstrom, 1993).

In Drosophila, three Cecropin genes have been characterized: CecA1, CecA2, and CecB, all in a dense cluster at 99E on the third chromosome. From the same locus, a fourth member of the cecropin gene family, CecC, has been isolated; it is mainly expressed at the early pupal stage. CecC is induced in the anterior end of the larval hindgut and in other larval tissues that are undergoing histolysis. Within these other tissues it is often expressed in distinct foci that may correspond to hemocytes. A similar pattern of expression in the metamorphosing pupa is also observed for the CecA and CecB genes. Comparing the DNA sequences of the cecropin genes, a conserved region is observed about 30 bp upstream of the TATA box. It consists of three shorter motifs, two of which are reminiscent of a putative promoter element in immune protein genes from the cecropia moth (Tryselius, 1992).

The GATA motif is a well known positive cis-regulatory element in vertebrates. Experimental evidence is provided for the direct participation of a GATA motif in the expression of the Drosophila antibacterial peptide gene Cecropin A1. A kappaB-like site is necessary for Cecropin A1 gene expression. The Drosophila Rel protein which binds to the kappaB-like site, requires an intact GATA site for maximal Dif-mediated transactivation of the Cecropin A1 gene. A Drosophila blood cell line contains factors binding specifically to the GATA motif of the Cecropin A1 gene. The GATA binding activity is likely to include member(s) of the GATA family of transcriptional regulators. The promoters of several inducible insect immune genes possess GATA sites 0-12 base pairs away from kappaB-like sites in functionally important promoter regions. The serpent gene is expressed both in fat body and hemocytes, and embryos mutatnt for srp lack mature fat body and hemocytes. Like the srp gene, the Cec genes are also expressed in fat body and hemocytes. The overlapping expression pattern of srp and Cec genes makes serpent an interesting candidate for the GATA-binding activity. Clusters of GATA and kappaB sites are also observed in the promoters of two important mammalian immune genes: IL6 and IL3. The consistent proximity of GATA and kappaB sites appears to be a common theme in the immune gene expression of insects and mammals (Kadalayil, 1997).

The Drosophila cell line mbn-2 is of blood cell origin, derived from larval hemocytes of the mutant lethal (2) malignant blood neoplasm (l[2]mbn). The mbn-2 cells respond to microbial substances by the activation of cecropin genes, coding for bactericidal peptides. The response here is stronger than that found in other Drosophila cell lines, including four that were totally unresponsive. Bacterial lipopolysaccharide, algal laminarin (a beta-1,3-glucan), and bacterial flagellin are strong inducers, while bacterial peptidoglycan fragments give a weaker response. Experiments with different drugs indicate that the response may be mediated by a G protein, but not by protein kinase C or eicosanoids, and that it requires a protein factor with a high rate of turnover (Samakovlis, 1992).

DNaseI footprinting experiments were performed on the Diptericin gene combined with gel-shift assays in two inducible systems: the larval fat body and a tumorous Drosophila blood cell line. These results confirm the importance of kappa B-like elements in the immune response of insects and reveal for the first time the involvement of other regions containing sequences homologous to mammalian acute-phase response elements (Georgel, 1993 and Hoffmann, 1993).

Diptericins are 9 kDa inducible antibacterial peptides initially isolated from immune hemolymph of Phormia (Diptera). The Diptericin gene is inducible by injection of live bacteria or complete Freund's adjuvant and respects the tissue specific expression pattern of the resident diptericin gene. There are at least four distinct phases in the regulation of this gene: young larvae, late third instar larvae, pupae and adults. This complexity may be related to the presence in the upstream sequences of multiple copies of response elements previously characterized in genes encoding acute phase response proteins in mammals (e.g. NK-kappa B, NF-kappa B related, NF-IL6 response elements) (Reichhart, 1992).

Insect defensins are a family of 4-kDa, cationic, inducible antibacterial peptides which bear six cysteine residues engaged in three intramolecular disulfide bridges. They owe their name to certain sequence similarities with defensins from mammalian neutrophiles and macrophages. A novel Defensin isoform in Drosophila encoding a preprodefensin is intronless and present in a single copy/haploid genome; it maps at position 46CD on the right arm of the second chromosome. The upstream region of the gene contains multiple putative cis-regulatory sequences similar to mammalian regulatory motifs of acute-phase-response genes. Transcriptional profiles indicate that the Drosophila Defensin gene is induced by bacterial challenge with acute-phase kinetics. It is also expressed in the absence of immune challenge during metamorphosis. It is likely that insect and mammalian defensins have evolved independently (Dimarcq, 1994).

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).

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).


dorsal-related immunity factor: Biological Overview | Evolutionary Homologs | Protein Interactions | Effects of Mutation | References

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