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).

GENE STRUCTURE

cDNA clone length - 376bp

Bases in 5' UTR - 50

Exons - 1

Bases in 3' UTR - 113

PROTEIN STRUCTURE AND EVOLUTIONARY HOMOLOGS

Amino Acids - 70

Structural Domains

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).

date revised: 1 December 2006

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