InteractiveFly: GeneBrief

Metchnikowin: Biological Overview | References

Insect-derived antifungal peptides have a significant economic potential, particularly for the engineering of pathogen-resistant crops. However, the nonspecific antifungal activity of such peptides could result in detrimental effects against beneficial fungi, whose interactions with plants promote growth or increase resistance against biotic and abiotic stress. The antifungal peptide Metchnikowin (Mtk) from Drosophila melanogaster acts selectively against pathogenic Ascomycota, including Fusarium graminearum, without affecting Basidiomycota such as the beneficial symbiont Piriformospora indica. This study investigated the mechanism responsible for the selective antifungal activity of Mtk by using the peptide to probe a yeast two-hybrid library of F. graminearum cDNAs. Mtk was found to specifically target the iron-sulfur subunit (SdhB) of succinate-coenzyme Q reductase (SQR). A functional assay based on the succinate dehydrogenase (SDH) activity of mitochondrial complex II clearly demonstrated that Mtk inhibited the SDH activity of F. graminearum mitochondrial SQR by up to 52%, but that the equivalent enzyme in P. indica was unaffected. A phylogenetic analysis of the SdhB family revealed a significant divergence between the Ascomycota and Basidiomycota. SQR is one of the key targets of antifungal agents and Mtk is therefore proposed as an environmentally sustainable and more selective alternative to chemical fungicides (Moghaddam, 2017b).

Insects are well protected against pathogens by an immunity-related arsenal of effector molecules including antimicrobial peptides (AMPs). Some AMPs are active against a broad spectrum of microbes, whereas the activity of others is restricted to certain types of bacteria or fungi. Insects produce a large number of antifungal peptides to protect them against fungal pathogens and parasites, and these peptides often interact with intracellular targets to inhibit key physiological processes such as DNA and protein synthesis, cell cycle progression and metabolic activity. AMPs therefore offer significant potential as leads for the development of drugs and biocides, but the mechanisms of action must first be understood. A small number of natural antifungal peptides have been characterized in this regard, including termicin from termites, heliomicin from the tobacco budworm Heliothis virescens , gallerimycin from larvae of the greater wax moth Galleria mellonella 8, and drosomycin and metchnikowin (Mtk) from Drosophila melanogaster. The 26-residue proline-rich linear peptide Mtk is induced following microbial infection in D. melanogaster and its activity is remarkably specific. Whereas the activity of other antifungal peptides can affect both pathogens and beneficial endophytes, Mtk acts specifically against pathogenic Ascomycota such as Fusarium graminearum and Blumeria graminis f. sp. hordei, but is inactive against beneficial endophytic Basidiomycota such as Piriformospora indica, when expressed in barley. This is particularly important because plants benefit from their mutual interactions with fungal endophytes. Although the precise basis of this specificity is not understood, previous observations suggest that Mtk inhibits the ability of susceptible fungi to suppress plant defense responses. A recent study revealed that Mtk interferes with F. graminearum cell wall biosynthesis by targeting the β(1,3)-glucanosyltransferase Gel1, which is responsible for β(1,3)-glucan chain elongation in the cell wall (Moghaddam, 2017b).

AMPs often have multiple targets to reduce the likelihood of emerging microbial resistance. This study therefore sought additional Mtk intracellular targets by probing a yeast two-hybrid library of F. graminearum cDNAs with an artificial Mtk peptide, revealing a specific interaction with the iron-sulfur subunit (SdhB) of succinate-coenzyme Q reductase (SQR). The holoenzyme is a heterotetramer comprising two hydrophilic subunits (flavoprotein SdhA and iron-sulfur protein SdhB) and two hydrophobic subunits (SdhC and SdhD). SdhA contains the cofactor flavin adenine dinucleotide (FAD) and a succinate binding site, whereas SdhB contains three iron-sulfur clusters, and SdhC and SdhD are the membrane anchor subunits. SQR is a key enzyme in both the Krebs cycle (also known as the citric acid cycle or tricarboxylic acid cycle) and the electron transport chain, both of which are required for energy generation. In the Krebs cycle, SQR catalyzes the oxidation of succinate to fumarate via the reduction of ubiquinone to ubiquinol. The resulting electrons enter the respiratory chain complex III, reducing oxygen to water and thus providing the electrochemical gradient across the mitochondrial inner membrane which is needed for ATP synthesis (Moghaddam, 2017b).

Therefore this study investigated whether Mtk can selectively inhibit mitochondrial SQR (complex II) activity in F. graminearum but not in P. indica, based on the hypothesis that the specific activity of Mtk against Ascomycota reflects the selective inhibition of this enzyme. A phylogenetic analysis of SdhB homologs in different Ascomycota and Basidiomycota was conducted to determine whether the enzyme has diverged in these two fungal phyla (Moghaddam, 2017b).

Insect AMPs have often been shown to act against phytopathogenic fungi, and could therefore be suitable for expression as recombinant peptides in transgenic plants to reduce yield and quality losses. However, the nonspecific antifungal effects of many such peptides limit their plant protection applications because most crops establish beneficial mutualistic interactions with fungal endophytes, which promote growth by facilitating access to water and nutrients, or by inhibiting other pathogens. The deployment of AMPs with selective activity against pathogenic fungi offers an environmentally sustainable alternative to hazardous chemical fungicides, particularly given the emergence and spread of fungicide resistance. The proline-rich antifungal peptide Mtk from D. melanogaster acts selectively against phytopathogenic Ascomycota such as F. graminearum without affecting beneficial endophytic Basidiomycota such as P. indica. Recent findings indicate that this specific activity partly reflects the interaction between Mtk and the Ascomycota β(1,3)glucanosyltransferase Gel1, which is only distantly related to Gel homologs in the Basidiomycota (Moghaddam, 2017b).

The specificity of Mtk against Ascomycota was considered in more detail by screening a yeast two-hybrid library of F. graminearum cDNAs using Mtk as the probe, which identified the iron-sulfur subunit of SQR (UniProtKB-I1RNM7) as an additional Mtk target. The F2H and BiFC systems were used to verify the interaction. The advantage of protein-protein interaction assays based on two-hybrid systems rather than in vitro biophysical or biochemical methods is their ability to detect weak or transient interactions. Some of these interactions need species-dependent post-translational modifications and/or particular cofactors, therefore verification in mammalian and/or plant cells is highly recommended. The F2H assay reported a positive interaction but only in a few cells, suggesting the ability of BHK-21 cells to support the interaction was limited. Potential explanations include the nuclear targeting of the bait and prey, forcing them to interact in a non-native compartment, or changes in conformation caused by the fluorescent protein tags, obscuring the interaction site. The BiFC assay was also unsatisfactory, which may reflect the non-optimal stoichiometry of the interacting components caused by differences in transformation efficiency by the separate Agrobacterium tumefaciens strains, or non-specific reassembly due to the prevention of translational read-through in the native Gateway cloning cassettes. Furthermore, background signals in BiFC assays can obscure weak interactions. The possibility cannot be excluded that the rare Mtk-SdhB interaction events in mammalian and tobacco cells indicate the lack of physical interaction. However, a functional assay demonstrated that Mtk reduces the SDH activity of F. graminearum SQR but not that of its P. indica homolog. Therefore, the absence of an expected physical interaction in the F2H and BiFC assays may reflect the requirement for additional components that are present in the S. cerevisiae strain used for the original Y2H assay but not in the BHK-21 or tobacco cells, bearing in mind that S. cerevisiae is also a member of the Ascomycota (Moghaddam, 2017b).

The mitochondrial enzyme SQR, which contains three further subunits in addition to the iron-sulfur subunit SdhB, plays an important role in both the Krebs cycle and the mitochondrial electron transport chain, both of which are essential for oxidative phosphorylation. SQR catalyzes the oxidation of succinate to fumarate in the mitochondrial matrix, which results in the reduction of ubiquinone to ubiquinol in the mitochondrial inner membrane. SdhB, which is located between SdhA and the two transmembrane subunits SdhC and SdhD, contains three iron-sulfur clusters that are required for tunneling the electrons through the complex. Coenzyme Q accepts the electrons from complexes I and II, and carries them to complex III, from where they are diverted to reduce the ubiquinone pool. The reducing equivalents reduce superoxide anions that accumulate from either exogenous sources or the respiratory chain. In contrast to other dehydrogenases in the Krebs cycle, SQR does not transport the succinate-derived electrons to soluble NAD+ intermediates but to the ubiquinone pool of the respiratory chain as an electron sink to provide antioxidants in the mitochondrial inner membrane. This study found that Mtk diminishes the overall mitochondrial SDH activity by up to 52%, which may lead to (1) higher levels of reactive oxygen species such as superoxide due to the depleted ubiquinone pool, and (2) loss of the electron gradient, thus compromising the survival of the fungus. The inhibition of SdhB by Mtk may arrest the delivery of electrons required for the full reduction of ubiquinone to ubiquinol, and may therefore increase oxygen toxicity in the mitochondria. On the other hand, a deficient electrochemical gradient across the mitochondrial inner membrane would inhibit the generation of ATP, which is coupled to the oxidation of nicotinamide adenine dinucleotide (NADH)/FADH2 and the reduction of oxygen to water within the respiratory chain25. Therefore, the inhibition of SDH activity by Mtk would impose a high fitness cost on F. graminearum, explaining the resistance of transgenic barley plants expressing Mtk against this pathogen. The ability of Mtk to inhibit SdhB and thus perturb the Krebs cycle could also disturb the equilibrium between the malate-aspartate shuttle and reducing equivalents, further restricting electron delivery from the mitochondrial inner membrane to the electron transport chain. Mutations in the subunits of mitochondrial complex II therefore increase oxidative stress and decrease longevity (Moghaddam, 2017b).

Interestingly, Mtk did not inhibit the SDH activity of mitochondrial SQR in P. indica, providing a further explanation for the selective activity of Mtk against Ascomycota11. Although it was not possible to comment on the relative activities of SQR enzymes in F. graminearum and P. indica, the mitochondrial respiratory activity in the latter is so high that it can be detected in colonized plant roots using the SDH assay. Previous studies have already revealed that the selective activity of Mtk is partly due to the specific interaction between Mtk and the Ascomycota β(1,3)-glucanosyltransferase Gel1, which is only distantly related to the equivalent enzyme in the Basidiomycota. The phylogenetic analysis described in this study also revealed a significant divergence between the Ascomycota and Basidiomycota SdhB homologs, which provides a mechanistic basis for the selective inhibition that was observed. Mtk is therefore a highly promising antifungal candidate that is active against pathogenic Ascomycota but not against beneficial endophytic Basidiomycota such as P. indica due to its phylum-specific activity against at least two distinct enzymes (Moghaddam, 2017b).

SQRs are useful targets for fungicide development. SDH inhibitors (SDHIs) that occupy either the succinate-binding pocket (e.g. malonate) or the ubiquinone-binding pocket (e.g. carboxamides) are highly effective against diverse fungal species. Amide fungicides target SQR in the mitochondrial respiratory chain resulting in growth arrest and even cell death by disrupting the mitochondrial Krebs cycle and interfering with respiration. All SDHIs currently used for crop protection target the ubiquinone-binding pocket, which is structurally defined by the interface among the subunits SdhB, SdhC and SdhD. However, mutations in SQR subunits have conferred SDHI resistance in 14 fungal species thus far, highlighting the need for new antifungal agents with diverse mechanisms of action. Although F. graminearum remains susceptible to chemical SDHIs for the time being, mutations that reduce susceptibility have been identified in a number of pathogens including B. cinerea, Podosphaera xanthii, Alternaria spp. and Sclerotinia sclerotiorum. Mtk could help to prevent or at least delay the emergence of resistant fungal pathogens because resistance would require the simultaneous mutation of multiple targets (Moghaddam, 2017b).

In conclusion, this study has shown that Mtk attacks F. graminearum using at least two distinct mechanisms. First the integrity of the cell wall is compromised when Mtk interacts with the β(1,3)glucanosyltransferase Gel1 to inhibit the synthesis of cell wall polymers, and then the general metabolic fitness of the fungus is targeted by inhibiting the SDH activity of mitochondrial SQR, resulting in suboptimal energy generation. The fungus suffers a loss of fitness due to the combined impact of a weak cell wall, compromised energy generation, oxidative stress and slow metabolism. Mtk represents an ideal lead for the development of environmentally sustainable fungicides due to its selectivity and multiple intracellular targets, which reduces the impact on beneficial fungi and discourages the emergence of resistant pathogens (Moghaddam, 2017b).

The insect-derived antimicrobial peptide metchnikowin targets Fusarium graminearum beta(1,3)glucanosyltransferase Gel1, which is required for the maintenance of cell wall integrity

Antimicrobial peptides (AMPs) are essential components of the insect innate immune system. Their diversity provides protection against a broad spectrum of microbes and they have several distinct modes of action. Insect-derived AMPs are currently being developed for both medical and agricultural applications, and their expression in transgenic crops confers resistance against numerous plant pathogens. The antifungal peptide Metchnikowin (Mtk), which was originally discovered in the fruit fly Drosophila melanogaster, is of particular interest because it has potent activity against economically important phytopathogenic fungi of the phylum Ascomycota, such as Fusarium graminearum, but it does not harm beneficial fungi such as the mycorrhizal basidiomycete Piriformospora indica. To investigate the specificity of Mtk, the peptide was used to screen a F. graminearum yeast two-hybrid library. This revealed that Mtk interacts with the fungal enzyme beta(1,3)-glucanosyltransferase Gel1 (FgBGT), which is one of the enzymes responsible for fungal cell wall synthesis. The interaction was independently confirmed in a second interaction screen using mammalian cells. FgBGT is required for the viability of filamentous fungi by maintaining cell wall integrity. This study therefore paves the way for further applications of Mtk in formulation of bio fungicides or as a supplement in food preservation (Moghaddam, 2017a).

Interaction of lncRNA-CR33942 with Dif/Dorsal Facilitates Antimicrobial Peptide Transcriptions and Enhances Drosophila Toll Immune Responses

The Drosophila Toll signaling pathway mainly responds to Gram-positive (G(+)) bacteria or fungal infection, which is highly conserved with mammalian TLR signaling pathway. Although many positive and negative regulators involved in the immune response of the Toll pathway have been identified in Drosophila, the roles of long noncoding RNAs (lncRNAs) in Drosophila Toll immune responses are poorly understood to date. In this study, the results demonstrate that lncRNA-CR33942 is mainly expressed in the nucleus and upregulatƒƒed after Micrococcus luteus infection. Especially, lncRNA-CR33942 not only modulates differential expressions of multiple antimicrobial peptide genes but also affects the Drosophila survival rate during response to G(+) bacterial infection based on the transiently overexpressing and the knockdown lncRNA-CR33942 assays in vivo. Mechanically, lncRNA-CR33942 interacts with the NF-κB transcription factors Dorsal-related immunity factor/Dorsal to promote the transcriptions of antimicrobial peptides drosomycin and metchnikowin, thus enhancing Drosophila Toll immune responses. Taken together, this study identifies lncRNA-CR33942 as a positive regulator of Drosophila innate immune response to G(+) bacterial infection to facilitate Toll signaling via interacting with Dorsal-related immunity factor/Dorsal. It would be helpful to reveal the roles of lncRNAs in Toll immune response in Drosophila and provide insights into animal innate immunity (Zhou, 2022).

Expression of antimicrobial peptides associated with different susceptibilities to environmental chemicals in Drosophila suzukii and Drosophila melanogaster

Drosophila suzukii is a serious agricultural pest. The evolved morphology of the female D. suzukii assists in penetrating the surface of fresh fruit and spawns eggs with its unique ovipositor. Conversely, Drosophila melanogaster, a taxonomically close species with D. suzukii, largely inhabits decaying and fermenting fruits and is consistently exposed to extensive environmental chemicals, such as 2-phenylethanol, ethanol, and acetic acid, produced by microorganisms. Considering the distinct habitats of the two flies, D. suzukii is thought to be more susceptible to environmental chemicals than D. melanogaster. This study investigated the significantly higher survival rate of D. melanogaster following exposure to 2-phenylethanol, ethanol, and acetic acid. A comparison of the expression of antimicrobial peptides (AMPs) between the two flies treated with chemicals established that AMPs were generally more abundantly induced in D. melanogaster than in D. suzukii, particularly in the gut and fat body. Among the AMPs, the induction of genes (Diptericin A, Diptericin B, and Metchnikowin), which are regulated by the immune deficiency (IMD) pathway, was significantly higher than that of Drosomycin, which belongs to the Toll pathway in chemical-treated D. melanogaster. A transgenic RNAi fly (D. melanogaster) with silenced expression of AMPs and Relish, a transcription factor of the IMD pathway, exhibited significantly reduced survival rates than the control fly. These results suggest that AMPs regulated by the IMD pathway play an important role in the chemical tolerance of D. melanogaster, and these flies are adapted to their habitats by physiological response (Kim, 2022).

LncRNA-CR11538 Decoys Dif/Dorsal to Reduce Antimicrobial Peptide Products for Restoring Drosophila Toll Immunity Homeostasis

Avoiding excessive or insufficient immune responses and maintaining homeostasis are critical for animal survival. Although many positive or negative modulators involved in immune responses have been identified, little has been reported to date concerning whether the long non-coding RNA (lncRNA) can regulate Drosophila immunity response. Firstly, this study discovered that the overexpression of lncRNA-CR11538 can inhibit the expressions of antimicrobial peptides Drosomycin (Drs) and Metchnikowin (Mtk) in vivo, thereby suppressing the Toll signaling pathway. Secondly, the results demonstrate that lncRNA-CR11538 can interact with transcription factors Dif/Dorsal in the nucleus based on both subcellular localization and RIP analyses. Thirdly, the findings reveal that lncRNA-CR11538 can decoy Dif/Dorsal away from the promoters of Drs and Mtk to repress their transcriptions by ChIP-qPCR and dual luciferase report experiments. Fourthly, the dynamic expression changes of Drs, Dif, Dorsal and lncRNA-CR11538 in wild-type flies (w(1118)) at different time points after M. luteus stimulation disclose that lncRNA-CR11538 can help Drosophila restore immune homeostasis in the later period of immune response. Overall, this study reveals a novel mechanism by which lncRNA-CR11538 serves as a Dif/Dorsal decoy to downregulate antimicrobial peptide expressions for restoring Drosophila Toll immunity homeostasis, and provides a new insight into further studying the complex regulatory mechanism of animal innate immunity (Zhou, 2021).

Loss of the Antimicrobial Peptide Mechnikowin Protects Against Traumatic Brain Injury Outcomes in Drosophila melanogaster

Neuroinflammation is a major pathophysiological feature of traumatic brain injury (TBI). Early and persistent activation of innate immune response signaling pathways by primary injuries is associated with secondary cellular injuries that cause TBI outcomes to change over time. A Drosophila melanogaster model was used to investigate the role of antimicrobial peptides (AMPs) in acute and chronic outcomes of closed-head TBI. AMPs are effectors of pathogen and stress defense mechanisms mediated by the evolutionarily conserved Toll Immune-deficiency (Imd) innate immune response pathways that activate Nuclear Factor kappa B (NF-kB) transcription factors. This study analyzed the effect of null mutations in 10 of the 14 known Drosophila AMP genes on TBI outcomes. Mutation of Metchnikowin (Mtk) was unique in protecting flies from mortality within the 24 h following TBI under two diet conditions that produce different levels of mortality. In addition, Mtk mutants had reduced behavioral deficits at 24 h following TBI and increased lifespan either in the absence or presence of TBI. Using a transcriptional reporter of gene expression, it was found that TBI increased Mtk expression in the brain. Quantitative analysis of mRNA in whole flies revealed that expression of other AMPs in the Toll and Imd pathways as well as NF-κB transcription factors were not altered in Mtk mutants. Overall, these results demonstrate that Mtk plays an infection-independent role in the fly nervous system, and TBI-induced expression of Mtk in the brain activates acute and chronic secondary injury pathways that are also activated during normal aging (Swanson, 2020).

Achilles is a circadian clock-controlled gene that regulates immune function in Drosophila

The circadian clock is a transcriptional/translational feedback loop that drives the rhythmic expression of downstream mRNAs. Termed "clock-controlled genes," these molecular outputs of the circadian clock orchestrate cellular, metabolic, and behavioral rhythms. This study identified a novel clock-controlled gene in Drosophila melanogaster, Achilles (Achl), which is rhythmic at the mRNA level in the brain and which represses expression of anti-microbial peptides in the immune system. Achilles knock-down in neurons dramatically elevates expression of crucial immune response genes, including IM1 (Immune induced molecule 1), Mtk (Metchnikowin), and Drs (Drosomysin). As a result, flies with knocked-down Achilles expression are resistant to bacterial challenges. Meanwhile, no significant change in core clock gene expression and locomotor activity is observed, suggesting that Achilles influences rhythmic mRNA outputs rather than directly regulating the core timekeeping mechanism. Notably, Achilles knock-down in the absence of immune challenge significantly diminishes the fly's overall lifespan, indicating a behavioral or metabolic cost of constitutively activating this pathway. Together, these data demonstrate that (1) Achilles is a novel clock-controlled gene that (2) regulates the immune system, and (3) participates in signaling from neurons to immunological tissues (Li, 2016).

Differential activation of immune factors in neurons and glia contribute to individual differences in resilience/vulnerability to sleep disruption

Individuals frequently find themselves confronted with a variety of challenges that threaten their wellbeing. While some individuals face these challenges efficiently and thrive (resilient) others are unable to cope and may suffer persistent consequences (vulnerable). Resilience/vulnerability to sleep disruption may contribute to the vulnerability of individuals exposed to challenging conditions. With that in mind this study exploited individual differences in a fly's ability to form short-term memory (STM) following 3 different types of sleep disruption to identify the underlying genes. The analysis showed that in each category of flies examined, there are individuals that form STM in the face of sleep loss (resilient) while other individuals show dramatic declines in cognitive behavior (vulnerable). Molecular genetic studies revealed that Antimicrobial Peptides, factors important for innate immunity, were candidates for conferring resilience/vulnerability to sleep deprivation. Specifically, Metchnikowin (Mtk), drosocin (dro) and Attacin (Att) transcript levels seemed to be differentially increased by sleep deprivation in glia (Mtk), neurons (dro) or primarily in the head fat body (Att). Follow-up genetic studies confirmed that expressing Mtk in glia but not neurons, and expressing dro in neurons but not glia, disrupted memory while modulating sleep in opposite directions. These data indicate that various factors within glia or neurons can contribute to individual differences in resilience/vulnerability to sleep deprivation (Dissel, 2015).

Forkhead, a new cross regulator of metabolism and innate immunity downstream of TOR in Drosophila

Antimicrobial peptides (AMPs) are conserved cationic peptides which both act as defense molecules of the host immune system and as regulators of the commensal microbiome. Expression of AMPs is induced in response to infection by the Toll and Imd pathway. Under non-infected conditions, the transcription factor dFOXO directly regulates a set of AMP expression at low levels when nutrients are limited. This study has analyzed whether Target of rapamycin (TOR), another major regulator of growth and metabolism, also modulates AMP responses in Drosophila. Downregulation of TOR by feeding the drug rapamycin or by overexpressing the negative TOR regulators TSC1/TSC2 was shown to result in a specific induction of the AMPs Diptericin (Dpt) and Metchnikowin (Mtk). In contrast, overexpression of Rheb, which positively regulates TOR led to a repression of the two AMPs. Genetic and pharmacological experiments indicate that Dpt and Mtk activation is controlled by the transcription factor Forkhead (Fkh), the founding member of the FoxO family. Shuttling of Fkh from the cytoplasm to the nucleus is induced in the fat body and in the posterior midgut in response to TOR downregulation. The Fkh-dependent induction of Dpt and Mtk can be triggered in dFOXO null mutants and in immune-compromised Toll and IMD pathway mutants indicating that FKH acts in parallel to these regulators. Together, this study has discovered that FKH is the second conserved member of the FoxO family cross-regulating metabolism and innate immunity. dFOXO and FKH, which are activated upon downregulation of insulin or TOR activities, respectively, act in parallel to induce different sets of AMPs, thereby modulating the immune status of metabolic tissues such as the fat body or the gut in response to the oscillating energy status of the organism (Varma, 2014).

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. 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).

GATA factors participate in tissue-specific immune responses in Drosophila larvae

Drosophila responds to infection by producing a broad range of antimicrobial agents in the fat body and more restricted responses in tissues such as the gut, trachea, and malpighian tubules. The regulation of antimicrobial genes in larval fat depends on linked Rel/NF-kappaB and GATA binding sites. Serpent functions as the major GATA transcription factor in the larval fat body. However, the transcriptional regulation of other tissue-specific responses is less well understood. This study presents evidence that dGATAe regulates antimicrobial gene expression in the midgut. Regulatory regions for antimicrobial genes Diptericin and Metchnikowin require GATA sites for activation in the midgut, where Grain (dGATAc), dGATAd, and dGATAe are expressed in overlapping domains. Ectopic expression of dGATAe in the larval fat body, where it is normally absent, causes dramatic up-regulation of numerous innate immunity and gut genes, as judged by microarray analysis and in situ hybridization. Ectopic dGATAe also causes a host of symptoms reminiscent of hyperactive Toll (Toll^10b) mutants, but without apparent activation of Toll signaling. Based on this evidence it is proposed that dGATAe mediates a Toll-independent immune response in the midgut, providing a window into the first and perhaps most ancient line of animal defense (Senger, 2006).

Previous studies have established the importance of Serpent in mediating the systemic immune response in the larval fat body. Serpent is also essential for the differentiation of the fat body during embryogenesis; srp mutants are lethal and lack fat cell differentiation. Serpent is not merely a transient determinant of fat body development. It is proposed that dGATAe plays an analogous role in the anterior midgut: it functions as a tissue determinant in the embryo but mediates immunity in larvae (Senger, 2006).

dGATAe is expressed throughout the developing midgut during embryogenesis. As seen for srp in the fat body, dGATAe expression persists in the definitive midgut of feeding larvae. Thus, both srp and dGATAe might have dual roles in development and physiology. Early expression is required for tissue differentiation, and late expression is required for the immune response. srp mediates immunity in the fat body, whereas dGATAe mediates expression of specific immunity genes in the midgut (Senger, 2006).

Evidence that dGATAe functions in the early development of the midgut stems from microarray assays. Misexpression of dGATAe in the fat body leads to ectopic induction of a number of genes required for digestion, including trypsin-like serine proteases, a sugar transporter, and genes involved in lipid metabolism. All of these genes display restricted expression in the midgut of developing embryos, in regions where dGATAe is also expressed. It is proposed that at least some of these genes are immediate and direct targets of dGATAe in the developing gut, and consequently, they are efficiently activated by dGATAe in the fat body (Senger, 2006).

dGATAe might also activate genes required for immunity gene expression in the anterior midgut of feeding larvae. By analogy to Serpent, dGATAe might activate different components of signaling pathways required for immunity. When larvae ingest pathogenic bacteria such as E. carotovora, these pathways are induced to trigger expression of Drs, Dpt, Mtk, and other immunity genes in the anterior midgut. When misexpressed in the fat body, dGATAe only moderately affects the levels of Dpt or Mtk, although the regulatory regions of these genes showed GATA-dependent activity in the midgut. This low activity is attributed to the presence of repressors in the fat body that cannot be overcome by ectopic dGATAe (Senger, 2006).

It is possible that dGATAe-mediated immunity in the anterior midgut does not depend on the Toll signaling pathway, because none of the signaling components of this pathway are up-regulated in the fat body on misexpresion of dGATAe. This misexpression is nonetheless sufficient to induce Drs and dro5 in the absence of infection or injury. It is proposed that dGATAe plays two roles in the differentiated midgut. One role is activating 'housekeeping' genes that are required for digestion. The second role of dGATAe in the midgut is triggering unknown signaling pathways that lead to the activation of immunity genes such as Drs, Dpt, and Mtk. Drs may be especially sensitive to dGATAe in the fat body because it is “poised” for induction (Senger, 2006).

In summary, it has been argued that dGATAe is critical for anterior midgut formation and function in a manner analogous to Serpent in the fat body. It is possible that the dGATAe immunity pathway is an evolutionarily ancient form of innate immunity. Under typical living conditions, the gut is the first line of defense, because ingestion is the most likely basis for contact with pathogens. The immunity signaling pathway(s) governing dGATAe activity is not yet known. However, ectopic expression of dGATAe in the fat body leads to the activation of a number of signaling components, including RhoL, Takl2, and Tetraspanins. The latter are integral membrane proteins that have been implicated in the immune responses of higher organisms, including antigen presentation. Future studies will assess the role, if any, of these genes in the gut-specific immune response (Senger, 2006).

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

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

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

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

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

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

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

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

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 TGF-ß activated kinase 1 (Tak1) gene that encodes Tak1, a homolog of TAK1. Tak1 mutant flies are viable and fertile, but they do not produce antibacterial peptides and are highly susceptible to Gram-negative bacterial infection. This phenotype is similar to the phenotypes generated by mutations in components of the Drosophila Imd pathway. Genetic studies also indicate that Tak1 functions downstream of the Imd protein and upstream of the IKK complex in the Imd pathway that controls the Rel/NF-kappaB like transactivator Relish. In addition, epistatic analysis places the caspase, Dredd, downstream of the IKK complex, which supports the idea that Relish is processed and activated by a caspase activity. This genetic demonstration of Tak1's role in the regulation of Drosophila antimicrobial peptide gene expression suggests an evolutionary conserved role for TAK1 in the activation of Rel/NF-kappaB-mediated host defense reactions (Vidal, 2001).

The Toll signaling pathway, which was first identified as a regulator of embryonic dorsal-ventral patterning, is one regulator of antimicrobial peptide gene expression in Drosophila. Upon infection, the Spaetzle (Spz) protein is cleaved to generate a ligand for the Toll transmembrane receptor protein; Toll binding by Spz stimulates the degradation of the IkappaB homolog, Cactus, and the nuclear translocation of the Rel proteins Dorsal and Dorsal-like immunity factor (Dif). A second pathway regulating antimicrobial peptide gene expression in flies was initially identified by a mutation in the immune deficiency (imd) gene that results in susceptibility to Gram-negative bacterial infection and an impairment of antibacterial peptide gene expression. imd encodes a homolog of the mammalian Receptor Interacting Protein (RIP). Genetic placement of imd suggests that Imd has a conserved function in flies as part of a receptor-adaptor complex that responds to Gram-negative bacterial infection. Molecular studies have isolated four additional factors that appear to define the Imd pathway: Relish; two members of a Drosophila IkappaB kinase (IKK) complex, that is, the kinase DmIKKß and a structural component DmIKKgamma and Dredd, a caspase. Like imd, mutations in DmIKKß, DmIKKgamma, Dredd, and Relish affect antibacterial peptide gene expression after infection and induce susceptibility to Gram-negative bacterial infections. However, mutations in these genes do not induce susceptibility to fungal infections, demonstrating that the immune responses regulated by the Imd pathway are required to resist Gram-negative bacterial but not fungal infections (Vidal, 2001 and references therein).

Some significant conclusions of recent studies on the regulation of Drosophila antimicrobial peptide gene expression are that the Toll and Imd pathways do not share any components and that each pathway regulates specific Rel proteins. The only evidence of interactions between the two pathways is the observation that both pathways are required to fully induce some of the antimicrobial peptide genes, suggesting that these genes respond to combinations of Rel proteins controlled by the two pathways. The influence of each pathway on the expression of each antimicrobial peptide gene is apparent in flies carrying mutations that affect either the Toll or the Imd pathway: Drosomycin is mainly controlled by the Toll pathway; Diptericin and Drosocin can be fully activated by the Imd pathway; and full Metchnikowin, Defensin, Cecropin A and Attacin activation requires both pathways (Vidal, 2001 and references therein).

None of the antimicrobial peptide genes are induced in imd;Toll double mutant flies, demonstrating that Imd and Toll are two essential pathways that regulate antimicrobial gene expression pathways. Despite an increased understanding of the regulation of antimicrobial peptide gene expression in flies, various intermediates in the Toll and Imd pathways remain uncharacterized: for example, neither the kinase that targets Cactus for degradation in the Toll pathway nor the receptor-adaptor complex that regulates the Imd pathway have been identified. Following the observations that null mutations affecting the Imd pathway are not required for viability, a search for additional members of the Imd pathway was initiated by screening for nonlethal mutations that induce susceptibility to Gram-negative bacterial infection in adult flies. Null mutations in the Drosophila transforming growth factor activated kinase 1 gene (Tak1) encoding the Drosophila homolog of the mammalian mitogen-activated protein kinase kinase kinase (MAPKKK) TAK1 induce high susceptibility to Gram-negative bacterial infection and block antibacterial peptide gene expression. These results indicate that Drosophila Tak1 codes for a new component of the Imd pathway (Vidal, 2001).

To compare the Tak1 (D10) phenotype with the phenotypes generated by mutations in other genes that regulate Drosophila immune responses, the susceptibility of D10 and other mutant lines to infection by four microorganisms was assayed: flies were pricked with the Gram-negative bacteria Escherichia coli (E. coli), the Gram-positive bacteria Micrococcus luteus (M. luteus), or the fungus Aspergillus fumigatus, and flies were naturally infected with the entomopathogenic fungus Beauveria bassiana (B. bassiana). The D10 phenotype is similar to the imd and Relish phenotypes; flies carrying the D10, imd, and Relish mutations are susceptible to Gram-negative bacterial infection and resistant to Gram-positive bacterial and fungal infections, although D10 flies, like imd flies, exhibit slightly lower susceptibility to Gram-negative bacterial infection compared to Relish mutants. In contrast, mutations in the spz gene render flies susceptible to fungal infections, and only flies carrying mutations in both spz and imd are susceptible to Gram-positive bacterial infection. This survival analysis demonstrates that the D10 gene product, like Imd and Relish, is required to resist Gram-negative bacterial infection (Vidal, 2001).

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

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

The loss-of-function Tak1 mutations display immune response phenotypes that are very similar to the phenotypes generated by mutations in imd, DmIKKß, DmIKKgamma, Dredd, and Relish, suggesting that these genes function together in the Imd pathway. Previous studies indicated that DmIKKgamma and DmIKKß directly regulate Relish activity; 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).

The antibacterial arm of the Drosophila innate immune response requires an IkappaB kinase

The ird5 gene was identified in a genetic screen for Drosophila immune response mutants. Mutations in ird5 prevent induction of six antibacterial peptide genes in response to infection but do not affect the induction of an antifungal peptide gene. Consistent with this finding, Escherichia coli survive 100 times better in ird5 adults than in wild-type animals. The ird5 gene encodes a Drosophila homolog of mammalian IkappaB kinases (IKKs) and is the catalytic subunit of the IKK complex that contains IKKγ (Kenny) as a regulatory subunit. The ird5 phenotype and sequence suggest that the gene is specifically required for the activation of Relish, a Drosophila NF-kappaB family member (Lu, 2001).

Mutations in ird5 prevent induction of a diptericin-lacZ reporter gene in response to infection and also prevent transcriptional induction of the endogenous diptericin gene. E. coli-induced expression of all seven classes of antimicrobial peptide genes in ird5 mutant larvae were examined by RNA blot hybridization, including the genes encoding antibacterial (Diptericin, Cecropin A, Defensin, Attacin, Drosocin, and Metchnikowin) and antifungal (Drosomycin) peptides. In wild-type larvae, all the antimicrobial genes are strongly induced after bacterial challenge. In contrast, in larvae homozygous for either ird5 allele, there is no detectable induction of the diptericin, cecropin A, defensin, drosocin, or metchnikowin genes. The attacin gene is induced in the mutants to ~30% of normal levels, while drosomycin is induced to normal levels. The same effects on the induction of antimicrobial peptide genes are seen in ird5¹/Df and ird5²/Df animals, suggesting that both alleles cause a complete loss of gene function (Lu, 2001).

Mutations in three other genes, imd, Relish, and Death related ced-3/Nedd2-like protein, have been shown to prevent normal induction of antibacterial peptide genes in adult Drosophila. The pattern of antimicrobial peptide gene induction in ird5 mutants was compared with that in imd and Relish mutants in both larvae and adults. Mutations in all three genes have very similar effects on antimicrobial gene induction in larvae: diptericin and cecropin A are not induced; attacin induction is reduced and drosomycin induction is normal. In adult animals, the antimicrobial gene expression phenotypes of ird5 and Relish mutants are very similar: diptericin induction is blocked, cecropin A and attacin induction is reduced, and drosomycin induction is normal. The antimicrobial gene expression phenotype of imd adults is slightly different, with some residual diptericin expression. Mutations in Dredd, a Drosophila caspase, prevent normal induction of diptericin and attacin and allow induction of drosomycin. These comparisons suggest that ird5, Dredd, Relish, and probably imd act in the same signaling pathway to control the induction of antibacterial peptide genes in response to infection (Lu, 2001).

To assess the importance of the ird5 gene in controlling the growth of invading bacteria, bacterial survival and growth were compared in wild-type, ird5, imd, and Relish animals. In wild-type larvae, most of the E. coli injected into the animal are killed by 6 h after infection. At this same time point, there are four to 15 times as many E. coli in ird5 mutant larvae as in wild-type animals. The effects of the ird5 mutations are more striking in experiments with adults: at 24 h after infection, there are 20-350 times as many bacteria per animal in ird5 mutants compared to wild type. The bacterial growth phenotype of ird5 mutants is similar to that seen in Relish mutant larvae and adults and somewhat stronger than that of imd mutants. This is consistent with the stronger effects of ird5 and Relish on the antimicrobial peptide genes: Mutations in either ird5 or Relish prevent normal induction of diptericin, cecropin, drosocin, attacin, and metchnikowin, while induction of metchnikowin is induced in imd mutants (Lu, 2001).

The mutations responsible for the failure to induce the diptericin-lacZ reporter gene for both ird5 alleles were mapped between the visible markers cu (86D1-4) and sr (90D2-F7) on the right arm of the third chromosome. The deficiency Df(3R)sbd⁴⁵(89B4-10) fails to complement the immune response defect of either ird5 allele. Further deficiency-complementation tests and male recombination mapping narrowed the ird5 interval to 89B4-9, between pannier and Stubble. Two molecular-defined genes in this interval were considered as candidates responsible for the ird5 phenotype, Akt and a gene defined by an EST that is related to mammalian IkappaB kinases (IKKs). Mutant alleles of Akt cause recessive lethality, and ird5/Akt[l(3)89Bq] heterozygous animals are viable and showed normal induction of the diptericin-lacZ reporter gene, indicating that the ird5 phenotypes are not caused by mutations in Akt (Lu, 2001).

Mammalian IKKbeta is required for activation of NF-kappaB in response to inflammatory signals such as TNF-alpha and IL-1; the IKK homolog was therefore considered as a candidate gene for ird5. A full-length cDNA was cloned for the IKK homolog, termed DmIkkbeta. The same gene has also identified molecularly as encoding a kinase activated by LPS in a Drosophila cell line. Based on genomic DNA sequence, DmIkkbeta is located between pannier and mini-spindles. Two size classes of transcripts, 2.7 and 4.2 kb, were detected from the DmIkkbeta gene; the cDNA corresponds to the 2.7-kb transcript. Both transcripts are expressed at higher levels after infection. The complete open reading frame of DmIkkbeta was sequenced from the ird5¹ and ird5² chromosomes. A single C-to-T nucleotide substitution was found in ird5¹ that would change a glutamine codon (CAA) at amino acid 266 of the open reading frame to a stop codon (TAA) within the conserved kinase domain. No sequence changes were identified in the open reading frame in ird5²; however, neither DmIkkbeta transcript was detectable in ird5² homozygotes. This analysis indicates that both ird5 alleles are associated with mutations that should abolish DmIkkbeta activity (Lu, 2001).

The ird5 immune response phenotype shows striking specificity: all of the antibacterial peptide genes are strongly affected by the ird5 mutations, but the antifungal peptide gene drosomycin is induced normally in ird5 mutants. The specific immune response phenotype of ird5/DmIkkbeta in vivo contrasts with the global effects on antimicrobial peptide genes seen in cell lines when a dominant negative form of the same gene is expressed in cultured cells. The ird5/DmIkkbeta mutant phenotype implies that, in vivo, ird5 is not an essential component of the Toll pathway, which is required for the induction of drosomycin. The ird5/DmIkkbeta gene is therefore a component of an independent signaling pathway, which could be activated by another member of the Drosophila Toll-like receptor family (Lu, 2001).

Mammalian IKKalpha and IKKbeta phosphorylate serine residues in the N-terminal domain of IkappaB; these serine residues target IkappaB for degradation, thereby allowing the nuclear localization and activation of NF-kappaB. The ird5/DmIkkbeta sequence suggests that the protein encoded by this gene phosphorylates an IkappaB-like protein. There are two known Drosophila IkappaB-like proteins that could act as inhibitor proteins in the immune response: Cactus, and the C-terminal ankyrin repeat domain of Relish. In cactus mutants, drosomycin is expressed constitutively, but the antibacterial peptide genes are not, which indicates that Cactus is not involved in the pathways that regulate the antibacterial peptide genes. Furthermore, ird5/DmIkkbeta homozygous mutant females are fertile, demonstrating that this gene is not required for degradation of Cactus during dorsal-ventral patterning in the embryo (Lu, 2001).

The ird5/DmIkkbeta phenotype is similar to the phenotype of Relish mutants. For both genes, homozygous mutant flies are viable and fertile, indicating that the two genes are not essential for development. Mutations in either Relish or ird5/DmIkkbeta completely prevent induction of diptericin and cecropin but allow some induction of attacin and drosomycin. Mutations in either gene produce comparable effects on bacterial growth. These results argue that ird5/DmIkkbeta and Relish act in the same signaling pathway and suggest that Ird5/DmIkkbeta activates Relish-containing dimers. Relish activation requires proteolytic cleavage of Relish protein into an N-terminal Rel domain that translocates to the nucleus and a C-terminal ankyrin repeat domain that remains in the cytoplasm. Recent biochemical experiments have shown that DmIkkbeta can phosphorylate Relish protein, which is consistent with the model that phosphorylation of Relish by DmIkkbeta leads to targeted proteolysis and activation of Relish (Lu, 2001).

Although ird5/DmIkkbeta is expressed maternally, ird5 mutant females are fertile, demonstrating that the gene is not required for embryonic dorsal-ventral patterning. However, a small fraction of embryos (~0.5%) produced by homozygous ird5¹ or ird5¹/ird5² females show a weakly dorsalized phenotype, suggesting that ird5/DmIkkbeta does have a minor role in the maternal pathway that activates Dorsal. It is suggested that there is another kinase in the early embryo that is primarily responsible for phosphorylation and degradation of Cactus. The normal induction of drosomycin in ird5/DmIkkbeta mutants suggests that there will also be another kinase activated by the Toll pathway in the immune response -- perhaps the same kinase that acts downstream of Toll to activate Dorsal in the embryo. The genome sequence indicates that there is one additional IkappaB kinase gene in Drosophila. Future experiments will test whether this gene plays a role in embryonic patterning and the antifungal immune response (Lu, 2001).

The data suggest that different Drosophila Rel dimers are activated by homologous but distinct signaling pathways. Given the similarities of innate immune response pathways in Drosophila and mammals, it is likely that similar pathway-specific signaling components will mediate the activities of the members of the mammalian Rel proteins (Lu, 2001).

The Drosophila caspase Dredd is required to resist Gram-negative bacterial infection

The Drosophila innate immune system discriminates between pathogens and responds by inducing the expression of specific antimicrobial peptide-encoding genes through distinct signaling cascades. Fungal infection activates NF-kappaB-like transcription factors via the Toll pathway, which also regulates innate immune responses in mammals. The pathways that mediate antibacterial defenses, however, are less defined. Loss-of-function mutations are reported in the caspase encoding gene dredd, which block the expression of all genes that code for peptides with antibacterial activity. These mutations also render flies highly susceptible to infection by Gram-negative bacteria. These results demonstrate that Dredd regulates antibacterial peptide gene expression, and it is proposed that Dredd, Immune Deficiency and the P105-like rel protein Relish define a pathway that is required to resist Gram-negative bacterial infections (Leulier, 2000).

To identify genes that control Drosophila antibacterial immune responses, a screen was carried out for mutations on the X chromosome that affect the expression of the antibacterial peptide gene diptericin after bacterial infection. Among 2500 EMS mutagenized lines, five viable, recessive mutations (named B118, F64, L23, D55, D44) were isolated of a gene that is required for the expression of a diptericin-GFP reporter gene in larvae after bacterial infection. In addition, Northern blot analysis shows that adults homozygous for each of the five alleles do not express the diptericin gene after bacterial injection. The B118 allele was mapped to cytological region 1B9-1B13 on the proximal tip of the X chromosome and a small deficiency, Df(1)R194, was identified that does not complement B118. Deficiency Df(1)R194 spans four previously identified genes: rpL36, l(1)1Bi, dredd and su(s). Several results demonstrate that B118 is a mutation in dredd: (1) B118 is allelic to a viable P element insertion (EP-1412) inserted 50 bp upstream of dredd coding sequences; (2) the two genes flanking dredd, su(s) and l(1)1Bi complement B118; (3) a small deficiency, Df(1)dredd^D3, 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)dredd^D3, 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 dredd^B118 mutant, however, mixed Gram-positive/Gram-negative infections only induce the expression of the antifungal gene drosomycin and the gene coding for Metchnikowin, which has both antifungal and antibacterial activity; diptericin, cecropin A, attacin A and defensin are expressed at <5% of wild-type levels and metchnikowin is expressed at 50% of the wild-type level. Antimicrobial gene expression is similarly affected in flies homozygous for rel^E20, 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 dredd^B118 and rel^E20 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, dredd^B118 and rel^E20 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 Tl^10b 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. dredd^B118, rel^E20 and imd;spz^rm7 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;spz^rm7 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 spz^rm7 and imd;spz^rm7 mutants are highly sensitive to natural infection by the entomopathogenic fungus Beauveria bassiana or injection of Aspergillus fumigatus spores, confirming that responses to fungi are largely activated by the Toll pathway (Leulier, 2000).

The dredd immune phenotype is similar to the relish and imd phenotypes; it is predicted that the Imd, Dredd and Relish proteins function in a common signaling pathway that regulates antibacterial peptide gene expression. Based on the respective activites of Dredd as a caspase and Relish as a transcriptional transactivator, it is also hypothesized that Dredd functions upstream of Relish in the control of antimicrobial gene expression. This hypothesis is supported by the observation that Dredd is required for Relish activation via endoproteolytic cleavage. It is believed that the weaker effects of the imd mutation on antibacterial gene expression place the imd gene product at an early stage of the antibacterial cascade where multiple responses, some of which bypass imd, trigger the activation of the pathway. Alternatively, the imd mutation may represent a hypomorphic allele (Leulier, 2000).

Caspases were originally identified as effectors of apoptosis, but there is increasing evidence that caspases also function in other physiological processes. Recent studies suggest that the recruitment of the caspase-8 precursor to the TNF-R1 signaling complex either activates NF-kappaB through a Traf2-, RIP-, NIK- and IKK-dependent pathway or, after proteolytic processing of caspase-8, induces apoptosis. The data indicate that Dredd, a close homolog of caspase-8, may also have dual functions in NF-kappaB signaling and apoptosis in Drosophila. Further biochemical analysis is necessary to determine whether Dredd participates directly in Relish activation or functions further upstream (Leulier, 2000).

Deciphering the mechanisms that enable Drosophila to differentiate between pathogens and mount specific immune responses is essential for understanding innate immunity. Recent studies indicate that the Toll pathway is mainly activated in response to fungal and Gram-positive bacterial infection. Several observations suggest that imd, dredd and relish mediate most of the responses to Gram-negative bacterial infection: (1) these genes regulate the antimicrobial peptide genes that are most highly induced by Gram-negative bacterial infection; (2) dredd and relish control the induction of metchnikowin and drosomycin after Gram-negative bacterial infection, and (3) these three genes are required for resistance to Gram-negative bacterial infection. A model is proposed whereby antimicrobial gene expression in Drosophila adults is regulated by a balance of inputs from the Toll pathway and the Imd pathway, which includes Imd, Dredd and Relish, and that these two pathways are differentially activated by different classes of microorganisms. Identifying the receptors that discriminate between invading microbes and stimulate these pathways presents an exciting challenge in the study of innate immunity (Leulier, 2000).

Two distinct pathways can control expression of the gene encoding the Drosophila antimicrobial peptide metchnikowin

Metchnikowin is a recently discovered proline-rich peptide from Drosophila with antibacterial and antifungal properties. Like most other antimicrobial peptides from insects, its expression is immune-inducible. Evidence is presented that induction of metchnikowin gene expression can be mediated either by the Toll pathway or by the imd gene product. The gene remains inducible in Toll-deficient mutants, in which the antifungal response is blocked, as well as in imd mutants, which fail to mount an antibacterial response. However, in Toll-deficient;imd double mutants, metchnikowin gene expression can no longer be detected after immune challenge. These results suggest that expression of this peptide with dual activity can be triggered by signals generated by either bacterial or fungal infection. Cloning of the metchnikowin gene revealed the presence in the 5' flanking region of several putative cis-regulatory motifs characterized in the promoters of insect immune genes: namely, Rel sites, GATA motifs, interferon consensus response elements and NF-IL6 response elements. Establishment of transgenic fly lines in which the GFP reporter gene was placed under the control of 1.5 kb of metchnikowin gene upstream sequences indicates that this fragment is able to confer full immune inducibility and tissue specificity of expression on the transgene (Levashina, 1998).

Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms.

Insects respond to microbial infection by the rapid and transient expression of several genes encoding potent antimicrobial peptides. This antimicrobial response of Drosophila is specific and can discriminate between various classes of microorganisms. The genes encoding antibacterial and antifungal peptides are differentially expressed after injection of distinct microorganisms. The level of induction of the diptericin gene in immune-challenged adults varies strikingly with the microorganism tested. Gram-negative bacteria are potent inducers. In contrast, Gram-positives do not induce expression above the level of a simple injury. The pattern of cecropin A, drosocin, defensin, and attacin induction roughly corresponds to the pattern of diptericin induction Drosophila that are naturally infected by entomopathogenic fungi exhibit an adapted response by producing only peptides (especially drosomycin) with antifungal activities. The expression of metchnikowin combines both patterns, as this gene is strongly induced by all microorganisms. These responses is mediated through the selective activation of the Toll pathway. Genes whose expression levels are most strongly affected by the immune deficiency, imd, mutation and that code for strictly antibacterial peptides are also those that are most strongly induced by challenge with Gram-negative as compared with Gram-positive bacteria. In contrast, the metchnikowin and drosomycin genes that are strongly induces by Gram-positive bacteria retain most of their inducibility in imd mutants (Lemaitre, 1997).

Search PubMed for articles about Drosophila Mechnikowin

Dissel, S., Seugnet, L., Thimgan, M. S., Silverman, N., Angadi, V., Thacher, P. V., Burnham, M. M. and Shaw, P. J. (2015). Differential activation of immune factors in neurons and glia contribute to individual differences in resilience/vulnerability to sleep disruption. Brain Behav Immun 47: 75-85. PubMed ID: 25451614

Kim, Y., Goh, G. and Kim, Y. H. (2022). Expression of antimicrobial peptides associated with different susceptibilities to environmental chemicals in Drosophila suzukii and Drosophila melanogaster. Pestic Biochem Physiol 187: 105210. PubMed ID: 36127054

Lemaitre, B., Reichhart, J. M. and Hoffmann, J. A. (1997). Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms. Proc. Natl. Acad. Sci. 94(26): 14614-14619. PubMed Citation: 9405661

Leulier, F., et al. (2000). The Drosophila caspase Dredd is required to resist Gram-negative bacterial infection. EMBO Reports 1: 353-358. 11269502

Levashina, E. A., et al. (1998). Two distinct pathways can control expression of the gene encoding the Drosophila antimicrobial peptide metchnikowin. J. Mol. Biol. 278(3): 515-27. 9600835

Li, J., Terry, E.E., Fejer, E., Gamba, D., Hartmann, N., Logsdon, J., Michalski, D., Rois, L.E., Scuderi, M.J., Kunst, M. and Hughes, M.E. (2016). Achilles is a circadian clock-controlled gene that regulates immune function in Drosophila. Brain Behav Immun [Epub ahead of print]. PubMed ID: 27856350

Lu, Y., Wu, L. P. and Anderson, K. V. (2001). The antibacterial arm of the Drosophila innate immune response requires an IkappaB kinase. Genes Dev 15: 104-110. 11156609

Moghaddam, M. B., Gross, T., Becker, A., Vilcinskas, A. and Rahnamaeian, M. (2017b). The selective antifungal activity of Drosophila melanogaster metchnikowin reflects the species-dependent inhibition of succinate-coenzyme Q reductase. Sci Rep 7(1): 8192. PubMed ID: 28811531

Moghaddam, M. R. B., Vilcinskas, A. and Rahnamaeian, M. (2017a). The insect-derived antimicrobial peptide metchnikowin targets Fusarium graminearum beta(1,3)glucanosyltransferase Gel1, which is required for the maintenance of cell wall integrity. Biol Chem 398(4): 491-498. PubMed ID: 27811341

Peng, J., Zipperlen, P. and Kubli, E. (2005). Drosophila Sex-peptide stimulates female innate immune system after mating via the Toll and Imd pathways. Curr. Biol. 15: 1690-1694. 16169493

Reed, D. E., et al. (2008). DEAF-1 regulates immunity gene expression in Drosophila. Proc. Natl. Acad. Sci. 105(24): 8351-6. PubMed ID: 18550807

Senger, K., Harris, K. and Levine, M. (2006). GATA factors participate in tissue-specific immune responses in Drosophila larvae. Proc. Natl. Acad. Sci. 103(43): 15957-62. PubMed Citation: 17032752

Swanson, L. C., Rimkus, S. A., Ganetzky, B. and Wassarman, D. A. (2020). Loss of the Antimicrobial Peptide Mechnikowin Protects Against Traumatic Brain Injury Outcomes in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 32631949

Varma, D., Bulow, M. H., Pesch, Y. Y., Loch, G. and Hoch, M. (2014). Forkhead, a new cross regulator of metabolism and innate immunity downstream of TOR in Drosophila. J Insect Physiol. PubMed ID: 24842780

Vidal, S., et al. (2001). Mutations in the Drosophila Tak1 gene reveal a conserved function for MAPKKKs in the control of rel/NF-kappaB-dependent innate immune responses. Genes Dev. 15: 1900-1912. 11485985

Zhou, H., Li, S., Pan, W., Wu, S., Ma, F. and Jin, P. (2022). Interaction of lncRNA-CR33942 with Dif/Dorsal Facilitates Antimicrobial Peptide Transcriptions and Enhances Drosophila Toll Immune Responses. J Immunol 208(8): 1978-1988. PubMed ID: 35379744

date revised: 13 April, 2022

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