Expression Of Drosomycin Research Articles

PAR-008 - Methotrexate hinders the expression of Drosomycin induced via Toll/NF-KB pathway in both acute and chronic inflammation

PAR-008 - Methotrexate hinders the expression of Drosomycin induced via Toll/NF-KB pathway in both acute and chronic inflammation

Differential Requirements for Mediator Complex Subunits in Drosophila melanogaster Host Defense Against Fungal and Bacterial Pathogens.

The humoral immune response to bacterial or fungal infections in Drosophila relies largely on a transcriptional response mediated by the Toll and Immune deficiency NF-κB pathways. Antimicrobial peptides are potent effectors of these pathways and allow the organism to attack invading pathogens. Dorsal-related Immune Factor (DIF), a transcription factor regulated by the Toll pathway, is required in the host defense against fungal and some Gram-positive bacterial infections. The Mediator complex is involved in the initiation of transcription of most RNA polymerase B (PolB)-dependent genes by forming a functional bridge between transcription factors bound to enhancer regions and the gene promoter region and then recruiting the PolB pre-initiation complex. Mediator is formed by several modules that each comprises several subunits. The Med17 subunit of the head module of Mediator has been shown to be required for the expression of Drosomycin, which encodes a potent antifungal peptide, by binding to DIF. Thus, Mediator is expected to mediate the host defense against pathogens controlled by the Toll pathway-dependent innate immune response. Here, we first focus on the Med31 subunit of the middle module of Mediator and find that it is required in host defense against Aspergillus fumigatus, Enterococcus faecalis, and injected but not topically-applied Metarhizium robertsii. Thus, host defense against M. robertsii requires Dif but not necessarily Med31 in the two distinct infection models. The induction of some Toll-pathway-dependent genes is decreased after a challenge of Med31 RNAi-silenced flies with either A. fumigatus or E. faecalis, while these flies exhibit normal phagocytosis and melanization. We have further tested most Mediator subunits using RNAi by monitoring their survival after challenges to several other microbial infections known to be fought off through DIF. We report that the host defense against specific pathogens involves a distinct set of Mediator subunits with only one subunit for C. glabrata or Erwinia carotovora carotovora, at least one for M. robertsii or a somewhat extended repertoire for A. fumigatus (at least eight subunits) and E. faecalis (eight subunits), with two subunits, Med6 and Med11 being required only against A. fumigatus. Med31 but not Med17 is required in fighting off injected M. robertsii conidia. Thus, the involvement of Mediator in Drosophila innate immunity is more complex than expected.

Tissue-autonomous immune response regulates stress signaling during hypertrophy.

Postmitotic tissues are incapable of replacing damaged cells through proliferation, but need to rely on buffering mechanisms to prevent tissue disintegration. By constitutively activating the Ras/MAPK-pathway via RasV12-overexpression in the postmitotic salivary glands (SGs) of Drosophila larvae, we overrode the glands adaptability to growth signals and induced hypertrophy. The accompanied loss of tissue integrity, recognition by cellular immunity, and cell death are all buffered by blocking stress signaling through a genuine tissue-autonomous immune response. This novel, spatio-temporally tightly regulated mechanism relies on the inhibition of a feedback-loop in the JNK-pathway by the immune effector and antimicrobial peptide Drosomycin. While this interaction might allow growing SGs to cope with temporary stress, continuous Drosomycin expression in RasV12-glands favors unrestricted hypertrophy. These findings indicate the necessity to refine therapeutic approaches that stimulate immune responses by acknowledging their possible, detrimental effects in damaged or stressed tissues.

MiR-958 inhibits Toll signaling and Drosomycin expression via direct targeting of Toll and Dif in Drosophila melanogaster.

Drosophila melanogaster is widely used as a model system to study innate immunity and signaling pathways related to innate immunity, including the Toll signaling pathway. Although this pathway is well studied, the precise mechanisms of posttranscriptional regulation of key components of the Toll signaling pathway by microRNAs (miRNAs) remain obscure. In this study, we used an in silico strategy in combination with the Gal80ts-Gal4 driver system to identify microRNA-958 (miR-958) as a candidate Toll pathway regulating miRNA in Drosophila We report that overexpression of miR-958 significantly reduces the expression of Drosomycin, a key antimicrobial peptide involved in Toll signaling and the innate immune response. We further demonstrate in vitro and in vivo that miR-958 targets the Toll and Dif genes, key components of the Toll signaling pathway, to negatively regulate Drosomycin expression. In addition, a miR-958 sponge rescued the expression of Toll and Dif, resulting in increased expression of Drosomycin. These results, not only revealed a novel function and modulation pattern of miR-958, but also provided a new insight into the underlying molecular mechanisms of Toll signaling in regulation of innate immunity.

Genome-wide miRNA screening reveals miR-310 family members negatively regulate the immune response in Drosophila melanogaster via co-targeting Drosomycin

Although innate immunity mediated by Toll signaling has been extensively studied in Drosophila melanogaster, the role of miRNAs in regulating the Toll-mediated immune response remains largely unknown. In this study, following Gram-positive bacterial challenge, we identified 93 differentially expressed miRNAs via genome-wide miRNA screening. These miRNAs were regarded as immune response related (IRR). Eight miRNAs were confirmed to be involved in the Toll-mediated immune response upon Gram-positive bacterial infection through genetic screening of 41 UAS-miRNA lines covering 60 miRNAs of the 93 IRR miRNAs. Interestingly, four out of these eight miRNAs, miR-310, miR-311, miR-312 and miR-313, are clustered miRNAs and belong to the miR-310 family. These miR-310 family members were shown to target and regulate the expression of Drosomycin, an antimicrobial peptide produced by Toll signaling. Taken together, our study implies important regulatory roles of miRNAs in the Toll-mediated innate immune response of Drosophila upon Gram-positive bacterial infection.

Functional screening of mammalian mechanosensitive genes using Drosophila RNAi library- Smarcd3/Bap60 is a mechanosensitive pro-inflammatory gene.

Disturbed blood flow (d-flow) induces atherosclerosis by altering the expression of mechanosensitive genes in the arterial endothelium. Previously, we identified >580 mechanosensitive genes in the mouse arterial endothelium, but their role in endothelial inflammation is incompletely understood. From this set, we obtained 84 Drosophila RNAi lines that silences the target gene under the control of upstream activation sequence (UAS) promoter. These lines were crossed with C564-GAL4 flies expressing GFP under the control of drosomycin promoter, an NF-κB target gene and a marker of pathogen-induced inflammation. Silencing of psmd12 or ERN1 decreased infection-induced drosomycin expression, while Bap60 silencing significantly increased the drosomycin expression. Interestingly, knockdown of Bap60 in adult flies using temperature-inducible Bap60 RNAi (C564ts-GAL4-Bap60-RNAi) enhanced drosomycin expression upon Gram-positive bacterial challenge but the basal drosomycin expression remained unchanged compared to the control. In the mammalian system, smarcd3 (mammalian ortholog of Bap60) expression was reduced in the human- and mouse aortic endothelial cells exposed to oscillatory shear in vitro as well as in the d-flow regions of mouse arterial endothelium in vivo. Moreover, siRNA-mediated knockdown of smarcd3 induced endothelial inflammation. In summary, we developed an in vivo Drosophila RNAi screening method to identify flow-sensitive genes that regulate endothelial inflammation.

Multiple targets of the microRNA miR-8 contribute to immune homeostasis in Drosophila

MicroRNAs (miRNAs) are noncoding, around 22-nucleotide-long RNAs that exert significant modulatory roles in gene expression throughout the genome. As such, miRNAs take part in and modulate almost all biological processes like cell growth, development, and immunity. We previously showed that miR-8 miRNA plays a role in maintaining immune homeostasis in Drosophila. Here, we further discovered that targeting of multiple coding genes by miR-8 contributes to the maintenance of immune homeostasis. Toll and Dorsal, respectively the receptor and transcription factor in the Toll immune pathway, were found to be miR-8 targets, as shown by reporter assays and miR-8 null flies. Moreover, U-shaped (Ush), a previously verified miR-8 target, was seen to mediate miR-8 regulation of immune homeostasis. Consistently, overexpression of either Dorsal or Ush in the fat body led to increased Drosomycin expression, mimicking that induced by deletion of miR-8. Furthermore, mutation in Toll immune pathway or Ush rescues the abnormal expression of Drosomycin and lethality in miR-8 mutant. Thus, miR-8 regulates Drosophila immune homeostasis by targeting multiple immune genes, thereby contributing to survival.

Drosomycin, an essential component of antifungal defence in Drosophila

Drosomycin is an inducible antifungal peptide of 44 residues initially isolated from bacteria-challenged Drosophila melanogaster. The systemic expression of drosomycin is regulated by the Toll pathway present in fat body, whereas inducible local expression in the respiratory tract is controlled by the Immune Deficiency (IMD) pathway. Drosomycin belongs to the cysteine-stabilized alpha-helical and beta-sheet (CSalphabeta) superfamily and is composed of an alpha-helix and a three-stranded beta-sheet stabilized by four disulphide bridges. Drosomycin exhibits a narrow antimicrobial spectrum and is only active against some filamentous fungi. However, recent work using recombinant drosomycin expressed in Escherichia coli revealed its antiparasitic and anti-yeast activities. Two evolutionary epitopes (alpha- and gamma-patch) and the m-loop have been proposed as putative functional regions of drosomycin for interaction with fungi and parasites, respectively. Similarity in sequence, structure and biological activity suggests that drosomycin and some defensin molecules from plants and fungi could originate from a common ancestor.

Unpaired ( upd)-3 expression and other immune-related functions are stimulated by interleukin-8 in Drosophila melanogaster SL2 cell line

Invertebrate innate immunity relies on both cellular and humoral components. Among humoral factors, there is less information on soluble molecules able to act as signals during the immune response (i.e. cytokines). In Drosophila melanogaster, the cytokine Unpaired (Upd)-3, is known to activate the JAK/STAT pathway, but it is still not clear which molecules and pathways are responsible for its induction and secretion. It has been proposed that highly chemotactic factors may increase the expression of upd-3. In this respect, we have studied the effects of the chemotactic human recombinant (hr) interleukin (IL)-8 on the immune functions of Drosophila SL2 macrophage-like cells. The hrIL-8 increases the percentage of phagocytic cells with a specific timing and enhances the expression of the cytokine, upd-3, as well as that of the putative cytokine Drosophila helical factor ( dhf). The antimicrobial peptides defensin, cecropin A1 and diptericin, are all influenced in their expression by the human chemokine, while hrIL-8 leaves unaffected the expression of drosomycin, i.e. the antimicrobial peptide more strictly connected with the Toll pathway. Similar effects to those registered for hrIL-8 are also provoked by a specific activator of the Imd pathway, i.e. the Escherichia coli peptidoglycan. RNAi experiments demonstrated that the silencing of the Imd pathway-associated kinase dTAK1, leaves unaffected the induction of upd-3, while it completely abolishes the effects of hrIL-8-on the expression of dhf. Our data suggest that the Imd pathway is not fundamental in regulating the levels of upd-3, whereas it controls the expression of the putative cytokine dhf.

Differential potency of drosomycin to Neurospora crassa and its mutant: implications for evolutionary relationship between defensins from insects and plants

Drosomycin, the first inducible antifungal peptide isolated from Drosophila, belongs to the superfamily of CSalphabeta-type defensins. In the present study we report a modified approach for high-level expression of drosomycin, which allows us to evaluate its differential potency on the filamentous fungus Neurospora crassa WT (wild type) and N. crassa MUT16, a specific resistance mutant strain to plant defensins, by using different approaches. The results presented here show for the first time that N. crassa MUT16 is resistant to our recombinant drosomycin. Differential survival rates of Drosophila larvae infected by N. crassa WT and MUT16 further confirm the key antifungal role of drosomycin in vivo. The absence of activity against MUT16 suggests a mechanical commonality between drosomycin and plant defensins, which provides additional evidence in favor of their homologous relationship. Furthermore, the existence of drosomycin-like molecules in fungi suggests that all these peptides could originate from a common ancestry rather than horizontal gene transfer between plants and insects, which is further strengthened by the monophyletic origin of these peptides from plants, fungi and insects.

Fungal Peptide Destruxin A Plays a Specific Role in Suppressing the Innate Immune Response in Drosophila melanogaster

Destruxins are a class of insecticidal, anti-viral, and phytotoxic cyclic depsipeptides that are also studied for their toxicity to cancer cells. They are produced by various fungi, and a direct relationship has been established between Destruxin production and the virulence of the entomopathogen Metarhizium anisopliae. Aside from opening calcium channels, their in vivo mode of action during pathogenesis remains largely uncharacterized. To better understand the effects of a Destruxin, we looked at changes in gene expression following injection of Destruxin A into the fruit fly Drosophila melanogaster. Microarray results revealed reduced expression of various antimicrobial peptides that play a major role in the humoral immune response of the fly. Flies co-injected with a non-lethal dose of Destruxin A and the normally innocuous Gram-negative bacteria Escherichia coli, showed increased mortality and an accompanying increase in bacterial titers. Mortality due to sepsis was rescued through ectopic activation of components in the IMD pathway, one of two signal transduction pathways that are responsible for antimicrobial peptide induction. These results demonstrate a novel role for Destruxin A in specific suppression of the humoral immune response in insects.

Dual Detection of Fungal Infections in Drosophila via Recognition of Glucans and Sensing of Virulence Factors

The Drosophila immune system discriminates between various types of infections and activates appropriate signal transduction pathways to combat the invading microorganisms. The Toll pathway is required for the host response against fungal and most Gram-positive bacterial infections. The sensing of Gram-positive bacteria is mediated by the pattern recognition receptors PGRP-SA and GNBP1 that cooperate to detect the presence of infections in the host. Here, we report that GNBP3 is a pattern recognition receptor that is required for the detection of fungal cell wall components. Strikingly, we find that there is a second, parallel pathway acting jointly with GNBP3. The Drosophila Persephone protease activates the Toll pathway when proteolytically matured by the secreted fungal virulence factor PR1. Thus, the detection of fungal infections in Drosophila relies both on the recognition of invariant microbial patterns and on monitoring the effects of virulence factors on the host.

Sensing of Gram-positive bacteria in Drosophila: GNBP1 is needed to process and present peptidoglycan to PGRP-SA

Genetic evidence indicates that Drosophila defense against Gram-positive bacteria is mediated by two putative pattern recognition receptors acting upstream of Toll, namely Gram-negative binding protein 1 (GNBP1) and peptidoglycan recognition protein SA (PGRP-SA). Until now however, the molecular recognition proceedings for sensing of Gram-positive pathogens were not known. In the present, we report the physical interaction between GNBP1 and PGRP-SA using recombinant proteins. GNBP1 was able to hydrolyze Gram-positive peptidoglycan (PG), while PGRP-SA bound highly purified PG fragments (muropeptides). Interaction between these proteins was enhanced in the presence of PG or muropeptides. PGRP-SA binding depended on the polymerization status of the muropeptides, pointing to constraints in the number of PGRP-SA molecules bound for signaling initiation. We propose a model whereby GNBP1 presents a processed form of PG for sensing by PGRP-SA and that a tripartite interaction between these proteins and PG is essential for downstream signaling.

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

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

DUbc9 negatively regulates the Toll-NF-κB pathways in larval hematopoiesis and drosomycin activation in Drosophila

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

The Drosophila Toll-9 activates a constitutive antimicrobial defense.

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

Tissue-Specific Inducible Expression of Antimicrobial Peptide Genes in Drosophila Surface Epithelia

The production of antimicrobial peptides is an important aspect of host defense in multicellular organisms. In Drosophila, seven antimicrobial peptides with different spectra of activities are synthesized by the fat body during the immune response and secreted into the hemolymph. Using GFP reporter transgenes, we show here that all seven Drosophila antimicrobial peptides can be induced in surface epithelia in a tissue-specific manner. The imd gene plays a critical role in the activation of this local response to infection. In particular, drosomycin expression, which is regulated by the Toll pathway during the systemic response, is regulated by imd in the respiratory tract, thus demonstrating the existence of distinct regulatory mechanisms for local and systemic induction of antimicrobial peptide genes in Drosophila.

What's the DIF? A Role in Antifungal Defense

In Drosophila , the Toll receptor pathway controls the response to infection by fungi and bacteria. The Rel family of transcription factors including Dorsal, Relish, and DIF are activated by Toll and mediate the expression of antimicrobial peptides that defend Drosophila from biological invasion. Rutschmann et al . demonstrate the requirement for DIF but not Relish or Dorsal in antifungal defense. The authors generated flies that carried mutations in Dif , and found that Dif flies are susceptible to fungal infection by B . bassiana . Expression of drosomycin, an antifungal peptide, but not several other antimicrobial peptides, was reduced in Dif flies challenged with fungal infection. Subsequent genetic analyses confirmed that Dif is required for drosomycin expression. These experimental data suggest that specific Rel family members may mediate the response to particular microbial pathogens. Rutschmann, S., Jung, A.C., Hetru, C., Reichhart, J.-M., Hoffmann, J.A., and Ferrandon, D. (2000) The Rel protein DIF mediates the antifungal but not the antibacterial host defense in Drosophila . Immunity 12 : 569-580. [Online Journal]

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

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

Malaria infection of the mosquitoAnopheles gambiaeactivates immune-responsive genes during critical transition stages of the parasite life cycle

Six gene markers have been used to map the progress of the innate immune response of the mosquito vector, Anopheles gambiae, upon infection by the malaria parasite, Plasmodium berghei. In addition to four previously reported genes, the set of markers included NOS (a nitric oxide synthase gene fragment) and ICHIT (a gene encoding two putative chitin-binding domains separated by a polythreonine-rich mucin region). In the midgut, a robust response occurs at 24 h post-infection, at a time when malaria ookinetes traverse the midgut epithelium, but subsides at later phases of malaria development. In contrast, the salivary glands show no significant response at 24 h, but are activated in a prolonged late phase when sporozoites are released from the midgut into the haemolymph and invade the glands, between 10 and 25 days after blood feeding. Furthermore, the abdomen of the mosquito minus the midgut shows significant activation of immune markers, with complex kinetics that are distinct from those of both midgut and salivary glands. The parasite evidently elicits immune responses in multiple tissues of the mosquito, two of which are epithelia that the parasite must traverse to complete its development. The mechanisms of these responses and their significance for malaria transmission are discussed.