Carbonic anhydrases in metazoan model organisms: molecules, mechanisms, and physiology.

During the past three decades, mice, zebrafish, fruit flies, and Caenorhabditis elegans have been the primary model organisms used for the study of various biological phenomena. These models have also been adopted and developed to investigate the physiological roles of carbonic anhydrases (CAs) and carbonic anhydrase-related proteins (CARPs). These proteins belong to eight CA families and are identified by Greek letters: α, ß, γ, δ, ζ, η, θ, and ι. Studies using model organisms have focused on two CA families, α-CAs and ß-CAs, which are expressed in both prokaryotic and eukaryotic organisms with species-specific distribution patterns and unique functions. This review covers the biological roles of CAs and CARPs in light of investigations performed in model organisms. Functional studies demonstrate that CAs are not only linked to the regulation of pH homeostasis, the classical role of CAs, but also contribute to a plethora of previously undescribed functions.

The Drosophila Toll Pathway in Innate Immunity: from the Core Pathway toward Effector Functions.

The fruit fly Drosophila melanogaster Toll signaling pathway has an evolutionarily conserved role in controlling immune responses. Whereas the microbial recognition mechanisms and the core signaling pathway leading to activation of the humoral immune response via the NF-κB transcription factors have been well established for many years, the mechanistic understanding of the effector functions at the molecular level is currently rapidly evolving. In this review, we discuss the current developments in elucidating the role of the Drosophila Toll signaling pathway in immunity. We discuss the emerging role of Toll in viral infections and sex-specific differences in immunity. Mainly, we focus on Toll pathway regulation, the effector molecules, and cellular immunity.

Osa-Containing Brahma Complex Regulates Innate Immunity and the Expression of Metabolic Genes in Drosophila.

Negative regulation of innate immunity is essential to avoid autoinflammation. In Drosophila melanogaster, NF-κB signaling-mediated immune responses are negatively regulated at multiple levels. Using a Drosophila RNA interference in vitro screen, we identified a set of genes inhibiting immune activation. Four of these genes encode members of the chromatin remodeling Osa-containing Brahma (BAP) complex. Silencing additional two genes of the BAP complex was shown to have the same phenotype, confirming its role in immune regulation in vitro. In vivo, the knockdown of osa and brahma was shown to enhance the expression of the Toll pathway-mediated antimicrobial peptides when the flies were challenged with Gram-positive bacteria Micrococcus luteus In this setting, osa knockdown had a particularly strong effect on immune effectors that are predominantly activated by the Imd pathway. Accordingly, Drosophila NF-κB Relish expression was increased by osa silencing. These transcriptional changes were associated with enhanced survival from M. luteus + E. faecalis infection. Besides regulating the expression of immune effector genes, osa RNA interference decreased the expression of a large group of genes involved in metabolism, particularly proteolysis. Of note, the expression of the recently characterized, immune-inducible gene Induced by Infection (IBIN) was diminished in osa knockdown flies. Although IBIN has been shown to modulate metabolism upon infection, the expression of selected Osa-regulated metabolism genes was not rescued by overexpressing IBIN. We conclude that the BAP complex regulates expression of genes involved in metabolism at least partially independent or downstream of IBIN Moreover, Osa affects the NF-κB-mediated immune response by regulating Drosophila NF-κB factor Relish expression.

Correction: Immune-inducible non-coding RNA molecule lincRNA-IBIN connects immunity and metabolism in Drosophila melanogaster.

[This corrects the article DOI: 10.1371/journal.ppat.1007504.].

Immune-inducible non-coding RNA molecule lincRNA-IBIN connects immunity and metabolism in Drosophila melanogaster.

Non-coding RNAs have important roles in regulating physiology, including immunity. Here, we performed transcriptome profiling of immune-responsive genes in Drosophila melanogaster during a Gram-positive bacterial infection, concentrating on long non-coding RNA (lncRNA) genes. The gene most highly induced by a Micrococcus luteus infection was CR44404, named Induced by Infection (lincRNA-IBIN). lincRNA-IBIN is induced by both Gram-positive and Gram-negative bacteria in Drosophila adults and parasitoid wasp Leptopilina boulardi in Drosophila larvae, as well as by the activation of the Toll or the Imd pathway in unchallenged flies. We show that upon infection, lincRNA-IBIN is expressed in the fat body, in hemocytes and in the gut, and its expression is regulated by NF-κB signaling and the chromatin modeling brahma complex. In the fat body, overexpression of lincRNA-IBIN affected the expression of Toll pathway -mediated genes. Notably, overexpression of lincRNA-IBIN in unchallenged flies elevated sugar levels in the hemolymph by enhancing the expression of genes important for glucose retrieval. These data show that lncRNA genes play a role in Drosophila immunity and indicate that lincRNA-IBIN acts as a link between innate immune responses and metabolism.

Proprotein convertase Furin1 expression in the Drosophila fat body is essential for a normal antimicrobial peptide response and bacterial host defense.

Invading pathogens provoke robust innate immune responses in Dipteran insects, such as Drosophila melanogaster In a systemic bacterial infection, a humoral response is induced in the fat body. Gram-positive bacteria trigger the Toll signaling pathway, whereas gram-negative bacterial infections are signaled via the immune deficiency (IMD) pathway. We show here that the RNA interference-mediated silencing of Furin1-a member of the proprotein convertase enzyme family-specifically in the fat body, results in a reduction in the expression of antimicrobial peptides. This, in turn, compromises the survival of adult fruit flies in systemic infections that are caused by both gram-positive and -negative bacteria. Furin1 plays a nonredundant role in the regulation of immune responses, as silencing of Furin2, the other member of the enzyme family, had no effect on survival or the expression of antimicrobial peptides upon a systemic infection. Furin1 does not directly affect the Toll or IMD signaling pathways, but the reduced expression of Furin1 up-regulates stress response factors in the fat body. We also demonstrate that Furin1 is a negative regulator of the Janus kinase/signal transducer and activator of transcription signaling pathway, which is implicated in stress responses in the fly. In summary, our data identify Furin1 as a novel regulator of humoral immunity and cellular stress responses in Drosophila-Aittomäki, S., Valanne, S., Lehtinen, T., Matikainen, S., Nyman, T. A., Rämet, M., Pesu, M. Proprotein convertase Furin1 expression in the Drosophila fat body is essential for a normal antimicrobial peptide response and bacterial host defense.

Edin Expression in the Fat Body Is Required in the Defense Against Parasitic Wasps in Drosophila melanogaster.

The cellular immune response against parasitoid wasps in Drosophila involves the activation, mobilization, proliferation and differentiation of different blood cell types. Here, we have assessed the role of Edin (elevated during infection) in the immune response against the parasitoid wasp Leptopilina boulardi in Drosophila melanogaster larvae. The expression of edin was induced within hours after a wasp infection in larval fat bodies. Using tissue-specific RNAi, we show that Edin is an important determinant of the encapsulation response. Although edin expression in the fat body was required for the larvae to mount a normal encapsulation response, it was dispensable in hemocytes. Edin expression in the fat body was not required for lamellocyte differentiation, but it was needed for the increase in plasmatocyte numbers and for the release of sessile hemocytes into the hemolymph. We conclude that edin expression in the fat body affects the outcome of a wasp infection by regulating the increase of plasmatocyte numbers and the mobilization of sessile hemocytes in Drosophila larvae.

The Drosophila imd signaling pathway.

The fruit fly, Drosophila melanogaster, has helped us to understand how innate immunity is activated. In addition to the Toll receptor and the Toll signaling pathway, the Drosophila immune response is regulated by another evolutionarily conserved signaling cascade, the immune deficiency (Imd) pathway, which activates NF-κB. In fact, the Imd pathway controls the expression of most of the antimicrobial peptides in Drosophila; thus, it is indispensable for normal immunity in flies. In this article, we review the current literature on the Drosophila Imd pathway, with special emphasis on its role in the (patho)physiology of different organs. We discuss the systemic response, as well as local responses, in the epithelial and mucosal surfaces and the nervous system.

ß carbonic anhydrase is required for female fertility in Drosophila melanogaster.

BACKGROUND: Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous enzymes that catalyze the reversible hydration reaction of carbon dioxide. CAs are present as six structurally divergent enzyme families: α, ß, γ, δ, ζ and η. ß-CAs have a wide distribution across different species including invertebrates. Previously, we showed that Drosophila melanogaster ß-CA is a highly active mitochondrial enzyme. In this study, we investigated the function of Drosophila ß-CA by silencing the expression of the ß-CA gene using UAS/GAL4-based RNA interference (RNAi) in Drosophila in vivo. RESULTS: Crossing ß-CA RNAi lines over ubiquitous Actin driver flies did not produce any viable progeny, indicating that ß-CA expression is required for fly development. RNAi silencing of ß-CA ubiquitously in adult flies did not affect their survival rate or function of mitochondrial electron transport chain. Importantly, ß-CA RNAi led to impaired reproduction. All ß-CA knockdown females were sterile, and produced few or no eggs. Whole ovaries of knockdown females looked normal but upon cadherin staining, there was an apparent functional defect in migration of border cells, which are considered essential for normal fertilization. CONCLUSIONS: These results indicate that although Drosophila ß-CA is dispensable for survival of adult flies, it is essential for female fertility.

Not4 enhances JAK/STAT pathway-dependent gene expression in Drosophila and in human cells.

The JAK/STAT pathway is essential for organogenesis, innate immunity, and stress responses in Drosophila melanogaster. The JAK/STAT pathway and its associated regulators have been highly conserved in evolution from flies to humans. We have used a genome-wide RNAi screen in Drosophila S2 cells to identify regulators of the JAK/STAT pathway, and here we report the characterization of Not4 as a positive regulator of the JAK/STAT pathway. Overexpression of Not4 enhanced Stat92E-mediated gene responses in vitro and in vivo in Drosophila. Specifically, Not4 increased Stat92E-mediated reporter gene activation in S2 cells; and in flies, Not4 overexpression resulted in an 8-fold increase in Turandot M (TotM) and in a 4-fold increase in Turandot A (TotA) stress gene activation when compared to wild-type flies. Drosophila Not4 is structurally related to human CNOT4, which was found to regulate interferon-γ- and interleukin-4-induced STAT-mediated gene responses in human HeLa cells. Not4 was found to coimmunoprecipitate with Stat92E but not to affect tyrosine phosphorylation of Stat92E in Drosophila cells. However, Not4 is required for binding of Stat92E to its DNA recognition sequence in the TotM gene promoter. In summary, Not4/CNOT4 is a novel positive regulator of the JAK/STAT pathway in Drosophila and in humans.

The Drosophila Toll signaling pathway.

The identification of the Drosophila melanogaster Toll pathway cascade and the subsequent characterization of TLRs have reshaped our understanding of the immune system. Ever since, Drosophila NF-κB signaling has been actively studied. In flies, the Toll receptors are essential for embryonic development and immunity. In total, nine Toll receptors are encoded in the Drosophila genome, including the Toll pathway receptor Toll. The induction of the Toll pathway by gram-positive bacteria or fungi leads to the activation of cellular immunity as well as the systemic production of certain antimicrobial peptides. The Toll receptor is activated when the proteolytically cleaved ligand Spatzle binds to the receptor, eventually leading to the activation of the NF-κB factors Dorsal-related immunity factor or Dorsal. In this study, we review the current literature on the Toll pathway and compare the Drosophila and mammalian NF-κB pathways.

Genome-wide RNA interference in Drosophila cells identifies G protein-coupled receptor kinase 2 as a conserved regulator of NF-kappaB signaling.

Because NF-kappaB signaling pathways are highly conserved in evolution, the fruit fly Drosophila melanogaster provides a good model to study these cascades. We carried out an RNA interference (RNAi)-based genome-wide in vitro reporter assay screen in Drosophila for components of NF-kappaB pathways. We analyzed 16,025 dsRNA-treatments and identified 10 novel NF-kappaB regulators. Of these, nine dsRNA-treatments affect primarily the Toll pathway. G protein-coupled receptor kinase (Gprk)2, CG15737/Toll pathway activation mediating protein, and u-shaped were required for normal Drosomycin response in vivo. Interaction studies revealed that Gprk2 interacts with the Drosophila IkappaB homolog Cactus, but is not required in Cactus degradation, indicating a novel mechanism for NF-kappaB regulation. Morpholino silencing of the zebrafish ortholog of Gprk2 in fish embryos caused impaired cytokine expression after Escherichia coli infection, indicating a conserved role in NF-kappaB signaling. Moreover, small interfering RNA silencing of the human ortholog GRK5 in HeLa cells impaired NF-kappaB reporter activity. Gprk2 RNAi flies are susceptible to infection with Enterococcus faecalis and Gprk2 RNAi rescues Toll(10b)-induced blood cell activation in Drosophila larvae in vivo. We conclude that Gprk2/GRK5 has an evolutionarily conserved role in regulating NF-kappaB signaling.

Eye transformer is a negative regulator of Drosophila JAK/STAT signaling.

JAK/STAT signaling pathway is evolutionarily conserved and tightly regulated. We carried out a reporter-based genome-wide RNAi in vitro screen to identify genes that regulate Drosophila JAK/STAT pathway and found 5 novel regulators. Of these, CG14225 is a negative regulator structurally related to the Drosophila JAK/STAT pathway receptor Domeless, especially in the extracellular domain, and to the mammalian IL-6 receptor and the signal transducer gp130. CG14225 coimmunoprecipitates with Domeless and its associated kinase hopscotch in S2 cells. CG14225 RNAi caused hyperphosphorylation of the transcription factor Stat92E in S2 cells on stimulation with the Drosophila JAK/STAT pathway ligand unpaired. CG14225 RNAi in vivo hyperactivated JAK/STAT target genes on septic injury and enhanced unpaired-induced eye overgrowth, and was thus named the eye transformer (ET). In the gastrointestinal infection model, where JAK/STAT signaling is important for stem cell renewal, CG14225/ET RNAi was protective in vivo. In conclusion, we have identified ET as a novel negative regulator of the Drosophila JAK/STAT pathway both in vitro and in vivo, and it functions in regulating Stat92E phosphorylation.

[Fruit fly--genetically excellent model animal for research]. / Banaanikärpänen--geneettisesti verraton mallieläin tutkimukseen.

Drosophila melanogaster, i.e., the fruit fly is a familiar nuisance in late summer. For those living in a country plagued by myriads of flying insects, it is difficult to think any other model animal as an ethically more suitable research tool. Due to its genetic manipulability, the fruit fly is an excellent research model, but mainly due to lack of experience, has been utilized only to a small extent in medical research in Finland. For instance, the readily available transgenic RNAi strains have made functional, genome-wide screening possible in a complete organism or in a designated tissue.

Proteasome α6 Subunit Negatively Regulates the JAK/STAT Pathway and Blood Cell Activation in Drosophila melanogaster.

JAK/STAT signaling regulates central biological functions such as development, cell differentiation and immune responses. In Drosophila, misregulated JAK/STAT signaling in blood cells (hemocytes) induces their aberrant activation. Using mass spectrometry to analyze proteins associated with a negative regulator of the JAK/STAT pathway, and by performing a genome-wide RNAi screen, we identified several components of the proteasome complex as negative regulators of JAK/STAT signaling in Drosophila. A selected proteasome component, Prosα6, was studied further. In S2 cells, Prosα6 silencing decreased the amount of the known negative regulator of the pathway, ET, leading to enhanced expression of a JAK/STAT pathway reporter gene. Silencing of Prosα6 in vivo resulted in activation of the JAK/STAT pathway, leading to the formation of lamellocytes, a specific hemocyte type indicative of hemocyte activation. This hemocyte phenotype could be partially rescued by simultaneous knockdown of either the Drosophila STAT transcription factor, or MAPKK in the JNK-pathway. Our results suggest a role for the proteasome complex components in the JAK/STAT pathway in Drosophila blood cells both in vitro and in vivo.

Iap2 is required for a sustained response in the Drosophila Imd pathway.

Fruit flies have effective immune response against Gram-negative bacteria. Upon infection, early JNK-signaling pathway mediated response is followed by the action of the Immune deficiency (Imd) signaling cascade, a Drosophila equivalent of mammalian TNF-receptor pathway, leading to the release of antimicrobial peptides. Recently, Tak1-binding protein 2 (Tab2) and Inhibitor of apoptosis 2 (Iap2) were identified as components of the Imd pathway. In this study, we carried out a genome-wide kinetic analysis of the role of Tab2 and Iap2 for immune response in Drosophila S2 cells using oligonucleotide microarrays. Tab2 RNAi abolished the induction of all immune response genes in S2 cells indicating its requirement for signaling both via the Imd and the JNK pathway. The role of Iap2 was more specific. Kinetic analysis indicated that Iap2 is required to sustain antimicrobial peptide gene expression in S2 cells. Furthermore, inactivation of Iap2 by RNAi resulted in impaired microbial resistance in Drosophila in vivo.

Cytokines in Drosophila immunity.

Cytokines are a large and diverse group of small proteins that can affect many biological processes, but most commonly cytokines are known as mediators of the immune response. In the event of an infection, cytokines are produced in response to an immune stimulus, and they function as key regulators of the immune response. Cytokines come in many shapes and sizes, and although they vary greatly in structure, their functions have been well conserved in evolution. The immune signaling pathways that respond to cytokines are remarkably conserved from fly to man. Therefore, Drosophila melanogaster, provides an excellent platform for studying the biology and function of cytokines. In this review, we will describe the cytokines and cytokine-like molecules found in the fly and discuss their roles in host immunity.

Genetic Screen in Drosophila Larvae Links ird1 Function to Toll Signaling in the Fat Body and Hemocyte Motility.

To understand how Toll signaling controls the activation of a cellular immune response in Drosophila blood cells (hemocytes), we carried out a genetic modifier screen, looking for deletions that suppress or enhance the mobilization of sessile hemocytes by the gain-of-function mutation Toll10b (Tl10b). Here we describe the results from chromosome arm 3R, where five regions strongly suppressed this phenotype. We identified the specific genes immune response deficient 1 (ird1), headcase (hdc) and possibly Rab23 as suppressors, and we studied the role of ird1 in more detail. An ird1 null mutant and a mutant that truncates the N-terminal kinase domain of the encoded Ird1 protein affected the Tl10b phenotype, unlike mutations that affect the C-terminal part of the protein. The ird1 null mutant suppressed mobilization of sessile hemocytes, but enhanced other Tl10b hemocyte phenotypes, like the formation of melanotic nodules and the increased number of circulating hemocytes. ird1 mutants also had blood cell phenotypes on their own. They lacked crystal cells and showed aberrant formation of lamellocytes. ird1 mutant plasmatocytes had a reduced ability to spread on an artificial substrate by forming protrusions, which may explain why they did not go into circulation in response to Toll signaling. The effect of the ird1 mutation depended mainly on ird1 expression in hemocytes, but ird1-dependent effects in other tissues may contribute. Specifically, the Toll receptor was translocated from the cell membrane to intracellular vesicles in the fat body of the ird1 mutant, and Toll signaling was activated in that tissue, partially explaining the Tl10b-like phenotype. As ird1 is otherwise known to control vesicular transport, we conclude that the vesicular transport system may be of particular importance during an immune response.