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1.
Curr Biol ; 33(23): 5132-5146.e5, 2023 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-37992718

RESUMO

The mechanisms underlying the construction of an air-liquid interface in respiratory organs remain elusive. Here, we use live imaging and genetic analysis to describe the morphogenetic events generating an extracellular lipid lining of the Drosophila airways required for their gas filing and animal survival. We show that sequential Rab39/Syx1A/Syt1-mediated secretion of lysosomal acid sphingomyelinase (Drosophila ASM [dASM]) and Rab11/35/Syx1A/Rop-dependent exosomal secretion provides distinct components for lipid film assembly. Tracheal inactivation of Rab11 or Rab35 or loss of Rop results in intracellular accumulation of exosomal, multi-vesicular body (MVB)-derived vesicles. On the other hand, loss of dASM or Rab39 causes luminal bubble-like accumulations of exosomal membranes and liquid retention in the airways. Inactivation of the exosomal secretion in dASM mutants counteracts this phenotype, arguing that the exosomal secretion provides the lipid vesicles and that secreted lysosomal dASM organizes them into a continuous film. Our results reveal the coordinated functions of extracellular vesicle and lysosomal secretions in generating a lipid layer crucial for airway gas filling and survival.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Tensoativos , Endossomos , Traqueia , Lipídeos , Proteínas do Tecido Nervoso , Proteínas de Drosophila/genética
2.
Front Biosci (Landmark Ed) ; 28(6): 124, 2023 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-37395037

RESUMO

BACKGROUND: Bruton's tyrosine kinase (BTK) is a non-receptor type tyrosine kinase originally identified as the genetic signature responsible for X-linked agammaglobulinemia (XLA) when mutated. Its functional form is required for B lymphocyte maturation in both humans and mice, whereas loss-of-function causes a different form of developmental defect in the fruit fly, Drosophila melanogaster. METHODS: Ibrutinib and other therapeutic inhibitors of BTK have been extensively used to successfully treat various leukemias and lymphomas. Btk29A type 2 is the ortholog of BTK in the fruit fly. We show that feeding wild-type flies an ibrutinib-containing diet induces phenocopying of Btk29A mutants, i.e., failure in the fusion of left and right halves of the dorsal cuticles, partial loss of wing tissues and dysregulation of germ cell production. RESULTS: We have previously reported that Btk29A phosphorylates Drosophila Arm (ß-catenin), and ibrutinib reduces phosphorylation at Tyrosine142 of endogenously expressed ß-catenin in Cos7 cells transfected with Btk29A type 2 cDNA. CONCLUSIONS: Thus, Drosophila is suitable for screens of novel BTK inhibitor candidates and offers a unique in vivo system in which the mode of action of BTK inhibitors can be examined at the molecular, cellular, and organismal levels.


Assuntos
Drosophila melanogaster , Proteínas Tirosina Quinases , Humanos , Animais , Camundongos , Drosophila melanogaster/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , beta Catenina/metabolismo , Drosophila/metabolismo , Tirosina Quinase da Agamaglobulinemia/genética , Tirosina Quinase da Agamaglobulinemia/metabolismo
3.
Curr Biol ; 32(24): 5354-5363.e3, 2022 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-36347251

RESUMO

In Drosophila melanogaster, processing of gustatory information and controlling feeding behavior are executed by neural circuits located in the subesophageal zone (SEZ) of the brain.1 Gustatory receptor neurons (GRNs) project their axons in the primary gustatory center (PGC), which is located in the SEZ.1,2,3,4 To address the function of the PGC, we need detailed information about the different classes of gustatory interneurons that frame the PGC. In this work, we screened large collections of driver lines for SEZ interneuron-specific labeling and subsequently used candidate lines to access the SEZ neuroblast lineages. We converted 130 Gal4 lines to LexA drivers and carried out functional screening using calcium imaging. We found one neuroblast lineage, TRdm, whose neurons responded to both sweet and bitter tastants, and formed green fluorescent protein (GFP) reconstitution across synaptic partners (GRASP)-positive synapses with sweet sensory neurons. TRdm neurons express the inhibitory transmitter GABA, and silencing these neurons increases appetitive feeding behavior. These results demonstrate that TRdm generates a class of inhibitory local neurons that control taste sensitivity in Drosophila.


Assuntos
Proteínas de Drosophila , Paladar , Animais , Paladar/fisiologia , Drosophila melanogaster/fisiologia , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Células Receptoras Sensoriais/fisiologia
4.
BMC Biol ; 19(1): 185, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34479564

RESUMO

BACKGROUND: A number of cellular processes have evolved in metazoans that increase the proteome repertoire in relation to the genome, such as alternative splicing and translation recoding. Another such process, translational stop codon readthrough (SCR), generates C-terminally extended protein isoforms in many eukaryotes, including yeast, plants, insects, and humans. While comparative genome analyses have predicted the existence of programmed SCR in many species including humans, experimental proof of its functional consequences are scarce. RESULTS: We show that SCR of the Drosophila POU/Oct transcription factor Ventral veins lacking/Drifter (Vvl/Dfr) mRNA is prevalent in certain tissues in vivo, reaching a rate of 50% in the larval prothoracic gland. Phylogenetically, the C-terminal extension is conserved and harbors intrinsically disordered regions and amino acid stretches implied in transcriptional activation. Elimination of Vvl/Dfr translational readthrough by CRISPR/Cas9 mutagenesis changed the expression of a large number of downstream genes involved in processes such as chromatin regulation, neurogenesis, development, and immune response. As a proof-of-principle, we demonstrate that the C-terminal extension of Vvl/Dfr is necessary for correct timing of pupariation, by increasing the capacity to regulate its target genes. The extended Vvl/Dfr isoform acts in synergy with the transcription factor Molting defective (Mld) to increase the expression and biosynthesis of the steroid hormone ecdysone, thereby advancing pupariation. Consequently, late-stage larval development was prolonged and metamorphosis delayed in vvl/dfr readthrough mutants. CONCLUSIONS: We demonstrate that translational recoding of a POU/Oct transcription factor takes place in a highly tissue-specific and temporally controlled manner. This dynamic and regulated recoding is necessary for normal expression of a large number of genes involved in many cellular and developmental processes. Loss of Vvl/Dfr translational readthrough negatively affects steroid hormone biosynthesis and delays larval development and progression into metamorphosis. Thus, this study demonstrates how SCR of a transcription factor can act as a developmental switch in a spatiotemporal manner, feeding into the timing of developmental transitions between different life-cycle stages.


Assuntos
Drosophila , Animais , Códon de Terminação , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ecdisona , Regulação da Expressão Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Cell Rep ; 32(4): 107972, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32726635

RESUMO

Drosophila development is governed by distinct ecdysone steroid pulses that initiate spatially and temporally defined gene expression programs. The translation of these signals into tissue-specific responses is crucial for metamorphosis, but the mechanisms that confer specificity to systemic ecdysone pulses are far from understood. Here, we identify Bric-à-brac 2 (Bab2) as an ecdysone-responsive transcriptional repressor that controls temporal gene expression during larval to pupal transition. Bab2 is necessary to terminate Salivary gland secretion (Sgs) gene expression, while premature Bab2 expression blocks Sgs genes and causes precocious salivary gland histolysis. The timely expression of bab2 is controlled by the ecdysone-responsive transcription factor Broad, and manipulation of EcR/USP/Broad signaling induces inappropriate Bab2 expression and termination of Sgs gene expression. Bab2 directly binds to Sgs loci in vitro and represses all Sgs genes in vivo. Our work characterizes Bab2 as a temporal regulator of somatic gene expression in response to systemic ecdysone signaling.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Fatores de Transcrição/genética , Animais , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Ecdisona/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Larva/metabolismo , Metamorfose Biológica/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética
7.
Sci Rep ; 9(1): 7529, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101845

RESUMO

The absence of high-affinity potassium uptake in Candida glabrata, the consequence of the deletion of the TRK1 gene encoding the sole potassium-specific transporter, has a pleiotropic effect. Here, we show that in addition to changes in basic physiological parameters (e.g., membrane potential and intracellular pH) and decreased tolerance to various cell stresses, the loss of high affinity potassium uptake also alters cell-surface properties, such as an increased hydrophobicity and adherence capacity. The loss of an efficient potassium uptake system results in diminished virulence as assessed by two insect host models, Drosophila melanogaster and Galleria mellonella, and experiments with macrophages. Macrophages kill trk1Δ cells more effectively than wild type cells. Consistently, macrophages accrue less damage when co-cultured with trk1Δ mutant cells compared to wild-type cells. We further show that low levels of potassium in the environment increase the adherence of C. glabrata cells to polystyrene and the propensity of C. glabrata cells to form biofilms.


Assuntos
Candida glabrata/genética , Candida glabrata/patogenicidade , Proteínas de Transporte de Cátions/genética , Adesão Celular/fisiologia , Potássio/metabolismo , Animais , Biofilmes/crescimento & desenvolvimento , Candida glabrata/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Drosophila melanogaster/microbiologia , Regulação Fúngica da Expressão Gênica/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Transporte de Íons , Macrófagos/imunologia , Potenciais da Membrana/fisiologia , Mariposas/microbiologia , Antiportadores de Potássio-Hidrogênio/genética , Propriedades de Superfície , Células THP-1 , Virulência/genética
8.
Insect Biochem Mol Biol ; 109: 24-30, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30954681

RESUMO

The innate immune system of insects deploys both cellular and humoral reactions in immunocompetent tissues for protection of insects against a variety of infections, including bacteria, fungi, and viruses. Transcriptional regulation of genes encoding antimicrobial peptides (AMPs), cytokines, and other immune effectors plays a pivotal role in maintenance of immune homeostasis both prior to and after infections. The POU/Oct transcription factor family is a subclass of the homeodomain proteins present in all metazoans. POU factors are involved in regulation of development, metabolism and immunity. Their role in regulation of immune functions has recently become evident, and involves control of tissue-specific, constitutive expression of immune effectors in barrier epithelia as well as positive and negative control of immune responses in gut and fat body. In addition, they have been shown to affect the composition of gut microbiota and play a role in regulation of intestinal stem cell activities. In this review, we summarize the current knowledge of how POU transcription factors control Drosophila immune homeostasis in healthy and infected insects. The role of POU factor isoform specific regulation of stem cell activities in Drosophila and mammals is also discussed.


Assuntos
Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Regulação da Expressão Gênica/imunologia , Homeostase/imunologia , Fatores do Domínio POU/genética , Fatores do Domínio POU/imunologia , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/imunologia , Homeostase/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia
9.
Development ; 146(12)2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30642837

RESUMO

The variability in transcription factor concentration among cells is an important developmental determinant, yet how variability is controlled remains poorly understood. Studies of variability have focused predominantly on monitoring mRNA production noise. Little information exists about transcription factor protein variability, as this requires the use of quantitative methods with single-molecule sensitivity. Using Fluorescence Correlation Spectroscopy (FCS), we have characterized the concentration and variability of 14 endogenously tagged TFs in live Drosophila imaginal discs. For the Hox TF Antennapedia, we investigated whether protein variability results from random stochastic events or is developmentally regulated. We found that Antennapedia transitioned from low concentration/high variability early, to high concentration/low variability later, in development. FCS and temporally resolved genetic studies uncovered that Antennapedia itself is necessary and sufficient to drive a developmental regulatory switch from auto-activation to auto-repression, thereby reducing variability. This switch is controlled by progressive changes in relative concentrations of preferentially activating and repressing Antennapedia isoforms, which bind chromatin with different affinities. Mathematical modeling demonstrated that the experimentally supported auto-regulatory circuit can explain the increase of Antennapedia concentration and suppression of variability over time.


Assuntos
Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Discos Imaginais/metabolismo , Fatores de Transcrição/metabolismo , Alelos , Animais , Proteína do Homeodomínio de Antennapedia/metabolismo , Sítios de Ligação , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Elementos Facilitadores Genéticos , Feminino , Genes Homeobox , Genótipo , Homozigoto , Masculino , Modelos Biológicos , Modelos Teóricos , Fenótipo , Ligação Proteica , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Espectrometria de Fluorescência , Processos Estocásticos , Transgenes
10.
PLoS Pathog ; 14(7): e1007076, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30059535

RESUMO

Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells' oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells' hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress.


Assuntos
Candida albicans/patogenicidade , Candidíase/metabolismo , Estresse Oxidativo/fisiologia , Simportadores de Próton-Fosfato/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Transporte Biológico/fisiologia , Drosophila , Proteínas Fúngicas/metabolismo , Humanos , Camundongos , Fosfatos/metabolismo , Transdução de Sinais/fisiologia , Virulência
11.
Stem Cell Reports ; 10(5): 1565-1578, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29681543

RESUMO

Drosophila POU/Oct transcription factors are required for many developmental processes, but their putative regulation of adult stem cell activity has not been investigated. Here, we show that Nubbin (Nub)/Pdm1, homologous to mammalian OCT1/POU2F1 and related to OCT4/POU5F1, is expressed in gut epithelium progenitor cells. We demonstrate that the nub-encoded protein isoforms, Nub-PB and Nub-PD, play opposite roles in the regulation of intestinal stem cell (ISC) maintenance and differentiation. Depletion of Nub-PB in progenitor cells increased ISC proliferation by derepression of escargot expression. Conversely, loss of Nub-PD reduced ISC proliferation, suggesting that this isoform is necessary for ISC maintenance, analogous to mammalian OCT4/POU5F1 functions. Furthermore, Nub-PB is required in enteroblasts to promote differentiation, and it acts as a tumor suppressor of Notch RNAi-driven hyperplasia. We suggest that a dynamic and well-tuned expression of Nub isoforms in progenitor cells is required for maintaining gut epithelium homeostasis.


Assuntos
Diferenciação Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Proteínas de Homeodomínio/metabolismo , Intestinos/citologia , Fatores do Domínio POU/metabolismo , Células-Tronco/citologia , Envelhecimento/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Feminino , Modelos Biológicos , Mutação/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , Receptores Notch/metabolismo , Células-Tronco/metabolismo
12.
PLoS Pathog ; 14(3): e1006936, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29499056

RESUMO

Gut immunity is regulated by intricate and dynamic mechanisms to ensure homeostasis despite a constantly changing microbial environment. Several regulatory factors have been described to participate in feedback responses to prevent aberrant immune activity. Little is, however, known about how transcriptional programs are directly tuned to efficiently adapt host gut tissues to the current microbiome. Here we show that the POU/Oct gene nubbin (nub) encodes two transcription factor isoforms, Nub-PB and Nub-PD, which antagonistically regulate immune gene expression in Drosophila. Global transcriptional profiling of adult flies overexpressing Nub-PB in immunocompetent tissues revealed that this form is a strong transcriptional activator of a large set of immune genes. Further genetic analyses showed that Nub-PB is sufficient to drive expression both independently and in conjunction with nuclear factor kappa B (NF-κB), JNK and JAK/STAT pathways. Similar overexpression of Nub-PD did, conversely, repress expression of the same targets. Strikingly, isoform co-overexpression normalized immune gene transcription, suggesting antagonistic activities. RNAi-mediated knockdown of individual nub transcripts in enterocytes confirmed antagonistic regulation by the two isoforms and that both are necessary for normal immune gene transcription in the midgut. Furthermore, enterocyte-specific Nub-PB expression levels had a strong impact on gut bacterial load as well as host lifespan. Overexpression of Nub-PB enhanced bacterial clearance of ingested Erwinia carotovora carotovora 15. Nevertheless, flies quickly succumbed to the infection, suggesting a deleterious immune response. In line with this, prolonged overexpression promoted a proinflammatory signature in the gut with induction of JNK and JAK/STAT pathways, increased apoptosis and stem cell proliferation. These findings highlight a novel regulatory mechanism of host-microbe interactions mediated by antagonistic transcription factor isoforms.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/imunologia , Proteínas de Homeodomínio/metabolismo , Homeostase , Imunidade Inata/imunologia , Intestinos/imunologia , Fatores do Domínio POU/metabolismo , Animais , Animais Geneticamente Modificados , Padronização Corporal , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/microbiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Intestinos/microbiologia , Masculino , NF-kappa B/genética , NF-kappa B/metabolismo , Fatores do Domínio POU/genética , Pectobacterium carotovorum/patogenicidade , Isoformas de Proteínas
13.
J Innate Immun ; 8(4): 412-26, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27231014

RESUMO

Maintenance of a stable gut microbial community relies on a delicate balance between immune defense and immune tolerance. We have used Drosophila to study how the microbial gut flora is affected by changes in host genetic factors and immunity. Flies with a constitutively active gut immune system, due to a mutation in the POU transcriptional regulator Pdm1/nubbin (nub) gene, had higher loads of bacteria and a more diverse taxonomic composition than controls. In addition, the microbial composition shifted considerably during the short lifespan of the nub1 mutants. This shift was characterized by a loss of relatively few OTUs (operational taxonomic units) and a remarkable increase in a large number of Acetobacter spp. and Leuconostoc spp. Treating nub1 mutant flies with antibiotics prolonged their lifetime survival by more than 100%. Immune gene expression was also persistently high in the presence of antibiotics, indicating that the early death was not a direct consequence of an overactive immune defense but rather an indirect consequence of the microbial load and composition. Thus, changes in host genotype and an inability to regulate the normal growth and composition of the gut microbiota leads to a shift in the microbial community, dysbiosis and early death.


Assuntos
Acetobacter/imunologia , Proteínas de Drosophila/genética , Drosophila melanogaster/imunologia , Disbiose/imunologia , Microbioma Gastrointestinal/imunologia , Proteínas de Homeodomínio/genética , Mucosa Intestinal/imunologia , Leuconostoc/imunologia , Mutação/genética , Fatores do Domínio POU/genética , Animais , Animais Geneticamente Modificados , Carga Bacteriana , Proteínas de Drosophila/metabolismo , Proteínas de Homeodomínio/metabolismo , Interações Hospedeiro-Patógeno , Tolerância Imunológica , Imunidade Inata , Mucosa Intestinal/microbiologia , Longevidade , Fatores do Domínio POU/metabolismo
14.
BMC Biol ; 11: 99, 2013 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-24010524

RESUMO

BACKGROUND: Innate immune responses are evolutionarily conserved processes that provide crucial protection against invading organisms. Gene activation by potent NF-κB transcription factors is essential both in mammals and Drosophila during infection and stress challenges. If not strictly controlled, this potent defense system can activate autoimmune and inflammatory stress reactions, with deleterious consequences for the organism. Negative regulation to prevent gene activation in healthy organisms, in the presence of the commensal gut flora, is however not well understood. RESULTS: We show that the Drosophila homolog of mammalian Oct1/POU2F1 transcription factor, called Nubbin (Nub), is a repressor of NF-κB/Relish-driven antimicrobial peptide gene expression in flies. In nub1 mutants, which lack Nub-PD protein, excessive expression of antimicrobial peptide genes occurs in the absence of infection, leading to a significant reduction of the numbers of cultivatable gut commensal bacteria. This aberrant immune gene expression was effectively blocked by expression of Nub from a transgene. We have identified an upstream regulatory region, containing a cluster of octamer sites, which is required for repression of antimicrobial peptide gene expression in healthy flies. Chromatin immunoprecipitation experiments demonstrated that Nub binds to octamer-containing promoter fragments of several immune genes. Gene expression profiling revealed that Drosophila Nub negatively regulates many genes that are involved in immune and stress responses, while it is a positive regulator of genes involved in differentiation and metabolism. CONCLUSIONS: This study demonstrates that a large number of genes that are activated by NF-κB/Relish in response to infection are normally repressed by the evolutionarily conserved Oct/POU transcription factor Nub. This prevents uncontrolled gene activation and supports the existence of a normal gut flora. We suggest that Nub protein plays an ancient role, shared with mammalian Oct/POU transcription factors, to moderate responses to immune challenge, thereby increasing the tolerance to biotic stress.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/microbiologia , Trato Gastrointestinal/microbiologia , Proteínas de Homeodomínio/metabolismo , Microbiota , Fatores do Domínio POU/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Drosophila/genética , Proteínas de Homeodomínio/genética , Tolerância Imunológica/genética , Tolerância Imunológica/imunologia , Imunidade Inata/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Fatores do Domínio POU/genética , Regulação para Cima
15.
J Innate Immun ; 4(3): 273-83, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22237424

RESUMO

The barrier epithelia of multicellular organisms frequently come into direct contact with microorganisms and thus need to fulfill the important task of preventing the penetration of pathogens that could cause systemic infections. A functional immune defence in the epithelial linings of the digestive, respiratory and reproductive organs as well as the epidermis/skin of animals is therefore of crucial importance for survival. Epithelial defence reactions are likely to be evolutionarily ancient, and the use of invertebrate animal models, such as insects and nematodes, has been crucial in unravelling the mechanisms underlying epithelial immunity. This review addresses basic questions of epithelial immunity in animals and humans. It focuses on recent developments in the understanding of the immune responses in the fruit fly Drosophila melanogaster and how the innate immune system acts locally in the epidermis and cuticle, tracheae, gut and genital organs. Both basal immune activities in epithelia that are constantly exposed to microbes as well as positive and negative regulation in response to pathogenic organisms are covered. Important immuno-physiological aspects of epithelial defence mechanisms are also discussed, such as wound healing, re-epithelialization and intestinal homeostasis.


Assuntos
Drosophila melanogaster/imunologia , Epitélio/imunologia , Mucosa Intestinal/imunologia , Sistema Respiratório/imunologia , Pele/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Proteínas de Homeodomínio/imunologia , Homeostase/imunologia , Humanos , Imunidade Inata , Imunomodulação , Receptores de Reconhecimento de Padrão/imunologia , Transdução de Sinais , Ativação Transcricional
16.
PLoS One ; 6(11): e27434, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22110651

RESUMO

The fungal pathogen Candida albicans is a common cause of opportunistic infections in humans. We report that wild-type Drosophila melanogaster (OrR) flies are susceptible to virulent C. albicans infections and have established experimental conditions that enable OrR flies to serve as model hosts for studying C. albicans virulence. After injection into the thorax, wild-type C. albicans cells disseminate and invade tissues throughout the fly, leading to lethality. Similar to results obtained monitoring systemic infections in mice, well-characterized cph1Δ efg1Δ and csh3Δ fungal mutants exhibit attenuated virulence in flies. Using the OrR fly host model, we assessed the virulence of C. albicans strains individually lacking functional components of the SPS sensing pathway. In response to extracellular amino acids, the plasma membrane localized SPS-sensor (Ssy1, Ptr3, and Ssy5) activates two transcription factors (Stp1 and Stp2) to differentially control two distinct modes of nitrogen acquisition (host protein catabolism and amino acid uptake, respectively). Our results indicate that a functional SPS-sensor and Stp1 controlled genes required for host protein catabolism and utilization, including the major secreted aspartyl protease SAP2, are required to establish virulent infections. By contrast, Stp2, which activates genes required for amino acid uptake, is dispensable for virulence. These results indicate that nutrient availability within infected hosts directly influences C. albicans virulence.


Assuntos
Candida albicans/metabolismo , Candida albicans/patogenicidade , Drosophila melanogaster/microbiologia , Nitrogênio/metabolismo , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Feminino , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica/imunologia , Injeções , Masculino , Camundongos , Mutação , Fagocitose , Saccharomyces cerevisiae/imunologia , Transdução de Sinais/imunologia , Tórax/microbiologia
17.
Dev Comp Immunol ; 35(3): 263-6, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21075135

RESUMO

Innate immunity in Drosophila involves the inducible expression and synthesis of antimicrobial peptides. We have previously shown that not only Drosophila larvae and adults, but also embryos, are capable of mounting an immune response after injection of bacterial substances. To simplify genetic dissection of the signaling pathways involved in immune-gene regulation we developed a procedure for permeabilization of large number of embryos and subsequent infiltration with bacterial fragments. This approach, which promoted expression of CecropinA1- and Diptericin-driven ß-gal expression in the epidermis of more than 90% of the treated embryos, will enable analysis of mutants that are embryonic lethal. Thus, genes that are involved in essential pleiotrophic functions, in addition to being candidates in immune-regulation will be amenable for analysis of their involvement in the fly's immune defense.


Assuntos
Técnicas de Laboratório Clínico , Proteínas de Drosophila/imunologia , Drosophila/imunologia , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Imunidade Inata/imunologia , Animais , Drosophila/embriologia , Embrião não Mamífero , Larva , Permeabilidade
18.
Mol Cell Biol ; 30(14): 3672-84, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20457811

RESUMO

Innate immunity operates as a first line of defense in multicellular organisms against infections caused by different classes of microorganisms. Antimicrobial peptides (AMPs) are synthesized constitutively in barrier epithelia to protect against microbial attack and are also upregulated in response to infection. Here, we implicate Drifter/Ventral veinless (Dfr/Vvl), a class III POU domain transcription factor, in tissue-specific regulation of the innate immune defense of Drosophila. We show that Dfr/Vvl is highly expressed in a range of immunocompetent tissues, including the male ejaculatory duct, where its presence overlaps with and drives the expression of cecropin, a potent broad-spectrum AMP. Dfr/Vvl overexpression activates transcription of several AMP genes in uninfected flies in a Toll pathway- and Imd pathway-independent manner. Dfr/Vvl activates a CecA1 reporter gene both in vitro and in vivo by binding to an upstream enhancer specific for the male ejaculatory duct. Further, Dfr/Vvl and the homeodomain protein Caudal (Cad) activate transcription synergistically via this enhancer. We propose that the POU protein Dfr/Vvl acts together with other regulators in a combinatorial manner to control constitutive AMP gene expression in a gene-, tissue-, and sex-specific manner, thus promoting a first-line defense against infection in tissues that are readily exposed to pathogens.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/imunologia , Imunidade Inata/genética , Fatores do Domínio POU/metabolismo , Animais , Animais Geneticamente Modificados , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Sequência de Bases , Primers do DNA/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/imunologia , Elementos Facilitadores Genéticos , Feminino , Genes de Insetos , Genitália Masculina/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Proteínas de Homeodomínio/metabolismo , Masculino , Modelos Biológicos , Mutação , Fatores do Domínio POU/genética , Fatores do Domínio POU/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Ativação Transcricional
19.
J Immunol ; 184(11): 6188-98, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20421637

RESUMO

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.


Assuntos
Proteínas de Drosophila/imunologia , Quinase 2 de Receptor Acoplado a Proteína G/imunologia , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Imunidade Inata , NF-kappa B/imunologia , Transdução de Sinais/fisiologia , Animais , Western Blotting , Drosophila , Proteínas de Drosophila/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/imunologia , Bactérias Gram-Negativas Quimiolitotróficas/imunologia , Bactérias Gram-Negativas Quimiolitotróficas/metabolismo , Humanos , Imuno-Histoquímica , Imunoprecipitação , NF-kappa B/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Peixe-Zebra
20.
Dev Dyn ; 237(1): 196-208, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18069688

RESUMO

Drosophila Dappled (DPLD) is a member of the RBCC/TRIM superfamily, a protein family involved in numerous diverse processes such as developmental timing and asymmetric cell divisions. DPLD belongs to the LIN-41 subclade, several members of which are micro RNA (miRNA) regulated. We re-examined the LIN-41 subclade members and their relation to other RBCC/TRIMs and dpld paralogs, and identified a new Drosophila muscle specific RBCC/TRIM: Another B-Box Affiliate, ABBA. In silico predictions of candidate miRNA regulators of dpld identified let-7 as the strongest candidate. Overexpression of dpld led to abnormal eye development, indicating that strict regulation of dpld mRNA levels is crucial for normal eye development. This phenotype was sensitive to let-7 dosage, suggesting let-7 regulation of dpld in the eye disc. A cell-based assay verified let-7 miRNA down-regulation of dpld expression by means of its 3'-untranslated region. Thus, dpld seems also to be miRNA regulated, suggesting that miRNAs represent an ancient mechanism of LIN-41 regulation.


Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Metaloproteínas/genética , MicroRNAs/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte , Células Cultivadas , Drosophila/classificação , Drosophila/embriologia , Proteínas de Drosophila/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Metaloproteínas/metabolismo , MicroRNAs/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico
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