RESUMO
Viruses are infectious entities that make use of the replication machinery of their hosts to produce more progenies, causing disease and sometimes death. To counter viral infection, metazoan hosts are equipped with various defense mechanisms, from the rapid-evoking innate immune responses to the most advanced adaptive immune responses. Previous research demonstrated that cells in fruit flies and mice infected with Drosophila C virus and influenza, respectively, undergo apoptosis, which triggers the engulfment of apoptotic virus-infected cells by phagocytes. This process involves the recognition of eat-me signals on the surface of virus-infected cells by receptors of specialized phagocytes, such as macrophages and neutrophils in mice and hemocytes in fruit flies, to facilitate the phagocytic elimination of virus-infected cells. Inhibition of phagocytosis led to severe pathologies and death in both species, indicating that apoptosis-dependent phagocytosis of virus-infected cells is a conserved antiviral mechanism in multicellular organisms. Indeed, our understanding of the mechanisms underlying apoptosis-dependent phagocytosis of virus-infected cells has shed a new perspective on how hosts defend themselves against viral infection. This chapter explores the mechanisms of this process and its potential for developing new treatments for viral diseases.
Assuntos
Fagocitose , Viroses , Animais , Camundongos , Fagocitose/fisiologia , Fagócitos/fisiologia , Imunidade Inata , Apoptose/fisiologia , AntiviraisRESUMO
Immunity is considered to be involved in the prevention of cancer. Although both humoral and cellular immune reactions may participate, underlying mechanisms have yet to be clarified. The present study was conducted to clarify this issue using a Drosophila model, in which neoplastic transformation was induced through the simultaneous inhibition of cell-cycle checkpoints and apoptosis. We first determined the location of hemocytes, blood cells of Drosophila playing a role of immune cells, in neoplasia-induced and normal larvae, but there was no significant difference between the two groups. When gene expression pattern in larval hemocytes was determined, the expression of immunity-related genes including those necessary for phagocytosis was reduced in the neoplasia model. We then asked the involvement of phagocytosis in the prevention of neoplasia examining animals where the expression of engulfment receptors instead of apoptosis was retarded. We found that the inhibition of engulfment receptor expression augmented the occurrence of neoplasia induced by a defect in cell-cycle checkpoints. This suggested a role for phagocytosis in the prevention of neoplastic transformation in Drosophila.
Assuntos
Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/metabolismo , Fagocitose/imunologia , Animais , Apoptose/imunologia , Linhagem Celular , Transformação Celular Neoplásica/genética , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Drosophila melanogaster/metabolismo , Feminino , Hemócitos/citologia , Hemócitos/imunologia , Hemócitos/metabolismo , Larva/metabolismo , Masculino , Neoplasias/genética , Neoplasias/imunologia , Fagocitose/genética , Fagocitose/fisiologiaRESUMO
The phagocytic elimination of cells undergoing apoptosis is an evolutionarily conserved innate immune mechanism for eliminating unnecessary cells. Previous studies showed an increase in the level of engulfment receptors in phagocytes after the phagocytosis of apoptotic cells, which leads to the enhancement of their phagocytic activity. However, precise mechanisms underlying this phenomenon require further clarification. We found that the pre-incubation of a Drosophila phagocyte cell line with the fragments of apoptotic cells enhanced the subsequent phagocytosis of apoptotic cells, accompanied by an augmented expression of the engulfment receptors Draper and integrin αPS3. The DNA-binding activity of the transcription repressor Tailless was transiently raised in those phagocytes, depending on two partially overlapping signal-transduction pathways for the induction of phagocytosis as well as the occurrence of engulfment. The RNAi knockdown of tailless in phagocytes abrogated the enhancement of both phagocytosis and engulfment receptor expression. Furthermore, the hemocyte-specific RNAi of tailless reduced apoptotic cell clearance in Drosophila embryos. Taken together, we propose the following mechanism for the activation of Drosophila phagocytes after an encounter with apoptotic cells: two partially overlapping signal-transduction pathways for phagocytosis are initiated; transcription repressor Tailless is activated; expression of engulfment receptors is stimulated; and phagocytic activity is enhanced. This phenomenon most likely ensures the phagocytic elimination of apoptotic cells by stimulated phagocytes and is thus considered as a mechanism to prime phagocytes in innate immunity.
Assuntos
Apoptose , Fagócitos/citologia , Transdução de Sinais , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Cicloeximida/química , Proteínas do Citoesqueleto/metabolismo , DNA/análise , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Hemócitos/citologia , Imunidade Inata , Cadeias alfa de Integrinas/metabolismo , Proteínas de Membrana/metabolismo , Proteína Oncogênica v-crk/metabolismo , Fagocitose , Interferência de RNA , Proteínas Repressoras/metabolismoRESUMO
An RNA chaperone of Escherichia coli, called host factor required for phage Qß RNA replication (Hfq), forms a complex with small noncoding RNAs to facilitate their binding to target mRNA for the alteration of translation efficiency and stability. Although the role of Hfq in the virulence and drug resistance of bacteria has been suggested, how this RNA chaperone controls the infectious state remains unknown. In the present study, we addressed this issue using Drosophila melanogaster as a host for bacterial infection. In an assay for abdominal infection using adult flies, an E. coli strain with mutation in hfq was eliminated earlier, whereas flies survived longer compared with infection with a parental strain. The same was true with flies deficient in humoral responses, but the mutant phenotypes were not observed when a fly line with impaired hemocyte phagocytosis was infected. The results from an assay for phagocytosis in vitro revealed that Hfq inhibits the killing of E. coli by Drosophila phagocytes after engulfment. Furthermore, Hfq seemed to exert this action partly through enhancing the expression of σ(38), a stress-responsive σ factor that was previously shown to be involved in the inhibition of phagocytic killing of E. coli, by a posttranscriptional mechanism. Our study indicates that the RNA chaperone Hfq contributes to the persistent infection of E. coli by maintaining the expression of bacterial genes, including one coding for σ(38), that help bacteria evade host immunity.
Assuntos
Drosophila/microbiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Regulação Bacteriana da Expressão Gênica/fisiologia , Fator Proteico 1 do Hospedeiro/metabolismo , Animais , Western Blotting , Modelos Animais de Doenças , Hemócitos/microbiologia , Fagocitose/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Virulência/fisiologiaRESUMO
We investigated whether phagocytosis participates in the protection of insects from viral infection using the natural host-virus interaction between Drosophila melanogaster and Drosophila C virus (DCV). Drosophila S2 cells were induced to undergo apoptotic cell death upon DCV infection. However, UV-inactivated virus was unable to cause apoptosis, indicating the need for productive infection for apoptosis induction. S2 cells became susceptible to phagocytosis by hemocyte-derived l(2)mbn cells after viral infection, and the presence of phagocytes in S2 cell cultures reduced viral proliferation. Phagocytosis depended, in part, on caspase activity in S2 cells, as well as the engulfment receptors Draper and integrin ßν in phagocytes. To validate the in vivo situation, adult flies were abdominally infected with DCV, followed by the analysis of fly death and viral growth. DCV infection killed flies in a dose-responding manner, and the activation of effector caspases was evident, as revealed by the cleavage of a target protein ectopically expressed in flies. Furthermore, hemocytes isolated from infected flies contained DCV-infected cells, and preinjection of latex beads to inhibit the phagocytic activity of hemocytes accelerated fly death after viral infection. Likewise, viral virulence was exaggerated in flies lacking the engulfment receptors, and was accompanied by the augmented proliferation of virus. Finally, phagocytosis of DCV-infected cells in vitro was inhibited by phosphatidylserine-containing liposome, and virus-infected flies died early when a phosphatidylserine-binding protein was ectopically expressed. Collectively, our study demonstrates that the apoptosis-dependent, phosphatidylserine-mediated phagocytosis of virus-infected cells plays an important role in innate immune responses against viral infection in Drosophila.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/imunologia , Hemócitos/fisiologia , Vírus de Insetos/fisiologia , Cadeias beta de Integrinas/metabolismo , Proteínas de Membrana/metabolismo , Fagócitos/fisiologia , Viroses/imunologia , Animais , Apoptose/efeitos da radiação , Caspases Efetoras/genética , Caspases Efetoras/metabolismo , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/virologia , Hemócitos/virologia , Imunidade Inata , Vírus de Insetos/patogenicidade , Vírus de Insetos/efeitos da radiação , Cadeias beta de Integrinas/genética , Proteínas de Membrana/genética , Mutação/genética , Fagócitos/virologia , Fagocitose/genética , Fosfatidilserinas/metabolismo , Raios Ultravioleta , VirulênciaRESUMO
Cells that have become unwanted by the body need to be selectively, rapidly, and safely removed. The removal of these cells is achieved by apoptosis-dependent phagocytosis: unwanted cells are induced to undergo apoptosis and given susceptibility to phagocytosis. Phagocytes recognize these cells using engulfment receptors that bind substances expressed on the surface of target cells during the apoptotic process. The phagocytic elimination of cells undergoing apoptosis is a mechanism that is conserved among multicellular organisms. Malfunctions in this process may lead to structural and functional defects in morphogenesis and tissue homeostasis. Therefore, molecules involved in this phenomenon may be targeted in medical treatments. The mechanisms responsible for the apoptosis-dependent phagocytosis of unwanted cells as well as its physiological and pathological consequences are described herein.
Assuntos
Apoptose/fisiologia , Fagócitos/fisiologia , Fagocitose/fisiologia , Transdução de Sinais/fisiologia , Citoesqueleto de Actina/fisiologia , Animais , Apresentação de Antígeno/fisiologia , Humanos , Morfogênese/fisiologia , Fosfatidilserinas/fisiologiaRESUMO
Bacteria adapt themselves to host environments by altering the pattern of gene expression. The promoter-recognizing subunit σ of bacterial RNA polymerase plays a major role in the selection of genes to be transcribed. Among seven σ factors of Escherichia coli, σ(38) is responsible for the transcription of genes in the stationary phase and under stressful conditions. We found a transient increase of σ(38) when E. coli was injected into the hemocoel of Drosophila melanogaster. The loss of σ(38) made E. coli rapidly eliminated in flies, and flies infected with σ(38)-lacking E. coli stayed alive longer than those infected with the parental strain. This was also observed in fly lines defective in humoral immune responses, but not in flies in which phagocytosis was impaired. The lack of σ(38) did not influence the susceptibility of E. coli to phagocytosis, but made them vulnerable to killing after engulfment. The changes caused by the loss of σ(38) were recovered by the forced expression of σ(38)-encoding rpoS as well as σ(38)-regulated katE and katG coding for enzymes that detoxify reactive oxygen species. These results collectively suggested that σ(38) contributes to the prolonged survival of E. coli in Drosophila by inducing the production of enzymes that protect bacteria from killing in phagocytes. Considering the similarity in the mechanism of innate immunity against invading bacteria between fruit flies and humans, the products of these genes could be new targets for the development of more effective antibacterial remedies.
Assuntos
Drosophila melanogaster/microbiologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Fator sigma/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Catalase/genética , Catalase/imunologia , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/imunologia , Drosophila melanogaster/imunologia , Escherichia coli/imunologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/imunologia , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/imunologia , Imunidade Humoral/genética , Imunidade Humoral/imunologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Masculino , Fagocitose/genética , Fagocitose/imunologia , Espécies Reativas de Oxigênio/imunologia , Fator sigma/imunologiaRESUMO
Integrins exert a variety of cellular functions as heterodimers of two transmembrane subunits named α and ß. Integrin ßν, a ß-subunit of Drosophila integrin, is involved in the phagocytosis of apoptotic cells and bacteria. Here, we searched for an α-subunit that forms a complex and cooperates with ßν. Examinations of RNAi-treated animals suggested that αPS3, but not any of four other α-subunits, is required for the effective phagocytosis of apoptotic cells in Drosophila embryos. The mutation of αPS3-encoding scb, deficiency, insertion of P-element, or alteration of nucleotide sequences, brought about a reduction in the level of phagocytosis. The defect in phagocytosis by deficiency was reverted by the forced expression of scb. Furthermore, flies in which the expression of both αPS3 and ßν was inhibited by RNAi showed a level of phagocytosis almost equal to that observed in flies with RNAi for either subunit alone. A loss of αPS3 also decreased the activity of larval hemocytes in the phagocytosis of Staphylococcus aureus. Finally, a co-immunoprecipitation analysis using a Drosophila cell line treated with a chemical cross-linker suggested a physical association between αPS3 and ßν. These results collectively indicated that integrin αPS3/ßν serves as a receptor in the phagocytosis of apoptotic cells and bacteria by Drosophila phagocytes.
Assuntos
Apoptose/fisiologia , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Hemócitos/metabolismo , Cadeias alfa de Integrinas/metabolismo , Cadeias beta de Integrinas/metabolismo , Fagocitose/fisiologia , Staphylococcus aureus , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Embrião não Mamífero/citologia , Hemócitos/citologia , Cadeias alfa de Integrinas/genética , Cadeias beta de Integrinas/genética , MutaçãoRESUMO
It is widely acknowledged that smoking exacerbates the severity of infectious diseases. A presumed mechanism involves the damage inflicted by tobacco smoke on the organs of host organisms. In this study, an alternative hypothesis was explored: smoking enhances the virulence of bacteria. This possibility was investigated using Escherichia coli as the model bacteria and Drosophila as the host organism. Our inquiry focused on the potential gene expression changes in E. coli subsequent to exposure to tobacco smoke extracts. Analysis of the transcription promoter activity of genes encoding proteins within the E. coli two-component system, a regulatory machinery governing gene expression, revealed the suppression of thirteen out of 23 promoters in response to tobacco smoke extracts. Subsequently, Drosophila was infected with E. coli exposed to tobacco smoke extracts or left untreated. Interestingly, there were no significant differences observed in the survival periods of Drosophila following infection with E. coli, whether treated or untreated with tobacco smoke extracts. Contrary to the initial hypothesis, the findings suggest that while tobacco smoke extracts alter gene expression in E. coli, these changes do not appear to impact bacterial virulence. Although this study has illuminated the influence of tobacco smoke extracts on the gene expression of E. coli, further analyses are necessary to elucidate the implications of these changes. Nevertheless, the results imply that smoking affects not only host organisms but may also exert influence on invading bacteria.
Assuntos
Escherichia coli , Escherichia coli/genética , Escherichia coli/patogenicidade , Escherichia coli/efeitos dos fármacos , Animais , Virulência/genética , Nicotiana/efeitos adversos , Nicotiana/microbiologia , Drosophila/microbiologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Fumaça/efeitos adversos , Fatores de Virulência/genéticaRESUMO
Aging is commonly characterized by a decline in the physiological functioning of the body organs, with one hallmark being the impairment of intestinal function, leading to increased intestinal permeability known as leaky gut. The aim of this study was to investigate the potential of curcumin to prevent the development of leaky gut in Drosophila melanogaster utilizing the smurf fly method. In this study, flies aged 3-5 days underwent a 10-day dextran sulfate sodium (DSS) treatment to induce intestinal permeability, followed by a smurf assay using brilliant blue dye and locomotor testing the next day. Flies displaying the smurf phenotype were divided into four groups: untreated control and curcumin-treated (10 µM, 50 µM, and 250 µM). After 21 days of treatment, flies were reassessed for the smurf phenotype and underwent locomotor testing. On day 23, flies were subjected to RT-qPCR analysis. By inducing increased intestinal permeability through the administration of DSS, a higher proportion of flies exhibiting the smurf phenotype and a reduced survival rate in the DSS-treated group were observed. Such phenotypes were reversed, decreased number of flies displaying the smurf phenotype and improved fly survival, upon the incorporation of curcumin in the fly food at concentrations of 10, 50, and 250 µM. Subsequent molecular analysis revealed upregulated expression of sod1, cat, and pepck genes, while no significant changes were observed in the expression of sod2, indy, and srl genes following treatment with curcumin at high concentration. Overall, our findings provide insight into the potential effect of curcumin to alleviate the phenotypical features associated with DSS-induced leaky gut, possibly via the selective regulation of aging-related genes.
Assuntos
Curcumina , Drosophila melanogaster , Permeabilidade , Animais , Curcumina/farmacologia , Drosophila melanogaster/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Sulfato de Dextrana , FenótipoRESUMO
N-glycosylation and proper processing of N-glycans are required for the function of membrane proteins including cell surface receptors. Fibroblast growth factor receptor (FGFR) is involved in a wide variety of biological processes including embryonic development, osteogenesis, angiogenesis, and cell proliferation. Human FGFR3 contains six potential N-glycosylation sites, however, the roles of glycosylation have not been elucidated. The site-specific profiles of N-glycans of the FGFR3 extracellular domain expressed and secreted by CHO-K1 cells were examined, and glycan occupancies and structures of four sites were determined. The results indicated that most sites were fully occupied by glycans, and the dominant populations were the complex type. By examining single N-glycan deletion mutants of FGFR3, it was found that N262Q mutation significantly increased the population with oligomannose-type N-glycans, which was localized in the endoplasmic reticulum. Protein stability assay suggested that fraction with oligomannose-type N-glycans in the N262Q mutant is more stable than those in the wild type and other mutants. Furthermore, it was found that ligand-independent phosphorylation was significantly upregulated in N262Q mutants with complex type N-glycans. The findings suggest that N-glycans on N262 of FGFR3 affect the intracellular localization and phosphorylation status of the receptor.
Assuntos
Fenômenos Biológicos , Polissacarídeos , Cricetinae , Animais , Humanos , Fosforilação , Glicosilação , Células CHO , Cricetulus , Polissacarídeos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
Microbial ecosystems experience spatial and nutrient restrictions leading to the coevolution of cooperation and competition among cohabiting species. To increase their fitness for survival, bacteria exploit machinery to antagonizing rival species upon close contact. As such, the bacterial type VI secretion system (T6SS) nanomachinery, typically expressed by pathobionts, can transport proteins directly into eukaryotic or prokaryotic cells, consequently killing cohabiting competitors. Here, we demonstrate for the first time that oral symbiont Aggregatibacter aphrophilus possesses a T6SS and can eliminate its close relative oral pathobiont Aggregatibacter actinomycetemcomitans using its T6SS. These findings bring nearer the anti-bacterial prospects of symbionts against cohabiting pathobionts while introducing the presence of an active T6SS in the oral cavity.
RESUMO
Integrin ßν, one of two ß subunits of Drosophila integrin, acts as a receptor in the phagocytosis of apoptotic cells. We here examined the involvement of this receptor in defense against infection by Staphylococcus aureus. Flies lacking integrin ßν died earlier than control flies upon a septic but not oral infection with this bacterium. A loss of integrin ßν reduced the phagocytosis of S. aureus and increased bacterial growth in flies. In contrast, the level of mRNA of an antimicrobial peptide produced upon infection was unchanged in integrin ßν-lacking flies. The simultaneous loss of integrin ßν and Draper, another receptor involved in the phagocytosis of S. aureus, brought about a further decrease in the level of phagocytosis and accelerated death of flies compared with the loss of either receptor alone. A strain of S. aureus lacking lipoteichoic acid, a cell wall component serving as a ligand for Draper, was susceptible to integrin ßν-mediated phagocytosis. In contrast, a S. aureus mutant strain that produces small amounts of peptidoglycan was less efficiently phagocytosed by larval hemocytes, and a loss of integrin ßν in hemocytes reduced a difference in the susceptibility to phagocytosis between parental and mutant strains. Furthermore, a series of experiments revealed the binding of integrin ßν to peptidoglycan of S. aureus. Taken together, these results suggested that Draper and integrin ßν cooperate in the phagocytic elimination of S. aureus by recognizing distinct cell wall components, and that this dual recognition system is necessary for the host organism to survive infection.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Integrinas/metabolismo , Proteínas de Membrana/metabolismo , Staphylococcus aureus/fisiologia , Animais , Peptídeos Catiônicos Antimicrobianos/química , Apoptose , Cruzamentos Genéticos , Drosophila melanogaster/microbiologia , Hemócitos/citologia , Sistema Imunitário , Imunidade Inata , Masculino , Modelos Biológicos , Modelos Genéticos , Peptidoglicano/química , Peptidoglicano/metabolismo , Fagocitose , Staphylococcus aureus/químicaRESUMO
To elucidate the actions of Draper, a receptor responsible for the phagocytic clearance of apoptotic cells in Drosophila, we isolated proteins that bind to the extracellular region of Draper using affinity chromatography. One of those proteins has been identified to be an uncharacterized protein called Drosophila melanogaster calcium-binding protein 1 (DmCaBP1). This protein containing the thioredoxin-like domain resided in the endoplasmic reticulum and seemed to be expressed ubiquitously throughout the development of Drosophila. DmCaBP1 was externalized without truncation after the induction of apoptosis somewhat prior to chromatin condensation and DNA cleavage in a manner dependent on the activity of caspases. A recombinant DmCaBP1 protein bound to both apoptotic cells and a hemocyte-derived cell line expressing Draper. Forced expression of DmCaBP1 at the cell surface made non-apoptotic cells susceptible to phagocytosis. Flies deficient in DmCaBP1 expression developed normally and showed Draper-mediated pruning of larval axons, but a defect in the phagocytosis of apoptotic cells in embryos was observed. Loss of Pretaporter, a previously identified ligand for Draper, did not cause a further decrease in the level of phagocytosis in DmCaBP1-lacking embryos. These results collectively suggest that the endoplasmic reticulum protein DmCaBP1 is externalized upon the induction of apoptosis and serves as a tethering molecule to connect apoptotic cells and phagocytes for effective phagocytosis to occur.
Assuntos
Apoptose/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Retículo Endoplasmático/metabolismo , Fagócitos/metabolismo , Fagocitose/fisiologia , Animais , Axônios/metabolismo , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Retículo Endoplasmático/genética , Hemócitos/citologia , Hemócitos/metabolismo , Larva/citologia , Larva/genética , Larva/metabolismo , Fagócitos/citologiaRESUMO
Phagocytic removal of cells undergoing apoptosis is necessary for animal development and tissue homeostasis. Draper, a homologue of the Caenorhabditis elegans phagocytosis receptor CED-1, is responsible for the phagocytosis of apoptotic cells in Drosophila, but its ligand presumably present on apoptotic cells remains unknown. An endoplasmic reticulum protein that binds to the extracellular region of Draper was isolated. Loss of this protein, which we name Pretaporter, led to a reduced level of apoptotic cell clearance in embryos, and the overexpression of pretaporter in the mutant flies rescued this defect. Results from genetic analyses suggested that Pretaporter functionally interacts with Draper and the corresponding signal mediators. Pretaporter was exposed at the cell surface after the induction of apoptosis, and cells artificially expressing Pretaporter at their surface became susceptible to Draper-mediated phagocytosis. Finally, the incubation with Pretaporter augmented the tyrosine-phosphorylation of Draper in phagocytic cells. These results collectively suggest that Pretaporter relocates from the endoplasmic reticulum to the cell surface during apoptosis to serve as a ligand for Draper in the phagocytosis of apoptotic cells.
Assuntos
Apoptose , Proteínas de Drosophila/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Fagocitose , Animais , Membrana Celular/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Retículo Endoplasmático/metabolismo , Hemócitos/metabolismo , Ligantes , Microscopia de Fluorescência/métodos , Modelos Genéticos , Mutação , Fagócitos/metabolismo , Estrutura Terciária de ProteínaRESUMO
Bacteria adapt to environmental changes by altering gene expression patterns with the aid of signal transduction machinery called the two-component regulatory system (TCS), which consists of two protein components, a sensor kinase and response regulator. We examined the role of the TCS in bacterial adaptation to host environments using genetically tractable organisms, Escherichia coli as a pathogen and Drosophila melanogaster as a host. To determine the strength of the transcription promoters of TCS-encoding genes in Drosophila, adult flies were infected with a series of E. coli strains that expressed GFP driven by the promoters of genes coding for 27 sensor kinases and 32 response regulators of E. coli TCS followed by the measurement of fluorescence intensities. We further analyzed EnvZ-OmpR among the TCS encoded by genes having stronger promoters. A mutant E. coli strain lacking EnvZ-OmpR had a higher pathogenic effect on fly survival than that of the parental strain, and the forced expression of envZ and ompR in the mutant strain lowered its pathogenicity. The lack of EnvZ-OmpR did not affect the growth of E. coli in a culture medium as well as the level of colony-formable E. coli in flies. An increase in E. coli virulence with the loss of EnvZ-OmpR was observed in flies defective in an Imd-mediated humoral response, and both the mutant and parental strains were equally engulfed by hemocytes in vitro. These results suggest that EnvZ-OmpR mitigated the virulence of E. coli in Drosophila by a mechanism not accompanied by a change of bacterial burden. This behavior of E. coli is most likely a bacterial strategy to achieve persistent infection.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Drosophila melanogaster/microbiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Regulação Bacteriana da Expressão Gênica , Complexos Multienzimáticos/metabolismo , Transativadores/metabolismo , Animais , Escherichia coli/genética , Proteínas de Fluorescência Verde/metabolismo , Hemócitos/microbiologia , Masculino , Fagocitose , Regiões Promotoras Genéticas , Transdução de Sinais , VirulênciaRESUMO
Serpins are protease inhibitors that play essential roles in the down-regulation of extracellular proteolytic cascades. The core serpin domain is highly conserved, and typical serpins are encoded with a molecular size of 35-50 kDa. Here, we describe a novel 93-kDa protein that contains two complete, tandemly arrayed serpin domains. This twin serpin, SPN93, was isolated from the larval hemolymph of the large beetle Tenebrio molitor. The N-terminal serpin domain of SPN93 forms a covalent complex with the Spätzle-processing enzyme, a terminal serine protease of the Toll signaling cascade, whereas the C-terminal serpin domain of SPN93 forms complexes with a modular serine protease and the Spätzle-processing enzyme-activating enzyme, which are two different enzymes of the cascade. Consequently, SPN93 inhibited ß-1,3-glucan-mediated Toll proteolytic cascade activation in an in vitro system. Site-specific proteolysis of SPN93 at the N-terminal serpin domain was observed after activation of the Toll proteolytic cascade in vivo, and down-regulation of SPN93 by RNAi sensitized ß-1,3-glucan-mediated larval death. Therefore, SPN93 is the first serpin that contains twin tandemly arrayed and functionally active serpin domains that have a regulatory role in the larval Toll proteolytic signaling cascade.
Assuntos
Inibidores de Serina Proteinase/química , Serpinas/química , Serpinas/metabolismo , Receptores Toll-Like/metabolismo , Animais , Cromatografia/métodos , Clonagem Molecular , Besouros , Humanos , Melaninas/química , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Transdução de SinaisRESUMO
Pathogenic bacteria mitigate host immunity to establish infections, but the mechanism of this bacterial action has not been fully elucidated. To search for cell wall components that modulate innate immune responses in host organisms, we examined Staphylococcus aureus mutants, which were deficient in components of the cell wall, for pathogenicity in Drosophila. A mutation of dltA, which is responsible for the D-alanylation of teichoic acids, brought about an increase in the survival rate of adult flies that had received a septic infection with the bacteria. The growth of dltA-deficient S. aureus in adult flies was less efficient than that of the parental strain. The level of mRNA of Toll pathway-dependent antimicrobial peptides was higher in flies infected with the dltA mutant than that observed after the infection with the parental strain. The defective phenotype associated with the mutation of dltA, reduced pathogenicity and growth, was not evident in flies lacking the Toll pathway. Finally, a fraction of peptidoglycan prepared from the dltA mutant induced the expression of mRNA of a Toll-dependent antimicrobial peptide in flies and was bound by peptidoglycan recognition protein-SA in vitro more effectively than that obtained from the parental strain, and this difference was lost after the removal of wall teichoic acid from peptidoglycan. Taken together, we conclude that D-alanylated wall teichoic acid of S. aureus mitigates a Toll-mediated humoral response in Drosophila interfering with the recognition of peptidoglycan by a pattern recognition receptor.
Assuntos
Proteínas de Drosophila/metabolismo , Peptidoglicano/metabolismo , Transdução de Sinais , Staphylococcus aureus/metabolismo , Ácidos Teicoicos/metabolismo , Receptores Toll-Like/metabolismo , Alanina/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Western Blotting , Carbono-Oxigênio Ligases/genética , Carbono-Oxigênio Ligases/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/microbiologia , Interações Hospedeiro-Patógeno , Masculino , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Staphylococcus aureus/genética , Staphylococcus aureus/fisiologia , Ácidos Teicoicos/química , Fatores de Tempo , Receptores Toll-Like/genéticaRESUMO
Cells that have become unwanted need to be promptly, selectively, and safely removed. This is made possible by apoptosis-dependent phagocytosis, in which cells unnecessary, obstructive, or dangerous to organisms are induced to undergo apoptosis so that they are earmarked for phagocytosis. The phagocytic elimination occurs so quickly that cells with hallmarks of apoptosis are barely detectable in vivo. The removal of particular types of cells at appropriate stages of development not only contributes to the disposal of spent cells, the creation of space for morphogenesis, and the exclusion of pathogenic or noxious cells, but seems to actively control tissue renewal, tissue remodeling, tissue function, and pathogenic state. This event thus plays an indispensable role in the maintenance of animal development and tissue homeostasis.
Assuntos
Apoptose/fisiologia , Fagocitose/fisiologia , Animais , Apoptose/genética , Homeostase , Humanos , Imunidade Inata/genética , Imunidade Inata/fisiologia , Modelos Biológicos , Fagocitose/genéticaRESUMO
Phagocytosis is central to cellular immunity against bacterial infections. As in mammals, both opsonin-dependent and -independent mechanisms of phagocytosis seemingly exist in Drosophila. Although candidate Drosophila receptors for phagocytosis have been reported, how they recognize bacteria, either directly or indirectly, remains to be elucidated. We searched for the Staphylococcus aureus genes required for phagocytosis by Drosophila hemocytes in a screening of mutant strains with defects in the structure of the cell wall. The genes identified included ltaS, which encodes an enzyme responsible for the synthesis of lipoteichoic acid. ltaS-dependent phagocytosis of S. aureus required the receptor Draper but not Eater or Nimrod C1, and Draper-lacking flies showed reduced resistance to a septic infection of S. aureus without a change in a humoral immune response. Finally, lipoteichoic acid bound to the extracellular region of Draper. We propose that lipoteichoic acid serves as a ligand for Draper in the phagocytosis of S. aureus by Drosophila hemocytes and that the phagocytic elimination of invading bacteria is required for flies to survive the infection.