RESUMO
The proteinaceous elicitor cryptogein triggers defence reactions in Nicotiana tabacum (tobacco) through a signalling cascade, including the early production of reactive oxygen species (ROS) by the plasma membrane (PM)-located tobacco respiratory burst oxidase homologue D (NtRbohD). Sphingolipid long-chain bases (LCBs) are emerging as potent positive regulators of plant defence-related mechanisms. This led us to question whether both LCBs and their phosphorylated derivatives (LCB-Ps) are involved in the early signalling process triggered by cryptogein in tobacco BY-2 cells. Here, we showed that cryptogein-induced ROS production was inhibited by LCB kinase (LCBK) inhibitors. Additionally, Arabidopsis thaliana sphingosine kinase 1 and exogenously supplied LCB-Ps increased cryptogein-induced ROS production, whereas exogenously supplied LCBs had a strong opposite effect, which was not driven by a reduction in cellular viability. Immunogold-electron microscopy assay also revealed that LCB-Ps are present in the PM, which fits well with the presence of a high LCBK activity associated with this fraction. Our data demonstrate that LCBs and LCB-Ps differentially regulate cryptogein-induced ROS production in tobacco BY-2 cells, and support a model in which a cooperative synergism between LCBK/LCB-Ps and NtRbohD/ROS in the cryptogein signalling pathway is likely at the PM in tobacco BY-2 cells.
Assuntos
Proteínas Fúngicas/farmacologia , Nicotiana/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Esfingolipídeos/metabolismo , Morte Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Fosforilação/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Células Vegetais/efeitos dos fármacos , Células Vegetais/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Nicotiana/citologia , Nicotiana/efeitos dos fármacosRESUMO
A large body of evidence from the past decade supports the existence, in membrane from animal and yeast cells, of functional microdomains playing important roles in protein sorting, signal transduction, or infection by pathogens. In plants, as previously observed for animal microdomains, detergent-resistant fractions, enriched in sphingolipids and sterols, were isolated from plasma membrane. A characterization of their proteic content revealed their enrichment in proteins involved in signaling and response to biotic and abiotic stress and cell trafficking suggesting that these domains were likely to be involved in such physiological processes. In the present study, we used (14)N/(15)N metabolic labeling to compare, using a global quantitative proteomics approach, the content of tobacco detergent-resistant membranes extracted from cells treated or not with cryptogein, an elicitor of defense reaction. To analyze the data, we developed a software allowing an automatic quantification of the proteins identified. The results obtained indicate that, although the association to detergent-resistant membranes of most proteins remained unchanged upon cryptogein treatment, five proteins had their relative abundance modified. Four proteins related to cell trafficking (four dynamins) were less abundant in the detergent-resistant membrane fraction after cryptogein treatment, whereas one signaling protein (a 14-3-3 protein) was enriched. This analysis indicates that plant microdomains could, like their animal counterpart, play a role in the early signaling process underlying the setup of defense reaction. Furthermore proteins identified as differentially associated to tobacco detergent-resistant membranes after cryptogein challenge are involved in signaling and vesicular trafficking as already observed in similar studies performed in animal cells upon biological stimuli. This suggests that the ways by which the dynamic association of proteins to microdomains could participate in the regulation of the signaling process may be conserved between plant and animals.
Assuntos
Proteínas de Algas/farmacologia , Membrana Celular/metabolismo , Detergentes/farmacologia , Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Proteômica/métodos , Transdução de Sinais/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Proteínas Fúngicas , Medições Luminescentes , Espectrometria de Massas , Peptídeos/análise , Peptídeos/química , Proteínas de Plantas/química , Ligação Proteica/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Coloração e Rotulagem , Nicotiana/citologia , Nicotiana/efeitos dos fármacos , Nicotiana/microbiologiaRESUMO
Chemiluminescence detection of reactive oxygen species (ROS) triggered in tobacco BY-2 cells by the fungal elicitor cryptogein was previously demonstrated to be abolished in cells transformed with an antisense construct of the plasma membrane NADPH oxidase, NtrbohD. Here, using electron microscopy, it has been confirmed that the first hydrogen peroxide production occurring a few minutes after challenge of tobacco cells with cryptogein is plasma membrane located and NtrbohD mediated. Furthermore, the presence of NtrbohD in detergent-resistant membrane fractions could be associated with the presence of NtrbohD-mediated hydrogen peroxide patches along the plasma membrane. Comparison of the subcellular localization of ROS in wild-type tobacco and in plants transformed with antisense constructs of NtrbohD revealed that this enzyme is also responsible for the hydrogen peroxide production occurring at the plasma membrane after infiltration of tobacco leaves with cryptogein. Finally, the reactivity of wild-type and transformed plants to the elicitor and their resistance against the pathogenic oomycete Phytophthora parasitica were examined. NtrbohD-mediated hydrogen peroxide production does not seem determinant for either hypersensitive response development or the establishment of acquired resistance but it is most likely involved in the signaling pathways associated with the protection of the plant cell.
Assuntos
Nicotiana/metabolismo , Oxirredutases/fisiologia , Proteínas de Plantas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Células Cultivadas , DNA Antissenso , Proteínas Fúngicas/farmacologia , Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/metabolismo , Microscopia Eletrônica de Transmissão , Oxirredutases/análise , Oxirredutases/genética , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Proteínas de Plantas/análise , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/metabolismo , Espécies Reativas de Oxigênio/análise , Nicotiana/efeitos dos fármacos , Nicotiana/genética , Nicotiana/ultraestruturaRESUMO
BACKGROUND: It is unknown whether this burden of disease of lower respiratory tract infections is comparable across the Canadian Arctic. The objectives of this surveillance study were to compare the rates of hospital admission for lower respiratory tract infection and the severity of infection across Arctic Canada, and to describe the responsible viruses. METHODS: We performed a prospective multicentre surveillance study of infants less than 1 year of age admitted in 2009 with lower respiratory tract infection to all hospitals (5 regional, 4 tertiary) in the Northwest Territories, Nunavut and Nunavik to assess for regional differences. Nasopharyngeal aspirates were processed by means of a polymerase chain reaction respiratory viral panel, testing for 20 respiratory viruses and influenza A (H1N1). The role of coinfection was assessed by means of regression analysis for length of stay (short: < 7 d; long: > 14 d). Outcomes compared included rates of lower respiratory tract infection, respiratory syncytial virus infection, transfer to tertiary hospital and severe lower respiratory tract infection (respiratory failure, intubation and mechanical ventilation, and/or cardiopulmonary resuscitation). RESULTS: There were 348 admissions for lower respiratory tract infection in the population of interest in 2009. Rates of admission per 1000 live births varied significantly, from 39 in the Northwest Territories to 456 in Nunavik (p < 0.001). The rates of tertiary admissions and severe lower respiratory tract infection per 1000 live births in the Northwest Territories were 5.6 and 1.4, respectively, compared to 55.9 and 17.1, respectively, in Nunavut and 52.0 and 20.0, respectively, in Nunavik (p ≤ 0.001). Respiratory syncytial virus was the most common virus identified (124 cases [41.6% of those tested]), and coinfection was detected in 51 cases (41.1%) of infection with this virus. Longer length of stay was associated with coinfection (odds ratio [OR] 2.64) and underlying risk factors (OR 4.39). Length of stay decreased by 32.2% for every 30-day increase in age (OR 0.68). INTERPRETATION: Nunavut and Nunavik have very elevated rates of lower respiratory tract infection, with severe outcomes. Respiratory syncytial virus was the most common virus identified, and coinfection was associated with longer length of stay. Targeted public health interventions are required to reduce the burden of disease for infants residing in these Arctic regions.
RESUMO
UNLABELLED: BACKGROUND/ OBJECTIVE: Nunavut has the highest hospitalization rates for respiratory syncytial virus (RSV) worldwide, with rates of 166 per 1000 live births per year <1 year of age. Palivizumab was implemented in Nunavut primarily for premature infants, or those with hemodynamically significant cardiac or chronic lung disease; however, the effectiveness of the program is unknown. The objective of the present multisite, hospital-based surveillance study was to estimate the effectiveness of palivizumab in infants <6 months of age in Nunavut for the 2009 and 2010 RSV seasons. METHODS: Infants identified as palivizumab candidates who were <6 months of age were compared with all admissions for lower respiratory tract infection through multisite, hospital-based surveillance documenting the adequacy of palivizumab prophylaxis, admission for lower respiratory tract infection and the results of RSV testing. The OR for RSV admission in unprophylaxed infants was compared with those who were prophylaxed, and the effectiveness of palivizumab was estimated. RESULTS: Within the study cohort (n=101) during the two RSV seasons, five of the 10 eligible infants who did not receive adequate prophylaxis were admitted with RSV while two of the 91 infants <6 months of age eligible for palivizumab who were adequately prophylaxed were hospitalized with RSV (OR 22.3 [95% CI 3.8 to 130]; P=0.0005). The estimated effectiveness of palivizumab for the cohort was as high as 96%. Eight eligible infants were missed by the program and did not receive prophylaxis. CONCLUSION: Palivizumab was highly effective in reducing hospitalizations due to RSV infection in Nunavut. Further efforts need to be made to ensure that all eligible infants are identified.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antivirais/uso terapêutico , Hospitalização/estatística & dados numéricos , Doenças do Prematuro/prevenção & controle , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Humanos , Lactente , Recém-Nascido , Recém-Nascido Prematuro , Doenças do Prematuro/diagnóstico , Doenças do Prematuro/etnologia , Inuíte , Nunavut , Palivizumab , Vigilância da População , Estudos Prospectivos , Infecções por Vírus Respiratório Sincicial/diagnóstico , Infecções por Vírus Respiratório Sincicial/etnologia , Resultado do TratamentoRESUMO
A large body of evidence from the past decade supports the existence, in membrane from animal and yeast cells, of functional microdomains that play important roles in protein sorting, signal transduction, or infection by pathogens. Recent reports demonstrated the presence, in plants, of detergent-resistant fractions isolated from plasma membrane. Analysis of the lipidic composition of this fraction revealed its enrichment in sphingolipids and sterols and depletion in phospho- and glycerolipids as previously observed for animal microdomains. One-dimensional gel electrophoresis experiments indicated that these detergent-resistant fractions are able to recruit a specific set of plasma membrane proteins and exclude others. In the present study, we used mass spectrometry to give an extensive description of a tobacco plasma membrane fraction resistant to solubilization with Triton X-100. This led to the identification of 145 proteins whose functional and physicochemical characteristics were analyzed in silico. Parameters such as isoelectric point, molecular weight, number and length of transmembrane segments, or global hydrophobicity were analyzed and compared with the data available concerning plant plasma membrane proteins. Post-translational modifications, such as myristoylation, palmitoylation, or presence of a glycosylphosphatidylinositol anchor, were examined in relation to the presence of the corresponding proteins in these microdomains. From a functional point of view, this analysis indicated that if a primary function of the plasma membrane, such as transport, seems under-represented in the detergent-resistant fraction, others undergo a significant increase of their relative importance. Among these are signaling and response to biotic and abiotic stress, cellular trafficking, and cell wall metabolism. This suggests that these domains are likely to constitute, as in animal cells, signaling platforms involved in these physiological functions.
Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/análise , Nicotiana/metabolismo , Proteínas de Plantas/análise , Proteoma/análise , Transporte Biológico , Parede Celular/metabolismo , Células Cultivadas , Espectrometria de Massas , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Octoxinol/química , Proteínas de Plantas/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteoma/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Five cDNAs encoding Rac protein homologues to the Rho-related proteins from plants (Rop) were isolated in tobacco, and the function of one of them, Ntrac5, was studied. The Ntrac5 mRNA is repressed when tobacco leaves and cells are treated with the fungal elicitor cryptogein. Tobacco cells were transformed with sense constructs of Ntrac5 or Ntrac5V15, encoding the native GTP/GDP-bound form of this Rac protein homologue or the constitutively active mutant in its GTP-bound form, respectively. Immunological studies indicate that the corresponding protein is continuously located on the plasma membrane (PM). Both types of transformed cells show the same extra-cellular alkalinization as the control, but a high decrease in the active oxygen species (AOS) production after elicitation with cryptogein. Moreover, the regulation of NtrbohD, the oxidase involved in AOS production upon elicitation, is affected at both transcriptional and translational levels in cells overexpressing Ntrac5. Thus, Ntrac5 could be considered as a negative regulator of NtrbohD.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Nicotiana/fisiologia , Oxirredutases/genética , Proteínas de Plantas/genética , Explosão Respiratória/genética , Proteínas rac de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sequência Conservada , DNA Complementar/química , DNA Complementar/genética , Humanos , Dados de Sequência Molecular , Oxirredutases/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Nicotiana/enzimologia , Nicotiana/genética , Transformação Genética , Proteínas rac de Ligação ao GTP/classificaçãoRESUMO
A large body of evidence from the past decade supports the existence of functional microdomains in membranes of animal and yeast cells, which play important roles in protein sorting, signal transduction, or infection by pathogens. They are based on the dynamic clustering of sphingolipids and cholesterol or ergosterol and are characterized by their insolubility, at low temperature, in nonionic detergents. Here we show that similar microdomains also exist in plant plasma membrane isolated from both tobacco leaves and BY2 cells. Tobacco lipid rafts were found to be greatly enriched in a sphingolipid, identified as glycosylceramide, as well as in a mixture of stigmasterol, sitosterol, 24-methylcholesterol, and cholesterol. Phospho- and glycoglycerolipids of the plasma membrane were largely excluded from lipid rafts. Membrane proteins were separated by one- and two-dimensional gel electrophoresis and identified by tandem mass spectrometry or use of specific antibody. The data clearly indicate that tobacco microdomains are able to recruit a specific set of the plasma membrane proteins and exclude others. We demonstrate the recruitment of the NADPH oxidase after elicitation by cryptogein and the presence of the small G protein NtRac5, a negative regulator of NADPH oxidase, in lipid rafts.