RESUMO
Aspergillus fumigatus is a saprophytic fungus and opportunistic pathogen often causing fatal infections in immunocompromised patients. Recently AfKDNAse, an exoglycosidase hydrolyzing 3-deoxy-D-galacto-D-glycero-nonulosonic acid (KDN), a rare sugar from the sialic acid family, was identified and characterized. The principal function of AfKDNAse is still unclear, but a study suggests a critical role in fungal cell wall morphology and virulence. Potent AfKDNAse inhibitors are required to better probe the enzyme's biological role and as potential antivirulence factors. In this work, we developed a set of AfKDNAse inhibitors based on enzymatically stable thio-KDN motifs. C2, C9-linked heterodi-KDN were designed to fit into unusually close KDN sugar binding pockets in the protein. A polymeric compound with an average of 54 KDN motifs was also designed by click chemistry. Inhibitory assays performed on recombinant AfKDNAse showed a moderate and strong enzymatic inhibition for the two classes of compounds, respectively. The poly-KDN showed more than a nine hundred fold improved inhibitory activity (IC50 = 1.52 ± 0.37 µM, 17-fold in a KDN molar basis) compared to a monovalent KDN reference, and is to our knowledge, the best synthetic inhibitor described for a KDNase. Multivalency appears to be a relevant strategy for the design of potent KDNase inhibitors. Importantly, poly-KDN was shown to strongly decrease filamentation when co-cultured with A. fumigatus at micromolar concentrations, opening interesting perspectives in the development of antivirulence factors.
Assuntos
Aspergillus fumigatus , Glicosídeo Hidrolases , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/efeitos dos fármacos , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/metabolismo , Glicosídeo Hidrolases/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Relação Estrutura-Atividade , Modelos Moleculares , Estrutura MolecularRESUMO
Pneumococcal conjugate vaccines offer an excellent safety profile and high protection against the serotypes comprised in the vaccine. However, inclusion of protein antigens fromStreptococcus pneumoniaecombined with potent adjuvants and a suitable delivery system are expected to both extend protection to serotype strains not represented in the formulation and stimulate a broader immune response, thus more effective in young children, elderly, and immunocompromised populations. Along this line, nanoparticle (NP) delivery systems can enhance the immunogenicity of antigens by protecting them from degradation and increasing their uptake by antigen-presenting cells, as well as offering co-delivery with adjuvants. We report herein the encapsulation of a semisynthetic glycoconjugate (GC) composed of a synthetic tetrasaccharide mimicking theS. pneumoniae serotype 14 capsular polysaccharide (CP14) linked to the Pneumococcal surface protein A (PsaA) using chitosan NPs (CNPs). These GC-loaded chitosan nanoparticles (GC-CNPs) were not toxic to human monocyte-derived dendritic cells (MoDCs), showed enhanced uptake, and displayed better immunostimulatory properties in comparison to the naked GC. A comparative study was carried out in mice to evaluate the immune response elicited by the glycoconjugate-administered subcutaneously (SC), where the GC-CNPs displayed 100-fold higher IgG response as compared with the group treated with nonencapsulated GC. Overall, the study demonstrates the potential of this chitosan-based nanovaccine for efficient delivery of glycoconjugate antigens.
Assuntos
Quitosana , Criança , Idoso , Humanos , Animais , Camundongos , Pré-Escolar , Vacinas Pneumocócicas , Streptococcus pneumoniae , Adjuvantes Imunológicos , Glicoconjugados/uso terapêuticoRESUMO
Introduction: There is a need to improve public health interventions to promote youth social and emotional development in close collaboration with schools, families and local communities. A close intersectoral collaboration between the regional public health, schools and school boards was established to co-construct and implement "Positive Intervention (PI)" in the Eastern Townships region (Quebec, Canada). This paper describes its implementation according to the "Integrated Community Care (ICC)" framework. Description: PI is a collaborative and personalized intervention leaning toward an integrated community social care model. In fact, PI relies on the close proximity between Public Health and their educational counterpart as well as their individual temporality. However, PI offered mainly social services and its relationships with Primary Care services was not yet a priority. Discussion: The results show that it is possible to develop and implement an intervention promoting positive mental health in children, with and for local organisations. The level of integration between schools and Public Health services achieved after only 6 months of implementation is encouraging. Conclusion: More research is needed to thoroughly document the implementation, social validity, and effects of such an intervention by taking in the point of view of all stakeholders.
Introduction: Il est nécessaire d'améliorer les interventions de santé publique pour promouvoir le développement social et émotionnel des jeunes en étroite collaboration avec les écoles, les familles et les communautés locales. Une telle collaboration intersectorielle entre la santé publique régionale, les écoles et les commissions scolaires a été mise en place pour coconstruire et mettre en Åuvre l'Intervention positive (IP) en Estrie (Québec, Canada). Cet article décrit sa mise en Åuvre initiale selon le cadre conceptuel des soins de santé et services sociaux intégrés en proximité des communautés. Description: L'IP est une intervention collaborative et personnalisée qui s'inscrit dans un modèle de services sociaux intégrés en proximité des communautés. En fait, l'IP s'appuie sur une forte proximité entre les services de santé publique et les milieux scolaires, ainsi qu'un ajustement à la temporalité des partenaires. Cependant, l'IP offre principalement des services sociaux et ses relations avec les services de soins primaires n'étaient pas encore une priorité. Discussion: Les résultats montrent qu'il est possible de développer et de mettre en Åuvre une intervention de promotion de la santé mentale positive chez les enfants, avec et pour les organisations locales. Le niveau d'intégration entre les écoles et les services de santé publique atteint après seulement six mois de mise en Åuvre est encourageant. Conclusion: Des recherches supplémentaires sont nécessaires pour documenter de manière approfondie la mise en Åuvre, la validité sociale et les effets d'une telle intervention en prenant en compte le point de vue de toutes les parties prenantes.
RESUMO
In cellulo site-specific unnatural amino acid incorporation based on amber stop codon reassignment is a powerful tool to modify proteins at defined positions. This technique is herein applied to the selective functionalization of the Pneumococcal surface adhesin A protein at three distinct positions. Nϵ -propargyloxycarbonyl-l-lysine residues were incorporated and their alkyne groups reacted using click-chemistry with a synthetic azido-functionalized tetrasaccharide representative of one repeat unit of the Streptococcus pneumoniae serotype 14 capsular polysaccharide. Anti-PsaA antibody response induced in mice by the trivalent glycoconjugate was determined in comparison with corresponding monovalent and randomly functionalized conjugates. Our results suggest that controlled was superior to random conjugation for preserving antigenicity. In definitive, the reported strategy offers a unique opportunity to study the impact of carbohydrate antigen-carrier protein connectivity on immunogenicity.
Assuntos
Aminoácidos , Açúcares , Animais , Camundongos , Streptococcus pneumoniae , Vacinas Pneumocócicas , Glicoconjugados/químicaRESUMO
Virus-Like Particles (VLPs) have been used as immunogenic molecules in numerous recombinant vaccines. VLPs can also serve as vaccine platform to exogenous antigens, usually peptides incorporated within the protein sequences which compose the VLPs or conjugated to them. We herein described the conjugation of a synthetic tetrasaccharide mimicking the Streptococcus pneumoniae serotype 14 capsular polysaccharide to recombinant adenoviral type 3 dodecahedron, formed by the self-assembling of twelve penton bases and investigated the induced immune response when administered subcutaneously (s.c.). Whether formulated in the form of a dodecahedron or disassembled, the glycoconjugate induced an anti-protein response after two and three immunizations equivalent to that observed when the native dodecahedron was administered. On the other hand, the glycoconjugate induced a weak anti-IgM response which diminishes after two doses but no IgM-to-IgG switch was observed in mice against the serotype 14 capsular polysaccharide. In definitive, the whole conjugation process preserved both particulate nature and immunogenicity of the adenoviral dodecahedron. Further studies are needed to fully exploit adenoviral dodecahedron potential in terms of plasticity towards sequence engineering and of its capacity to stimulate the immune system via the intranasal route of administration as well as to shift the response to the carbohydrate antigen by playing both with the carbohydrate to protein ratio and the length of the synthetic carbohydrate antigen.
Assuntos
Adenoviridae , Glicoconjugados/química , Vacinas Pneumocócicas/química , Vacinas Pneumocócicas/imunologia , Modelos Moleculares , Conformação Proteica , Streptococcus pneumoniae , Vacinas Conjugadas/química , Vacinas Conjugadas/imunologiaRESUMO
Chemoenzymatic strategies are useful for providing both regio- and stereoselective access to bioactive oligosaccharides. We show herein that a glycosynthase mutant of a Thermus thermophilus α-glycosidase can react with unnatural glycosides such as 6-azido-6-deoxy-d-glucose/glucosamine to lead to ß-d-galactopyranosyl-(1â3)-d-glucopyranoside or ß-d-galactopyranosyl-(1â3)-2-acetamido-2-deoxy-d-glucopyranoside derivatives bearing a unique azide function. Taking advantage of the orthogonality between the azide and the hydroxyl functional groups, the former was next selectively reacted to give rise to a library of galectin-3 inhibitors. Combining enzyme substrate promiscuity and bioorthogonality thus appears as a powerful strategy to rapidly access to sugar-based ligands.
Assuntos
Galectina 3 , Oligossacarídeos , Sequência de Carboidratos , Glicosídeos , Espectroscopia de Ressonância MagnéticaRESUMO
Under nitrogen-limiting conditions, legumes are able to interact symbiotically with bacteria of the Rhizobiaceae family. This interaction gives rise to a new organ, named a root nodule. Root nodules are characterized by an increased glutathione (GSH) and homoglutathione (hGSH) content compared to roots. These low molecular thiols are very important in the biological nitrogen fixation. In order to characterize the modification of nodule activity induced by the microsymbiont glutathione deficiency, physiological, biochemical, and gene expression modifications were analyzed in nodules after the inoculation of Medicago truncatula with the SmgshB mutant of Sinorhizobium meliloti which is deficient in GSH production. The decline in nitrogen fixation efficiency was correlated to the reduction in plant shoot biomass. Flow cytometry analysis showed that SmgshB bacteroids present a higher DNA content than free living bacteria. Live/dead microscopic analysis showed an early bacteroid degradation in SmgshB nodules compared to control nodules which is correlated to a lower bacteroid content at 20 dpi. Finally, the expression of two marker genes involved in nitrogen fixation metabolism, Leghemoglobin and Nodule Cysteine Rich Peptide 001, decreased significantly in mutant nodules at 20 dpi. In contrast, the expression of two marker genes involved in the nodule senescence, Cysteine Protease 6 and Purple Acid Protease, increased significantly in mutant nodules at 10 dpi strengthening the idea that an early senescence process occurs in SmgshB nodules. In conclusion, our results showed that bacterial GSH deficiency does not impair bacterial differentiation but induces an early nodule senescence.
RESUMO
Galectins play key roles in numerous biological processes. Their mode of action depends on their localization which can be extracellular, cytoplasmic, or nuclear and is partly mediated through interactions with ß-galactose containing glycans. Galectins have emerged as novel therapeutic targets notably for the treatment of inflammatory disorders and cancers. This has stimulated the design of carbohydrate-based inhibitors targeting the carbohydrate recognition domains (CRDs) of the galectins. Pursuing this approach, we reasoned that linear oligogalactosides obtained by straightforward iterative click chemistry could mimic poly-lactosamine motifs expressed at eukaryote cell surfaces which the extracellular form of galectin-3, a prominent member of the galectin family, specifically recognizes. Affinities toward galectin-3 consistently increased with the length of the representative oligogalactosides but without reaching that of oligo-lactosamines. Elucidation of the X-ray crystal structures of the galectin-3 CRD in complex with a synthesized di- and tri-galactoside confirmed that the compounds bind within the carbohydrate-binding site. The atomic structures revealed that binding interactions mainly occur with the galactose moiety at the non-reducing end, primarily with subsites C and D of the CRD, differing from oligo-lactosamine which bind more consistently across the whole groove formed by the five subsites (A-E) of the galectin-3 CRD.
Assuntos
Biopolímeros/química , Galactosídeos/química , Galectinas/antagonistas & inibidores , Triazóis/química , Configuração de Carboidratos , Cristalografia por Raios X , Análise Espectral/métodosRESUMO
Gold(III) porphyrin presents an attractive alternative to the use of, for example, cisplatin in chemotherapy. However, approaches that allow to selectively target cancer cells are highly sought. Many plant and mammalian lectins have been shown to bind oligosaccharide sequences of the aberrant glycosylation pattern found on cancerous tumors. For example human galectin-3, of the galectin family specific for ß-galactoside, is overexpressed in the extracellular matrix of tumorigenous and metastatic tissues. We searched for non-carbohydrate ligands for galectin-3 that can guide a cytotoxic drug to the cancer cells by maintaining its affinity for tumor associated carbohydrate antigens. Previous findings showed that zinc tetrasulfonatophenylporphyrin can bind galectin-3 with sub-micromolar affinity without disturbing lactose binding. Gold(III) porphyrin is not only cytotoxic to cancer cells, it knows also a potential application as photosensitiser in photodynamic therapy. We investigated the binding of gold(III) porphyrin to galectin-3 using different biophysical interaction techniques and demonstrated a low micromolar affinity of human galectin-3 for the cytotoxic compound. Co-crystallization attempts in order to understand the binding mode of gold porphyrin to galectin-3 failed, but molecular docking emphasized a highly populated secondary binding site that does not hinder lactose or Thomsen Friendenreich disaccharide binding. This suggests that gold(III) porphyrin might significantly enhance its concentration and delivery to cancer cells by binding to human galectin-3 that keeps its orientation towards tumor associated carbohydrate antigens.
Assuntos
Antineoplásicos/química , Galectina 3/química , Ouro/química , Simulação de Acoplamento Molecular , Proteínas de Neoplasias/química , Porfirinas/química , Proteínas Sanguíneas , Galectina 3/metabolismo , Galectinas , Humanos , Metástase Neoplásica , Proteínas de Neoplasias/metabolismoRESUMO
Glycoconjugate vaccines are formed by covalently link a carbohydrate antigen to a carrier protein whose role is to achieve a long lasting immune response directed against the carbohydrate antigen. The nature of the sugar antigen, its length, its ratio per carrier protein and the conjugation chemistry impact on both structure and the immune response of a glycoconjugate vaccine. In addition it has long been assumed that the sites at which the carbohydrate antigen is attached can also have an impact. These important issue can now be addressed owing to the development of novel chemoselective ligation reactions as well as techniques such as site-selective mutagenesis, glycoengineering, or extension of the genetic code. The preparation and characterization of homogeneous bivalent pneumococcal vaccines is reported. The preparation and characterization of homogeneous bivalent pneumococcal vaccines is reported. A synthetic tetrasaccharide representative of the serotype 14 capsular polysaccharide of Streptococcus pneumoniae has been linked using the thiol/maleimide coupling chemistry to four different Pneumococcal surface adhesin A (PsaA) mutants, each harboring a single cysteine mutation at a defined position. Humoral response of these 1 to 1 carbohydrate antigen/PsaA conjugates have been assessed in mice. Our results showed that the carbohydrate antigen-PsaA connectivity impacts the anti-carrier response and raise questions about the design of glycoconjugate vaccine whereby the protein plays the dual role of immunogen and carrier.
RESUMO
Here, we report on the first combined one-pot use of the two so-called "click reactions": the thiol-ene coupling and the copper-catalyzed alkyne-azide cycloaddition. These reactions were employed in an alternating and one-pot fashion to combine appropriately functionalized monomeric carbohydrate building blocks to create mimics of trisaccharides and tetrasaccharides as single anomers, with only minimal purification necessary. The deprotected oligosaccharide mimics were found to bind both plant lectins and human galectin-3.
RESUMO
Pneumococcal infections remain a major public health concern worldwide. The currently available vaccines in the market are based on pneumococcal capsular polysaccharides but they still need to be improved to secure an optimal coverage notably in population at risk. To circumvent this, association of virulence pneumococcal proteins to the polysaccharide valencies has been proposed with the hope to observe an additive - if not synergistic - protective effect. Along this line, the use of the highly conserved and ubiquitous pneumococcal surface adhesin A (PsaA) as a protein carrier for a synthetic pneumococcal oligosaccharide is demonstrated herein for the first time. A tetrasaccharide mimicking functional antigenic determinants from the S. pneumoniae serotype 14 capsular polysaccharide (Pn14TS) was chemically synthesised. The mature PsaA (mPsaA) was expressed in E. coli and purified using affinity chromatography. The Pn14PS was conjugated to mPsaA using maleimide-thiol coupling chemistry to obtain mPsaA-Pn14PS conjugate (protein/sugar molar ratio: 1/5.4). The mPsaA retained the structural conformation after the conjugation and lyophilisation. The prepared glycoconjugate adjuvanted with α-galactosylceramide, a potent activator of invariant Natural Killer T cells, was tested in mice for its immunological response upon subcutaneous injection in comparison with mPsaA alone and a model BSA conjugate (BSA-Pn14PS, used here as a control). Mice immunised with the mPsaA-Pn14TS produced a robust IgG response against mPsaA and against the capsular polysaccharide from pneumococcal serotype 14. These data provide the basis for novel pneumococcal vaccine development.
Assuntos
Proteínas de Bactérias/química , Glicoconjugados/química , Vacinas Pneumocócicas/química , Animais , Proteínas de Bactérias/imunologia , Escherichia coli/imunologia , Feminino , Galactosilceramidas/química , Glicoconjugados/imunologia , Imunização/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infecções Pneumocócicas/imunologia , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Vacinação/métodosRESUMO
Glycan microarrays are useful tools for lectin glycan profiling. The use of a glycan microarray based on evanescent-field fluorescence detection was herein further extended to the screening of lectin inhibitors in competitive experiments. The efficacy of this approach was tested with 2/3'-mono- and 2,3'-diaromatic typeâ II lactosamine derivatives and galectins as targets and was validated by comparison with fluorescence anisotropy proposed as an orthogonal protein interaction measurement technique. We showed that subtle differences in the architecture of the inhibitor could be sensed that pointed out the preference of galectin-3 for 2'-arylamido derivatives over ureas, thioureas, and amines and that of galectin-7 for derivatives bearing an α substituent at the anomeric position of glucosamine. We eventually identified a diaromatic oxazoline as a highly specific inhibitor of galectin-3 versus galectin-1 and galectin-7.
Assuntos
Galectinas/antagonistas & inibidores , Análise em Microsséries , Amino Açúcares , Animais , Polarização de Fluorescência , Galectina 3/antagonistas & inibidores , Humanos , Oxazóis/química , Sensibilidade e EspecificidadeRESUMO
Galectins have been recognized as potential novel therapeutic targets for the numerous fundamental biological processes in which they are involved. Galectins are key players in homeostasis, and as such their expression and function are finely tuned in vivo. Thus, their modes of action are complex and remain largely unexplored, partly because of the lack of dedicated tools. We thus designed galectin inhibitors from a lactosamine core, functionalized at key C2 and C3' positions by aromatic substituents to ensure both high affinity and selectivity, and equipped with a spacer that can be modified on demand to further modulate their physico-chemical properties. As a proof-of-concept, galectin-3 was selectively targeted. The efficacy of the synthesized di-aromatic lactosamine tools was shown in cellular assays to modulate collective epithelial cell migration and to interfere with actin/cortactin localization.
Assuntos
Amino Açúcares/farmacologia , Galectina 3/antagonistas & inibidores , Cicatrização/efeitos dos fármacos , Amino Açúcares/síntese química , Amino Açúcares/química , Proteínas Sanguíneas , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Galectina 1/antagonistas & inibidores , Galectinas/antagonistas & inibidores , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/fisiologiaRESUMO
Legumes associate with rhizobia to form nitrogen (N2)-fixing nodules, which is important for plant fitness [1, 2]. Medicago truncatula controls the terminal differentiation of Sinorhizobium meliloti into N2-fixing bacteroids by producing defensin-like nodule-specific cysteine-rich peptides (NCRs) [3, 4]. The redox state of NCRs influences some biological activities in free-living bacteria, but the relevance of redox regulation of NCRs in planta is unknown [5, 6], although redox regulation plays a crucial role in symbiotic nitrogen fixation [7, 8]. Two thioredoxins (Trx), Trx s1 and s2, define a new type of Trx and are expressed principally in nodules [9]. Here, we show that there are four Trx s genes, two of which, Trx s1 and s3, are induced in the nodule infection zone where bacterial differentiation occurs. Trx s1 is targeted to the symbiosomes, the N2-fixing organelles. Trx s1 interacted with NCR247 and NCR335 and increased the cytotoxic effect of NCR335 in S. meliloti. We show that Trx s silencing impairs bacteroid growth and endoreduplication, two features of terminal bacteroid differentiation, and that the ectopic expression of Trx s1 in S. meliloti partially complements the silencing phenotype. Thus, our findings show that Trx s1 is targeted to the bacterial endosymbiont, where it controls NCR activity and bacteroid terminal differentiation. Similarly, Trxs are critical for the activation of defensins produced against infectious microbes in mammalian hosts. Therefore, our results suggest the Trx-mediated regulation of host peptides as a conserved mechanism among symbiotic and pathogenic interactions.
Assuntos
Medicago truncatula/crescimento & desenvolvimento , Bactérias Fixadoras de Nitrogênio/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Sinorhizobium meliloti/crescimento & desenvolvimento , Tiorredoxinas/antagonistas & inibidores , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Regulação da Expressão Gênica de Plantas , Medicago truncatula/microbiologia , Bactérias Fixadoras de Nitrogênio/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Nódulos Radiculares de Plantas/microbiologia , Transdução de Sinais , Sinorhizobium meliloti/efeitos dos fármacos , SimbioseRESUMO
OBJECTIVES: To demonstrate the pertinence of putting personalisation at the heart of mental health services. METHODS: Review of littérature of personalisation research and intervention in the United Kingdom, the country where the personalisation is one of the key themes of the health and social services reform agenda. RESULTS: Presentation of the key challenges in the personalisation agenda and also of web tool directly inspired by research and practices in the UK. CONCLUSION: We think that individuals want to be treated as citizens that want control and choice over their destiny.
Assuntos
Serviços de Saúde Mental , Medicina de Precisão , HumanosRESUMO
Legumes form a symbiotic interaction with bacteria of the Rhizobiaceae family to produce nitrogen-fixing root nodules under nitrogen-limiting conditions. We examined the importance of glutathione (GSH) and homoglutathione (hGSH) during the nitrogen fixation process. Spatial patterns of the expression of the genes involved in the biosynthesis of both thiols were studied using promoter-GUS fusion analysis. Genetic approaches using the nodule nitrogen-fixing zone-specific nodule cysteine rich (NCR001) promoter were employed to determine the importance of (h)GSH in biological nitrogen fixation (BNF). The (h)GSH synthesis genes showed a tissue-specific expression pattern in the nodule. Down-regulation of the γ-glutamylcysteine synthetase (γECS) gene by RNA interference resulted in significantly lower BNF associated with a significant reduction in the expression of the leghemoglobin and thioredoxin S1 genes. Moreover, this lower (h)GSH content was correlated with a reduction in the nodule size. Conversely, γECS overexpression resulted in an elevated GSH content which was correlated with increased BNF and significantly higher expression of the sucrose synthase-1 and leghemoglobin genes. Taken together, these data show that the plant (h)GSH content of the nodule nitrogen-fixing zone modulates the efficiency of the BNF process, demonstrating their important role in the regulation of this process.
Assuntos
Glutationa/análogos & derivados , Medicago truncatula/metabolismo , Fixação de Nitrogênio/fisiologia , Regulação para Baixo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glutationa/biossíntese , Glutationa/metabolismo , Glutationa Sintase/antagonistas & inibidores , Medicago truncatula/genética , Medicago truncatula/microbiologia , Fixação de Nitrogênio/genética , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismo , Nódulos Radiculares de Plantas/microbiologia , Sinorhizobium meliloti/metabolismo , Simbiose/genética , Simbiose/fisiologiaRESUMO
The plant plasma membrane-localized NADPH oxidases, known as respiratory burst oxidase homologues (RBOHs), appear to play crucial roles in plant growth and development. They are involved in important processes, such as root hair growth, plant defence reactions and abscisic acid signalling. Using sequence similarity searches, we identified seven putative RBOH-encoding genes in the Medicago truncatula genome. A phylogenetic reconstruction showed that Rboh gene duplications occurred in legume species. We analysed the expression of these MtRboh genes in different M. truncatula tissues: one of them, MtRbohA, was significantly up-regulated in Sinorhizobium meliloti-induced symbiotic nodules. MtRbohA expression appeared to be restricted to the nitrogen-fixing zone of the functional nodule. Moreover, using S. meliloti bacA and nifH mutants unable to form efficient nodules, a strong link between nodule nitrogen fixation and MtRbohA up-regulation was shown. MtRbohA expression was largely enhanced under hypoxic conditions. Specific RNA interference for MtRbohA provoked a decrease in the nodule nitrogen fixation activity and the modulation of genes encoding the microsymbiont nitrogenase. These results suggest that hypoxia, prevailing in the nodule-fixing zone, may drive the stimulation of MtRbohA expression, which would, in turn, lead to the regulation of nodule functioning.
Assuntos
Medicago truncatula/enzimologia , NADPH Oxidases/metabolismo , Nódulos Radiculares de Plantas/enzimologia , Simbiose/fisiologia , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Medicago truncatula/citologia , Medicago truncatula/genética , Medicago truncatula/microbiologia , Anotação de Sequência Molecular , NADPH Oxidases/genética , Fixação de Nitrogênio/genética , Fenótipo , Filogenia , Transporte Proteico , Interferência de RNA , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/microbiologia , Sinorhizobium meliloti/fisiologiaRESUMO
Under nitrogen-limiting conditions, legumes interact with symbiotic rhizobia to produce nitrogen-fixing root nodules. We have previously shown that glutathione and homoglutathione [(h)GSH] deficiencies impaired Medicago truncatula symbiosis efficiency, showing the importance of the low M(r) thiols during the nodulation process in the model legume M. truncatula. In this study, the plant transcriptomic response to Sinorhizobium meliloti infection under (h)GSH depletion was investigated using cDNA-amplified fragment length polymorphism analysis. Among 6,149 expression tags monitored, 181 genes displayed significant differential expression between inoculated control and inoculated (h)GSH depleted roots. Quantitative reverse transcription polymerase chain reaction analysis confirmed the changes in mRNA levels. This transcriptomic analysis shows a down-regulation of genes involved in meristem formation and a modulation of the expression of stress-related genes in (h)GSH-depleted plants. Promoter-beta-glucuronidase histochemical analysis showed that the putative MtPIP2 aquaporin might be up-regulated during nodule meristem formation and that this up-regulation is inhibited under (h)GSH depletion. (h)GSH depletion enhances the expression of salicylic acid (SA)-regulated genes after S. meliloti infection and the expression of SA-regulated genes after exogenous SA treatment. Modification of water transport and SA signaling pathway observed under (h)GSH deficiency contribute to explain how (h)GSH depletion alters the proper development of the symbiotic interaction.
Assuntos
Glutationa/análogos & derivados , Medicago truncatula/crescimento & desenvolvimento , Nodulação , Sinorhizobium meliloti/fisiologia , Simbiose , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glutationa/deficiência , Glutationa/metabolismo , Medicago truncatula/genética , Medicago truncatula/metabolismo , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Legumes/rhizobium biological N(2) fixation (BNF) is dramatically affected under abiotic stress such as drought, salt, cold and heavy metal stresses. Nodule response to drought stress at the molecular level was analysed using soybean (Glycine max) and Bradyrhizobium japonicum as a model, since this symbiotic partnership is extremely sensitive to this stress. To gain insight into molecular mechanisms involved in drought-induced BNF inhibition, we have constructed a SSH (Suppression Subtractive Hybridisation) cDNA library from nodular tissue of plants irrigated at field capacity or plants water deprived for 5 days. Sequence analysis of the first set of 128 non redundant ESTs using protein databases and the Blastx program indicated that 70% of ESTs could be classified into putative known functions. Using reverse northern hybridization, 56 ESTs were validated as up-regulated genes in response to drought. Interestingly, only a few of them had been previously described as involved in plant response to drought, therefore most of the ESTs could be considered as new markers of drought stress. Here we discuss the potential role of some of these up-regulated genes in response to drought. Our analysis focused on two genes, encoding respectively a ferritin and a metallothionein, which are known to be involved in homeostasis and detoxification of metals and in response to oxidative stress. Their spatiotemporal expression patterns showed a high accumulation of transcripts restricted to infected cells of nodules in response to drought.