RESUMO
The BarA/UvrY two-component signal transduction system mediates adaptive responses of Escherichia coli to changes in growth stage. At late exponential growth phase, the BarA sensor kinase autophosphorylates and transphosphorylates UvrY, which activates transcription of the CsrB and CsrC noncoding RNAs. CsrB and CsrC, in turn, sequester and antagonize the RNA binding protein CsrA, which posttranscriptionally regulates translation and/or stability of its target mRNAs. Here, we provide evidence that during stationary phase of growth, the HflKC complex recruits BarA to the poles of the cells and silences its kinase activity. Moreover, we show that during the exponential phase of growth, CsrA inhibits hflK and hflC expression, thereby enabling BarA activation upon encountering its stimulus. Thus, in addition to temporal control of BarA activity, spatial regulation is demonstrated.
Assuntos
Proteínas de Escherichia coli , Fatores de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fosfotransferases/metabolismo , Transdução de Sinais , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
The BarA/UvrY two-component signal transduction system is widely conserved in γ-proteobacteria and provides a link between the metabolic state of the cells and the Csr posttranscriptional regulatory system. In Escherichia coli, the BarA/UvrY system responds to the presence of acetate and other short-chain carboxylic acids by activating transcription of the noncoding RNAs, CsrB and CsrC, which sequester the RNA-binding protein CsrA, a global regulator of gene expression. However, the state of the carboxyl group in the acetate molecule, which serves as the BarA stimulus, and the signal reception site of BarA remain unknown. In this study, we show that the deletion or replacement of the periplasmic domain of BarA and also the substitution of certain hydroxylated and hydrophobic amino acid residues in this region, result in a sensor kinase that remains unresponsive to its physiological stimulus, demonstrating that the periplasmic region of BarA constitutes a functional detector domain. Moreover, we provide evidence that the protonated state of acetate or formate serves as the physiological stimulus of BarA. In addition, modeling of the BarA sensor domain and prediction of the signal-binding site, by blind molecular docking, revealed a calcium channels and chemotaxis receptors domain with a conserved binding pocket, which comprised uncharged polar and hydrophobic amino acid residues. Based on the comparative sequence and phylogenetic analyses, we propose that, at least, two types of BarA orthologues diverged and evolved separately to acquire distinct signal-binding properties, illustrating the wide adaptability of the bacterial sensor kinase proteins.
Assuntos
Acetatos/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Proteínas de Membrana/química , Simulação de Acoplamento Molecular , Fosfotransferases/química , Acetatos/metabolismo , Sítios de Ligação , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfotransferases/genética , Fosfotransferases/metabolismo , FilogeniaRESUMO
Elucidation of host-pathogen interaction is essential for developing effective strategies to combat bacterial infection. Dual RNA-Seq using cultured cells or tissues/organs as the host of pathogen has emerged as a novel strategy to understand the responses concurrently from both pathogen and host at cellular level. However, bacterial infection mostly causes systematic responses from the host at organism level where the interplay is urgently to be understood but inevitably being neglected by the current practice. Here, we developed an approach that simultaneously monitor the genome-wide infection-linked transcriptional alterations in both pathogenic Vibrio parahaemolyticus and the infection host nematode Caenorhabditis elegans. Besides the dynamic alterations in transcriptomes of both C. elegans and V. parahaemolyticus during infection, we identify a two-component system, BarA/UvrY, that is important for virulence in host. BarA/UvrY not only controls the virulence factors in V. parahaemolyticus including Type III and Type VI secretion systems, but also attenuates innate immune responses in C. elegans, including repression on the MAP kinase-mediated cascades. Thus, our study exemplifies the use of dual RNA-Seq at organism level to uncover previously unrecognized interplay between host and pathogen.
Assuntos
Proteínas de Bactérias/genética , Vibrio parahaemolyticus/genética , Fatores de Virulência/genética , Virulência/genética , Animais , Caenorhabditis elegans/microbiologia , Linhagem Celular Tumoral , Células HeLa , Interações Hospedeiro-Patógeno/genética , Humanos , Imunidade Inata/genética , Proteínas de Membrana/genética , RNA-Seq/métodos , Fatores de Transcrição/genética , Peixe-ZebraRESUMO
Several bacterial pathogens, including Salmonella enterica, can cause persistent infections in humans by mechanisms that are poorly understood. By comparing genomes of isolates longitudinally collected from 256 prolonged salmonellosis patients, we identified repeated mutations in global regulators, including the barA/sirA two-component regulatory system, across multiple patients and Salmonella serovars. Comparative RNA-seq analysis revealed that distinct mutations in barA/sirA led to diminished expression of Salmonella pathogenicity islands 1 and 4 genes, which are required for Salmonella invasion and enteritis. Moreover, barA/sirA mutants were attenuated in an acute salmonellosis mouse model and induced weaker transcription of host immune responses. In contrast, in a persistent infection mouse model, these mutants exhibited long-term colonization and prolonged shedding. Taken together, these findings suggest that selection of mutations in global virulence regulators facilitates persistent Salmonella infection in humans, by attenuating Salmonella virulence and inducing a weaker host inflammatory response.
Assuntos
Infecções por Salmonella , Transativadores , Animais , Camundongos , Humanos , Transativadores/metabolismo , Infecção Persistente , Salmonella typhimurium , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções por Salmonella/microbiologia , Mutação , Regulação Bacteriana da Expressão GênicaRESUMO
BarA/UvrY, a two-component system and global regulator that controls expression of more than a hundred of genes involved in virulence, motility, biofilm formation, and central carbon metabolism under various stress conditions. In this study, we investigated the function of BarA/UvrY system in Serratia marcescens FS14. The disruption of barA or/and uvrY results in the yield increase of secondary metabolite prodigiosin. We further demonstrated that BarA/UvrY system represses prodigiosin production by inhibiting the transcription level of pig gene cluster with direct binding to the pigA promoter. In addition, deletion of barA or/and uvrY abolished the swarming motility of FS14, but not the swimming motility. We revealed that BarA/UvrY activates swarming through directly upregulating the expression of the biosurfactant synthesis gene swrW rather than flagella system. We also observed that BarA/UvrY positively regulates the resistance to H2O2 same as in Escherichia coli highlighting the importance of BarA/UvrY on hydrogen peroxide resistance. Our results demonstrated that the BarA/UvrY system differentially regulates the biosynthesis of the secondary metabolite prodigiosin and swarming motility in S. marcescens FS14. Comparison of our results with those observed for Serratia sp. 39006 suggests that BarA/UvrY's role in regulation of secondary metabolite production is different among Serratia species.
Assuntos
Proteínas de Escherichia coli , Prodigiosina , Animais , Suínos , Prodigiosina/metabolismo , Serratia marcescens/genética , Fatores de Transcrição/genética , Peróxido de Hidrogênio/metabolismo , Proteínas de Membrana/genética , Escherichia coli/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Fosfotransferases/genética , Proteínas de Escherichia coli/metabolismoRESUMO
The effect of two different CO(2) concentrations (400 and 800 µmol mol(-1)) on the photosynthesis rate, primary and secondary metabolite syntheses and the antioxidant activities of the leaves, stems and rhizomes of two Zingiber officinale varieties (Halia Bentong and Halia Bara) were assessed in an effort to compare and validate the medicinal potential of the subterranean part of the young ginger. High photosynthesis rate (10.05 µmol CO(2) m(-2)s(-1) in Halia Bara) and plant biomass (83.4 g in Halia Bentong) were observed at 800 µmol mol(-1) CO(2). Stomatal conductance decreased and water use efficiency increased with elevated CO(2) concentration. Total flavonoids (TF), total phenolics (TP), total soluble carbohydrates (TSC), starch and plant biomass increased significantly (P ≤ 0.05) in all parts of the ginger varieties under elevated CO(2) (800 µmol mol(-1)). The order of the TF and TP increment in the parts of the plant was rhizomes > stems > leaves. More specifically, Halia Bara had a greater increase of TF (2.05 mg/g dry weight) and TP (14.31 mg/g dry weight) compared to Halia Bentong (TF: 1.42 mg/g dry weight; TP: 9.11 mg/g dry weight) in average over the whole plant. Furthermore, plants with the highest rate of photosynthesis had the highest TSC and phenolics content. Significant differences between treatments and species were observed for TF and TP production. Correlation coefficient showed that TSC and TP content are positively correlated in both varieties. The antioxidant activity, as determined by the ferric reducing/antioxidant potential (FRAP) activity, increased in young ginger grown under elevated CO(2). The FRAP values for the leaves, rhizomes and stems extracts of both varieties grown under two different CO(2) concentrations (400 and 800 µmol mol(-1)) were significantly lower than those of vitamin C (3107.28 µmol Fe (II)/g) and α-tocopherol (953 µmol Fe (II)/g), but higher than that of BHT (74.31 µmol Fe (II)/g). These results indicate that the plant biomass, primary and secondary metabolite synthesis, and following that, antioxidant activities of Malaysian young ginger varieties can be enhanced through controlled environment (CE) and CO(2) enrichment.
Assuntos
Dióxido de Carbono/metabolismo , Flavonoides/biossíntese , Amido/biossíntese , Zingiber officinale/metabolismo , Biomassa , Ecossistema , Oxirredução , Fenóis/metabolismo , Fotossíntese , Folhas de Planta/metabolismo , Caules de Planta/metabolismo , Rizoma/metabolismoRESUMO
Bacteria have to process several levels of gene regulation and coordination of interconnected regulatory networks to ensure the most adequate cellular response to specific growth conditions. Especially, expression of complex and costly fitness and pathogenicity-associated traits is coordinated and tightly regulated at multiple levels. We studied the interconnected regulation of the expression of the colibactin and yersiniabactin polyketide biosynthesis machineries, which are encoded by two pathogenicity islands found in many phylogroup B2 Escherichia coli isolates. Comparative phenotypic and genotypic analyses identified the BarA-UvrY two-component system as an important regulatory element involved in colibactin and yersiniabactin expression. The carbon storage regulator (Csr) system controls the expression of a wide range of central metabolic and virulence-associated traits. The availability of CsrA, the key translational regulator of the Csr system, depends on BarA-UvrY activity. We employed reporter gene fusions to demonstrate UvrY- and CsrA-dependent expression of the colibactin and yersiniabactin determinants and confirmed a direct interaction of CsrA with the 5' untranslated leader transcripts of representative genes of the colibactin and yersiniabactin operons by RNA electrophoretic mobility shift assays. This posttranscriptional regulation adds an additional level of complexity to control mechanisms of polyketide expression, which is also orchestrated at the level of ferric uptake regulator (Fur)-dependent regulation of transcription and phosphopantetheinyl transferase-dependent activation of polyketide biosynthesis. Our results emphasize the interconnection of iron- and primary metabolism-responsive regulation of colibactin and yersiniabactin expression by the fine-tuned action of different regulatory mechanisms in response to variable environmental signals as a prerequisite for bacterial adaptability, fitness, and pathogenicity in different habitats. IMPORTANCE Secondary metabolite expression is a widespread strategy among bacteria to improve their fitness in habitats where they constantly compete for resources with other bacteria. The production of secondary metabolites is associated with a metabolic and energetic burden. Colibactin and yersiniabactin are two polyketides, which are expressed in concert and promote the virulence of different enterobacterial pathogens. To maximize fitness, they should be expressed only in microenvironments in which they are required. Accordingly, precise regulation of colibactin and yersiniabactin expression is crucial. We show that the expression of these two polyketides is also interconnected via primary metabolism-responsive regulation at the posttranscriptional level by the CsrA RNA-binding protein. Our findings may help to optimize (over-)expression and further functional characterization of the polyketide colibactin. Additionally, this new aspect of concerted colibactin and yersiniabactin expression extends our knowledge of conditions that favor the expression of these virulence- and fitness-associated factors in different Enterobacterales members.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Policetídeos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Fosfotransferases/genética , Policetídeos/metabolismo , Proteínas Repressoras/genética , Proteínas de Ligação a RNA/genéticaRESUMO
Phosphatidylinositol-3-phosphate (PtdIns3P) is essential for generating autophagosomes and regulating endocytic trafficking. Recently, we have shown that the activities of human PIK3C3/VPS34-containing complexes I and II, which synthesize PtdIns3P, are greatly affected by three membrane physicochemical parameters: lipid unsaturation, membrane curvature, and negative charge. Both complexes are more active on membranes composed of unsaturated lipids than saturated lipids, and high membrane curvature can compensate for the negative effect of high lipid saturation. Negatively charged phosphatidylserine (PS) activates the complexes, as well as PIK3C3/VPS34 alone. The kinase activity of complex I depends critically on the ATG14 BATS domain, whereas complex II relies on the BECN1 BARA domain. Our findings highlight the importance of the membrane character as sensed by the unique membrane binding motifs/domain of the complexes for regulating PIK3C3/VPS34 activity.
Assuntos
Autofagia , Classe III de Fosfatidilinositol 3-Quinases , Autofagossomos , Proteínas Relacionadas à Autofagia , Endossomos , HumanosRESUMO
The relationship between phenolics and flavonoids synthesis/accumulation and photosynthesis rate was investigated for two Malaysian ginger (Zingiber officinale) varieties grown under four levels of glasshouse light intensity, namely 310, 460, 630 and 790 µmol m(-2)s(-1). High performance liquid chromatography (HPLC) was employed to identify and quantify the polyphenolic components. The results of HPLC analysis indicated that synthesis and partitioning of quercetin, rutin, catechin, epicatechin and naringenin were high in plants grown under 310 µmol m(-2)s(-1). The average value of flavonoids synthesis in leaves for both varieties increased (Halia Bentong 26.1%; Halia Bara 19.5%) when light intensity decreased. Photosynthetic rate and plant biomass increased in both varieties with increasing light intensity. More specifically, a high photosynthesis rate (12.25 µmol CO(2) m(-2)s(-1) in Halia Bara) and plant biomass (79.47 g in Halia Bentong) were observed at 790 µmol m(-2)s(-1). Furthermore, plants with the lowest rate of photosynthesis had highest flavonoids content. Previous studies have shown that quercetin inhibits and salicylic acid induces the electron transport rate in photosynthesis photosystems. In the current study, quercetin was an abundant flavonoid in both ginger varieties. Moreover, higher concentration of quercetin (1.12 mg/g dry weight) was found in Halia Bara leaves grown under 310 µmol m(-2)s(-1) with a low photosynthesis rate. Furthermore, a high content of salicylic acid (0.673 mg/g dry weight) was detected in Halia Bara leaves exposed under 790 µmol m(-2)s(-1) with a high photosynthesis rate. No salicylic acid was detected in gingers grown under 310 µmol m(-2)s(-1). Ginger is a semi-shade loving plant that does not require high light intensity for photosynthesis. Different photosynthesis rates at different light intensities may be related to the absence or presence of some flavonoid and phenolic compounds.
Assuntos
Flavonoides/metabolismo , Fotossíntese , Ácido Salicílico/metabolismo , Zingiber officinale/metabolismo , Flavonoides/biossínteseRESUMO
Nowadays, phytochemicals and antioxidants in plants are raising interest in consumers for their roles in the maintenance of human health. Phenolics and flavonoids are known for their health-promoting properties due to protective effects against cardiovascular disease, cancers and other disease. Ginger (Zingiber officinale) is one of the traditional folk medicinal plants and it is widely used in cooking in Malaysia. In this study, four levels of glasshouse light intensities (310, 460, 630 and 790 µmol m(-2)s(-1)) were used in order to consider the effect of light intensity on the production, accumulation and partitioning of total phenolics (TP), total flavonoids (TF) and antioxidant activities in two varieties of Malaysian young ginger (Zingiber officinale). TF biosynthesis was highest in the Halia Bara variety under 310 µmol m(-2)s(-1) and TP was high in this variety under a light intensity of 790 µmol m(-2)s(-1). The highest amount of these components accumulated in the leaves and after that in the rhizomes. Also, antioxidant activities determined by the 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) assay in both of varieties, increased significantly (p ≤ 0.01) with increasing TF concentration, and high antioxidant activity was observed in the leaves of Halia Bara grown under 310 µmol m(-2)s(-1). The ferric reducing (FRAP) activity of the rhizomes was higher than that of the leaves in 310 µmol m(-2)s(-1) of sun light. This study indicates the ability of different light intensities to enhance the medicinal components and antioxidant activities of the leaves and young rhizomes of Zingiber officinale varieties. Additionally, this study also validated their medicinal potential based on TF and TP contents.
Assuntos
Flavonoides/biossíntese , Luz , Fenóis/metabolismo , Zingiber officinale/efeitos da radiação , Flavonoides/metabolismo , Zingiber officinale/metabolismo , Rizoma/metabolismo , Rizoma/efeitos da radiação , Especificidade da EspécieRESUMO
The effects of different drying methods, including sun-, oven-, and freeze-drying on the changes in the antioxidant and anti-inflammatory activities of ginger (Zingiber officinale var. Rubra) rhizome were studied. Sun-, oven-, and freeze-dried ginger showed a significant (p < 0.05) increase in phenolic content by 1.79, 1.53, and 1.91-fold; flavonoid content increased by 6.06, 5.27, and 4.90-fold; FRAP increased by 3.95, 3.51, and 3.15-fold; ABTSâ¢+ scavenging activity increased by 2.07, 1.72, and 1.61-fold; and DPPH⢠inhibition increased by 78%, 58%, and 56%, respectively. Dried ginger also exhibited better inhibitory effects on the lipopolysaccharides-induced nitric oxide production in murine macrophage RAW 264.7. The drying process demonstrated a positive effect on the bioactivities of ginger. The sun-dried ginger exhibited the most potent antioxidant properties with the best enhanced anti-inflammatory activity followed by the oven-dried ginger and lastly, the freeze-dried ginger.
RESUMO
As representações sobre a juventude variam histórica e culturalmente. No Alto Rio Negro, vivem grupos indígenas com mais de três séculos de contato com o mundo não-indígena. Nos últimos anos, os rituais de iniciação masculina foram suprimidos e houve a introdução da escola formal. Estes acontecimentos geraram ressignificações nas formas de representar as diferentes fases da vida. Neste trabalho objetivou-se compreender a construção das representações sobre a juventude indígena; como elas se correlacionam com as demais fases do ciclo vital e como contribuem para configurar práticas sociais estruturadas em torno deste grupo social. Analisaram-se diversas histórias-míticas que tratam das relações intergeracionais; estas foram correlacionadas com os termos designativos das diversas da vida, com as outras narrativas nativas e com dados obtidos a partir da observação participante. O estudo conclui que as representações sobre juventude são configuradas pelos papeis sociais de iniciandos/iniciados, fundidos na atualidade, de modo contraditório, numa categoria genérica e prolongada de estudante, desprovida de estatuto sociológico próprio, alongando a condição liminar desta fase da vida.
Assuntos
Brasil , Indígenas Sul-Americanos , Saúde de Populações Indígenas , Ecossistema Amazônico , Antropologia Cultural , Pesquisa Qualitativa , Saúde do AdolescenteRESUMO
Relato de Silvio Barreto, do grupo indígena Bará, doutorando pelo PPGAS / UFAM e pesquisador do NEAI. Reside na Comunidade São João, BR174 - Manaus.