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Cellulosic microfibrils in plant cell walls are largely ensheathed and probably tethered by hydrogen-bonded hemicelluloses. Ensheathing may vary developmentally as hemicelluloses are peeled to enable cell expansion. We characterised a simple method to quantify ensheathed versus naked cellulosic surfaces based on the ability to adsorb a radiolabelled 'cellulose-complementary oligosaccharide', [3H]cellopentaitol. Filter-paper (cellulose) adsorbed 40% and >80% of aqueous 5nM [3H]cellopentaitol within ~1h and ~20h respectively. When [3H]cellopentaitol was rapidly dried onto filter-paper, ~50% of it was desorbable by water, whereas after ~1d annealing in aqueous medium the adsorption became too strong to be reversible in water. 'Strongly' adsorbed [3H]cellopentaitol was, however, ~98% desorbed by 6M NaOH, ~50% by 0.2M cellobiose, and ~30% by 8M urea, indicating a role for hydrogen-bonding reinforced by complementarity of shape. Gradual adsorption was promoted by kosmotropes (1.4M Na2SO4 or 30% methanol), and inhibited by chaotropes (8M urea), supporting a role for hydrogen-bonding. [3H]Cellopentaitol adsorption was strongly competed by non-radioactive cello-oligosaccharides (Cell2-6), the IC50 (half-inhibitory concentration) being highly size-dependent: Cell2, ~70 mM; Cell3, ~7 mM; and Cell4-6, ~0.05 mM. Malto-oligosaccharides (400mM) had no effect, confirming the role of complementarity. The quantity of adsorbed [3H]cellopentaitol was proportional to mass of cellulose. Of seven cottons tested, wild-type Gossypium arboreum fibres were least capable of adsorbing [3H]cellopentaitol, indicating ensheathment of their microfibrillar surfaces, confirmed by their resistance to cellulase digestion, and potentially attributable to a high glucuronoarabinoxylan content. In conclusion, [3H]cellopentaitol adsorption is a simple, sensitive and quantitative way of titrating 'naked' cellulose surfaces.
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Terminalia arjuna is an evergreen medicinal plant that belongs to the Combretaceae family of flowering plants. The bark of the plant exhibits antiviral, anticancer, hypocholesterolemic, antioxidant and antimicrobial properties. In this study, composition antibacterial activity, antioxidant activity and cytotoxicity of bark oil of Terminalia Arjuna (Roxb.) were reported. Oils were extracted by microwave assisted hydrodistillation where an oil yield of 0.18% was obtained followed by the identification of 35 compounds by gas chromatography mass spectrometry. The most abundant volatiles were furfural (11.11%), isoeugenol (9.99%), p-ethylguaiacol (9.97%), α-cadinol (9.57%), and estragole (9.47%). The oil was further evaluated against ten different drug resistant strains where oil showed significant activity against all pathogens and the highest activity was found against Acinetobacter baumannii (22mm), Klebsiella pneumoniae (22mm) and Staphylococcus aureus (22mm) in a concentration-dependent manner. Antioxidant activity evaluation demonstrated 68% radical scavenging activity by the volatile oil as compared to 81% of the standard, ascorbic acid at a concentration of 1000 µg. Cytotoxicity studies were conducted to see the effect of sample on the expression level of a housekeeping gene, Glyceraldehyde 3-phosphate dehydrogenase where it did not affect the normal transcription of the gene.
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In this study, small antimicrobial peptides of <10 kDa (AMPs) from Moringa oleifera L were separated from crude protein by Ultra-15 Centrifugal filter devices and partially purified. The potency of morintides to interfere with the virulence determinants like biofilms, siderophores and elastase of selected bacteria was investigated by spectrophotometric method and Pseudomonas quinolone signal (PQS) by Thin-layer chromatography (TLC). GraphPad Prism 5.0 was used for statistical purpose. Assays were subjected to a two-way analysis of variance with Bonferroni as post-test. Shigella flexneri, Klebsiella pneumoniae, Escherichia coli and Staphylococcus aureus, showed a decrease in the attachment of cells to form biofilm in the presence of the morintides. While Pseudomonas aeruginosa and Salmonella typhi showed an increase in the attachment of cells to form biofilms. Morintides were very effective in the disruption of developed biofilms in cases of S. aureus, E. coli, K. pneumoniae from surgical wounds and S. flexneri, while others remained ineffective in the disruption of developed biofilms. Siderophore production was decreased by all bacterial strains under investigation in the presence of morintides except P. aeruginosa. The outcome of the research may have a significant contribution to drug discovery against antibiotic resistant biofilms of bacteria.
Asunto(s)
Sideróforos , Staphylococcus aureus , Antibacterianos/metabolismo , Antibacterianos/farmacología , Bacterias , Biopelículas , Escherichia coli , Klebsiella pneumoniae , Pruebas de Sensibilidad Microbiana , Elastasa Pancreática/metabolismo , Pseudomonas aeruginosa , Sideróforos/metabolismo , Sideróforos/farmacología , Virulencia , Factores de Virulencia/metabolismoRESUMEN
Pseudomonas aeruginosa harbors pvcABCD operon that is responsible for the synthesis of paerucumarin. Here we report the involvement of pvcABCD operon in chloramphenicol and ciprofloxacin resistance. P. aeruginosa mutant defective in pvcB (PW4832) was more sensitive to chloramphenicol and ciprofloxacin in comparison with its parent strain (MPAO1). A mutation in pvcA gene in MPAO1 (PW4830) did not alter the sensitivity to either antibiotic. As chloramphenicol and ciprofloxacin are substrates of MexEF-OprN efflux pump, so we decided to investigate the modulation of MexEF-OprN and its transcriptional regulator MexT in PW4832, PW4830 and MPAO1 strains. We isolated and sequenced mexT gene from MPAO1, PW4830 and PW4832. The nucleotide sequence of mexT gene in all three strains was identical. Expression levels of mexEF-oprN, mexT and mexS genes were checked via quantitative real-time RT-PCR. All these genes showed significant repression in mRNA levels in PW4832 as compared to MPAO1. These results indicate that chloramphenicol and ciprofloxacin sensitivity in PW4832 is due to transcriptional repression of mexT and mexEF-oprN genes. Exogenous addition of paerucumarin resumed the expression of mexT and mexEF-oprN genes as well as resistance against chloramphenicol and ciprofloxacin in PW4832 strain. This is a novel finding linking pvcB gene of P. aeruginosa with chloramphenicol and ciprofloxacin resistance and MexEF-OprN pump modulation which needs to be further explored.
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Cloranfenicol , Pseudomonas aeruginosa , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Cloranfenicol/farmacología , Ciprofloxacina/farmacología , Regulación Bacteriana de la Expresión Génica , Mutación , Operón , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismoRESUMEN
The methanolic extract of leaves and stem of Cestrum nocturnum and Cestrum diurnum were investigated for their antioxidant and anticancer attribute through standard methods. 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was carried out to estimate the antioxidant activity of the extracts. Whereas, anticancer potential of extracts were tested against colon cancer cell line, HCT 116 and acute myeloid leukemia (AML) cell lines, THP-1. Results showed that extracts of both plants exhibited a very strong antioxidant activity in a dose dependent manner. In addition, both extracts efficiently increased the cell death in two different cancer cell lines. Moreover, DNA fragmentation analysis further strengthens the anticancer potential of extracts of both types of plants. Current study, therefore, provide a preliminary data highlighting the antioxidant and anticancer activities of methanolic extract of leaves and stem of Cestrum nocturnum and Cestrum diurnum.
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Antineoplásicos/farmacología , Antioxidantes/farmacología , Cestrum/química , Extractos Vegetales/farmacología , Compuestos de Bifenilo/química , Línea Celular , Línea Celular Tumoral , Células HCT116 , Humanos , Metanol/química , Picratos/química , Hojas de la Planta/química , Células THP-1RESUMEN
Paerucumarin synthesized by pvc operon pvcABCD is an iron binding molecule which modulates biofilm formation in Pseudomonas aeruginosa but its direct function in bacterial pathogenesis needs further investigation. pvcA synthesizes isonitrile functionalized tyrosine (IFT) which is converted to mature paerucumarin by the proteins encoded by pvcB, pvcC and pvcD genes. Interruption of pvcB in MPAO1 resulted in accumulation of IFT as it cannot be converted to mature molecule. The MPAO1 pvcB mutant (PW4832) showed enhanced swarming motility, while complementation with plasmid pLL2 carrying pvcB reduced swarming motility. Enhanced levels of rhlA expression and rhamnolipid production were observed in PW4832 compared to the parent strain. Overexpression of ptxR, the positive regulator of pvcABCD, in PW4832 caused accumulation of more IFT and further elevated the level of rhlA expression. Expression of the quorum sensing system transcriptional activators lasR and rhlR, as well as the synthase genes lasI and rhlI, was enhanced in PW4832 compared to MPAO1, as was PQS accumulation. Exogenously added IFT, but not paerucumarin, enhanced the production of rhamnolipids in P. aeruginosa. These results suggest that IFT enhances swarming motility in P. aeruginosa either directly by enhancing rhamnolipid production or indirectly through modulation of the quorum sensing systems. This is the first report assigning an independent function to IFT in P. aeruginosa.
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Locomoción/efectos de los fármacos , Redes y Vías Metabólicas/genética , Nitrilos/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/fisiología , Percepción de Quorum/efectos de los fármacos , Tirosina/metabolismo , Proteínas Bacterianas/genética , Expresión Génica , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Operón , PlásmidosRESUMEN
Microbial biofilms have gathered interest in recent years as they have become the major cause of nosocomial infections. The abuse and misuse of antibiotics have created a selective pressure that results in widespread formation of resistant bacterial strains and a need to devise novel plant based antimicrobials. In this study, antimicrobial peptides were isolated from Peganum harmala and their effect was examined on biofilm related colonization genes of Pseudomonas aeruginosa and Staphylococcus aureus isolated from burn and surgical wounds. Results showed that in P. aeruginosa isolated from burn wound, the expression of flagellar gene (flgK), pilin gene (pilA) and fimbriae gene (cupA1) was significantly down-regulated indicating that Peganum harmala antimicrobial peptides (PhAMP) damage locomotors of planktonic cells by affecting the gene expression while in resistant biofilm cells, the expression of flgK, cupA1 and polysaccharide synthesis gene (pslA) was enhanced in the presence of PhAMP. In P. aeruginosa isolated from surgical wounds which was more sensitive; the expression of flgK, pilA, cupA1 and pslA was significantly down-regulated in biofilms and planktonic cells in the presence of PhAMP thus disrupting locomotors of planktonic as well as biofilm cells. In S. aureus isolated from burn wounds; the expression of capsular polysaccharide synthesis gene (CPS5) and inter cellular adhesion gene (icaA) was significantly up-regulated in biofilms as well as in planktonic cells in response to PhAMP stress showing resistance mechanism. Thus these genes can be used as efficient resistance markers for bacterial pathogens against antimicrobial agents.
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Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Peganum , Péptidos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Fimbrias/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Humanos , Plancton/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiología , Staphylococcus aureus/genética , Staphylococcus aureus/fisiologíaRESUMEN
Metallo-ß-lactamase (MBL) producing Escherichia coli are an emerging and serious threat to public health sector around the globe. MBL are spreading via plasmids to the host pathogens and produce resistance against carbapenems and left limited or no treatment option. Therefore, we designed this study to determine the dissemination of MBL producing E. coli in our locality. E. coli (n=100) were collected from various clinical samples from different tertiary care hospitals, Faisalabad. Microbes were sub-cultured on MacConkey and UTI Chromo Select agar. Bacteria were identified on the basis of culture characteristics and biochemically confirmed by API 20E. Antimicrobial susceptibility testing, carbapenemase and MBL was performed as per CLSI 2018 guidelines. Molecular identification of MBL genes were performed using specific primers by PCR. Of 100 E. coli, majority of them isolated from urine (n=55) followed by pus (n=23) and blood (n=22). Antibiogram displayed that all the E. coli were resistant to ß-lactam drugs including carbapenems followed by 76% to ciprofloxacin and 60% to amikacin. Among these, 81% were MBL producers. Molecular characterization revealed that 18.4% were blaNDM and 15.3% were blaVIM producers. This study concluded that there is high prevalence of MBL producing E. coli in our clinical settings.
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Proteínas de Escherichia coli/genética , Escherichia coli/enzimología , beta-Lactamasas/genética , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Humanos , Pruebas de Sensibilidad MicrobianaRESUMEN
Many clinical-pathogens have developed resistance against known antibiotics and there is an urgent need for the discovery of novel antibiotics. In this study, low molecular weight peptides were isolated from seeds/leaves of 20 medicinal plants and tested for their antibacterial activity against laboratory strains of S. aureusand P. aeruginosa. Peptides isolated from Peganum harmala (PhAMP) exhibited maximum activity against laboratory strains. As clinical-isolates are more virulent and resistant to antibiotics, we tested the potential of PhAMP on these bacterial strains isolated from infected wounds. Pathogens isolated from burn-wounds (S. aureus, P. aeruginosa and K. pneumoniae) and surgical-wounds (P. aeruginosa and K. pneumoniae) exhibited zones of inhibition against PhAMP when tested by disc diffusion method. Biofilm formation of wound pathogens in the presence/absence of PhAMP was analyzed to check its effect. Surgical-wound pathogens and K. pneumoniae from burn-wound showed significant reduction in biofilm formation and planktonic bacteria. While biofilms of S. aureus and P. aeruginosa from burn-wound showed resistance against PhAMP. An effective antibiotic treatment should not only inhibit but should also disrupt already developed biofilms. PhAMP was very effective in the disruption of developed biofilm of all pathogens after 36 hours. This data unravels the potential of PhAMP as a novel, natural antibiotic against clinical-pathogens.
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Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Quemaduras/microbiología , Peganum , Extractos Vegetales/farmacología , Herida Quirúrgica/microbiología , Antibacterianos/aislamiento & purificación , Antibacterianos/uso terapéutico , Biopelículas/crecimiento & desarrollo , Quemaduras/tratamiento farmacológico , Humanos , Pruebas de Sensibilidad Microbiana , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/uso terapéutico , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/uso terapéutico , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/farmacología , Proteínas de Plantas/uso terapéutico , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas aeruginosa/fisiología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/fisiología , Herida Quirúrgica/tratamiento farmacológicoRESUMEN
In Pseudomonas aeruginosa, type IV pili (TFP)-dependent twitching motility is required for development of surface-attached biofilm (SABF), yet excessive twitching motility is detrimental once SABF is established. In this study, we show that mucin significantly enhanced twitching motility and decreased SABF formation in strain PAO1 and other P. aeruginosa strains in a concentration-dependent manner. Mucin also disrupted partially established SABF. Our analyses revealed that mucin increased the amount of surface pilin and enhanced transcription of the pilin structural gene pilA. Mucin failed to enhance twitching motility in P. aeruginosa mutants defective in genes within the pilin biogenesis operons pilGHI/pilJK-chpA-E. Furthermore, mucin did not enhance twitching motility nor reduce biofilm development by chelating iron. We also examined the role of the virulence factor regulator Vfr in the effect of mucin. In the presence or absence of mucin, PAOΔvfr produced a significantly reduced SABF. However, mucin partially complemented the twitching motility defect of PAOΔvfr. These results suggest that mucin interferes with SABF formation at specific concentrations by enhancing TFP synthesis and twitching motility, that this effect, which is iron-independent, requires functional Vfr, and only part of the Vfr-dependent effect of mucin on SABF development occurs through twitching motility.
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Biopelículas/crecimiento & desarrollo , Fimbrias Bacterianas/fisiología , Mucinas/fisiología , Pseudomonas aeruginosa/fisiología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Movimiento , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidadRESUMEN
In the era of global warming, stress combinations instead of individual stress are realistic threats faced by plants that can alter or trigger a wide range of plant responses. In the current study, the cumulative effect of charcoal rot disease caused by notorious fungal pathogen viz., Macrophomina phaseolina was investigated under toxic levels of copper (Cu) in mash bean, and farmyard manure (FYM) was employed to manage stress. Therefore, Cu-spiked soil (50 and 100 mg/kg) was inoculated with the pathogen, and amended with 2% FYM, to assess the effect of intricate interactions on mash bean plants through pot experiments. Results demonstrated that the individual stress of the pathogen or Cu was more severe for morpho-growth, physio-biochemical, and expression profiles of stress-related genes and total protein in mash bean plants as compared to stress combinations. Under single Cu stress, a significant amount of Cu accumulated in plant tissues, particularly in roots than in upper ground tissues, while, under stress combination less Cu accumulated in the plants. Nonetheless, 2% FYM in soil encountered the negative effect of stress responses provoked by the pathogen, Cu, or both by improving health markers (photosynthetic pigments, reducing sugar, total phenolics) and oxidative stress markers (catalase, peroxidase, and polyphenol oxidase), together with regulating the expression of stress-related genes (catalase, ascorbate peroxidase, and cytokinin-resistant genes), and proteins, besides decreasing Cu uptake in the plants. FYM worked better at lower concentrations (50 mg/kg) of Cu than at higher ones (100 mg/kg), hence could be used as a suitable option for better growth, yield, and crop performance under charcoal rot disease stress in Cu-contaminated soils.
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Cobre , Estiércol , Catalasa , PeroxidasaRESUMEN
A class of proteins, 1-aminocyclopropane-1-carboxylate oxidase (ACO), is required in the final step of production of ethylene from its immediate precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Despite the crucial and regulatory role of ACO gene family in the fiber development, it has not been thoroughly analyzed and annotated in G. barbadense genome. In the present study, we have identified and characterized all isoforms of ACO gene family from genomes of Gossypium arboreum, G. barbadense, G. hirsutum and G. raimondii. Phylogenetic analysis classified all ACO proteins into six distinct groups on the basis of maximum likelihood. Gene locus analysis and circos plots indicated the distribution and relationship of these genes in cotton genomes. Transcriptional profiling of ACO isoforms in G. arboreum, G. barbadense and G. hirsutum fiber development exhibited the highest expression in G. barbadense during early fiber elongation. Moreover, the accumulation of ACC was found highest in developing fibers of G. barbadense in comparison with other cotton species. ACO expression and ACC accumulation correlated with the fiber length in cotton species. Addition of ACC to the ovule cultures of G. barbadense significantly increased fiber elongation while ethylene inhibitors hindered fiber elongation. These findings will be helpful in dissecting the role of ACOs in cotton fiber development and pave a way towards genetic manipulations for fiber quality improvement.
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Fibra de Algodón , Gossypium , Filogenia , Gossypium/genética , Etilenos/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
Survival of living organisms depends on the availability of water resources required for agriculture. In the current scenario of limited water resources, it is our priority to maximise the yield potential of crops with a minimum supply of available water. In this study, we evaluated seven cultivated varieties of Gossypium hirsutum (FH-114, FH-152, FH-326, FH-492, FH-942, VH-327 and FH-NOOR) for their tolerance, yield potential and fibre quality under water shortages. We also studied the effect of drought stress on osmoregulation, chlorophyll content, antioxidant (peroxidase and catalase) activity, lipid peroxidation and secondary metabolite accumulation in the varieties under study. It was revealed that three varieties (FH-114, FH-152 and VH-327) exhibited a lower stress susceptibility index and more tolerance to drought stress. All the varieties demonstrated enhanced proline and malondialdehyde content, but no significant change in chlorophyll content was observed under limited water supply. Antioxidant activity offered by catalase and phenolic content was enhanced in FH-492 whilst peroxidase activity increased in FH-114 and FH-326. Phenolic content was highest in FH-942 and decreased significantly in the remaining varieties. Ginning outturn of the cotton varieties increased in VH-327 (19.8%) and FH-326 (3.7%), was not affected in FH-114 and FH-492 and was reduced in FH-152, FH-942 and FH-NOOR. All cotton varieties tested showed an increase in micronaire thickness when exposed to drought stress as early as the seedling stage. This study highlights the evaluation and screening of cotton varieties for their response to drought stress in terms of yield and fibre quality when exposed to water shortages during plant development and can help in devising irrigation plans.
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The plant genome has conserved small non-coding microRNAs (miRNAs) genes about 20-24 nucleotides long. They play a vital role in the gene regulation at various stages of plant life. Their conserved nature among the various organisms not only suggests their early evolution in eukaryotes but also makes them a good source of new miRNA discovery by homology search using bioinformatics tools. A systematic search approach was used for interspecies orthologues of miRNA precursors, from known sequences of Gossypium in GenBank. The study resulted in 22 miRNAs belonging to 13 families. We found 7 miRNA families (miR160, 164, 827, 829, 836, 845 and 865) for the first time in cotton. All 22 miRNA precursors form stable minimum free energy (mfe) stem loop structure as their orthologues form in Arabidopsis and the mature miRNAs reside in the stem portion of the stem loop structure. Fifteen miRNAs belong to the world's most commercial fiber producing upland cotton (Gossypium hirsutum), five are from Gossypium raimondii and one each is from Gossypium herbaceum and Gossypium arboreum. Their targets consist of transcription factors, cell division regulating proteins and virus response gene. The discovery of 22 miRNAs will be helpful in future for detection of precise function of each miRNA at a particular stage in life cycle of cotton.
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Genoma de Planta , Gossypium/genética , MicroARNs/genética , ARN de Planta/genética , Arabidopsis/genética , Secuencia de Bases , Biología Computacional , Secuencia Conservada , Bases de Datos de Ácidos Nucleicos , Etiquetas de Secuencia Expresada , Genes de Plantas , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Alineación de Secuencia , Análisis de Secuencia de ARNRESUMEN
Proteins encoded by the Pseudomonas aeruginosa pvcA-D operon synthesize a novel isonitrile functionalized cumarin termed paerucumarin. The pvcA-D operon enhances the expression of the P. aeruginosa fimbrial chaperone/usher pathway (cup) genes and this effect is mediated through paerucumarin. Whether pvcA-D and/or paerucumarin affect the expression of other P. aeruginosa genes is not known. In this study, we examined the effect of a mutation in pvcA-D operon the global transcriptome of the P. aeruginosa strain PAO1-UW. The mutation reduced the expression of several ironcontrolled genes including pvdS, which is essential for the expression of the pyoverdine genes. Additional transcriptional studies showed that the pvcA-D operon is not regulated by iron. Exogenously added paerucumarin enhanced pyoverdine production and pvdS expression in PAO1-UW. Iron-chelation experiments revealed that purified paerucumarin chelates iron. However, exogenously added paerucumarin significantly reduced the growth of a P. aeruginosa mutant defective in pyoverdine and pyochelin production. In contrast to other secondary metabolite, Pseudomonas quinolone signal (PQS), paerucumarin is not localized to the P. aeruginosa membrane vesicles. These results suggest that paerucumarin enhances the expression of iron-controlled genes by chelating iron within the P. aeruginosa extracellular environment. Although paerucumarin chelates iron, it does not function as a siderophore. Unlike PQS, paerucumarin is not associated with the P. aeruginosa cell envelope.
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Vesículas Extracelulares/metabolismo , Hierro/metabolismo , Pseudomonas aeruginosa/metabolismo , Metabolismo Secundario , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Vesículas Extracelulares/genética , Regulación Bacteriana de la Expresión Génica , Mutación , Oligopéptidos/metabolismo , Operón , Pseudomonas aeruginosa/genéticaRESUMEN
In response to diverse environmental stimuli at different infection sites, Pseudomonas aeruginosa, a serious nosocomial pathogen, coordinates the production of different virulence factors through a complicated network of the hierarchical quorum-sensing (QS) systems including the las, rhl, and the 2-alkyl-4-quinolone-related QS systems. We recently showed that at early stages of growth serum alters the expression of numerous P. aeruginosa genes. In this study, we utilized transcriptional analysis and enzyme assays to examine the effect of serum on the QS and QS-controlled virulence factors during early and late phases of growth of the P. aeruginosa strain PAO1. At early phase, serum repressed the transcription of lasI, rhlI, and pqsA but not lasR or rhlR. However, at late phase, serum enhanced the expression of all QS genes. Serum produced a similar effect on the synthesis of the autoinducers 3OC12-HSL, C4-HSL, and HHQ/PQS. Additionally, serum repressed the expression of several QS-controlled genes in the early phase, but enhanced them in the late phase. Furthermore, serum influenced the expression of different QS-positive (vqsR, gacA, and vfr) as well as QS-negative (rpoN, qscR, mvaT, and rsmA) regulatory genes at either early or late phases of growth. However, with the exception of PAOΔvfr, we detected comparable levels of lasI/lasR expression in PAO1 and PAO1 mutants defective in these regulatory genes. At late stationary phase, serum failed to enhance lasI/lasR expression in PAOΔvfr. These results suggest that depending on the phase of growth, serum differentially influenced the expression of P. aeruginosa QS and QS-controlled virulence genes. In late phase, serum enhanced the expression of las genes through vfr.
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Medios de Cultivo/farmacología , Regulación Bacteriana de la Expresión Génica/fisiología , Pseudomonas aeruginosa/genética , Percepción de Quorum/genética , Suero , Animales , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Bovinos/sangre , Genes Bacterianos , Pseudomonas aeruginosa/crecimiento & desarrollo , Factores de Tiempo , Transcripción Genética , Virulencia/genéticaRESUMEN
The Pseudomonas aeruginosa fimbrial structures encoded by the cup gene clusters (cupB and cupC) contribute to its attachment to abiotic surfaces and biofilm formation. The P. aeruginosa pvcABCD gene cluster encodes enzymes that synthesize a novel isonitrile functionalized cumarin, paerucumarin. Paerucumarin has already been characterized chemically, but this is the first report elucidating its role in bacterial biology. We examined the relationship between the pvc operon and the cup gene clusters in the P. aeruginosa strain MPAO1. Mutations within the pvc genes compromised biofilm development and significantly reduced the expression of cupB1-6 and cupC1-3, as well as different genes of the cupB/cupC two-component regulatory systems, roc1/roc2. Adjacent to pvc is the transcriptional regulator ptxR. A ptxR mutation in MPAO1 significantly reduced the expression of the pvc genes, the cupB/cupC genes, and the roc1/roc2 genes. Overexpression of the intact chromosomally-encoded pvc operon by a ptxR plasmid significantly enhanced cupB2, cupC2, rocS1, and rocS2 expression and biofilm development. Exogenously added paerucumarin significantly increased the expression of cupB2, cupC2, rocS1 and rocS2 in the pvcA mutant. Our results suggest that pvc influences P. aeruginosa biofilm development through the cup gene clusters in a pathway that involves paerucumarin, PtxR, and different cup regulators.
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Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Chaperonas Moleculares/genética , Operón , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Transducción de Señal , Proteínas Bacterianas/metabolismo , Biopelículas , Flagelos/genética , Flagelos/metabolismo , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Familia de Multigenes , Mutación , Pili Sexual/genética , Pili Sexual/metabolismo , Unión Proteica , Pseudomonas aeruginosa/crecimiento & desarrolloRESUMEN
The enigma that is Alzheimer's disease (AD) continues to present daunting challenges for effective therapeutic intervention. The lack of disease-modifying therapies may, in part, be attributable to the narrow research focus employed to understand this complex disease. Most studies into disease pathogenesis are based on a priori assumptions about the role of AD lesion-associated proteins such as amyloid-ß and tau. However, the complex disease processes at work may not be amenable to single-target therapeutic approaches. Genome-wide expression studies provide an unbiased approach for investigating the pathogenesis of complex diseases like AD. A growing literature suggests a role for cerebrovascular contributions to the pathogenesis of AD. The objective of the current study is to examine human brain microvessels isolated from AD patients and controls by microarray analysis. Differentially expressed genes with more than 2-fold change are used for further data analysis. Gene ontology analysis and pathway analysis algorithms within GeneSpringGX are employed to understand the regulatory networks of differentially expressed genes. Twelve matched pairs of AD and control brain microvessel samples are hybridized to Agilent Human 4 × 44 K arrays in replication. We document that more than 2,000 genes are differentially altered in AD microvessels and that a large number of these genes map to pathways associated with immune and inflammatory response, signal transduction, and nervous system development and function categories. These data may help elucidate heretofore unknown molecular alterations in the AD cerebromicrovasculature.