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1.
Nucleic Acids Res ; 48(4): 2156-2172, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31925429

RESUMO

H-NS proteins act as osmotic sensors translating changes in osmolarity into altered DNA binding properties, thus, regulating enterobacterial genome organization and genes transcription. The molecular mechanism underlying the switching process and its conservation among H-NS family members remains elusive. Here, we focus on the H-NS family protein MvaT from Pseudomonas aeruginosa and demonstrate experimentally that its protomer exists in two different conformations, corresponding to two different functional states. In the half-opened state (dominant at low salt) the protein forms filaments along DNA, in the fully opened state (dominant at high salt) the protein bridges DNA. This switching is a direct effect of ionic strength on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of MvaT. The asymmetric charge distribution and intramolecular interactions are conserved among the H-NS family of proteins. Therefore, our study establishes a general paradigm for the molecular mechanistic basis of the osmosensitivity of H-NS proteins.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , DNA/química , Transativadores/química , Proteínas de Bactérias/genética , DNA/genética , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética , Concentração Osmolar , Domínios Proteicos/genética , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Transativadores/genética
2.
Spectrochim Acta A Mol Biomol Spectrosc ; 224: 117417, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31362188

RESUMO

To ensure the food security and protect public health, development of rapid and reliable approaches to detecting foodborne pathogens is of great significance. In this study, polydopamine-polyethyleneimine (PDA-PEI) copolymer dots are prepared via the self-polymerization of dopamine and cross-linking with branched PEI at room temperature. The PDA-PEI copolymer dots are very stable against photobleaching, extreme pH, as well as high ionic strength. They are used as a fluorescent probe to fabricate a biosensor for rapid and sensitive detection and quantification of Pseudomonas aeruginosa (P. aeruginosa). In the biosensor, dual-aptamers of P. aeruginosa are used to label PDA-PEI copolymer dots. Compared to single aptamer labeled PDA-PEI dots, the dual-aptamers labeled PDA-PEI dots endow the biosensor with enhanced sensitivity for target pathogen. The fluorescence biosensor demonstrates a wide linear response to P. aeruginosa in the concentration range of 101-107 cfu mL-1 with acceptable selectivity. The limit of detection is calculated to be 1 cfu mL-1. The whole detection process can be finished in 1.5 h. The feasibility of the fabricated biosensor is verified by successful determination of P. aeruginosa in skim milk, orange juice, and popsicle samples. The biosensor provides an alternative and attractive platform for rapid and sensitive detection of bacteria in food products.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Indóis/química , Polietilenoimina/química , Polímeros/química , Pseudomonas aeruginosa , Animais , Técnicas de Tipagem Bacteriana/métodos , Sucos de Frutas e Vegetais/microbiologia , Leite/microbiologia , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/isolamento & purificação , Pontos Quânticos , Espectrometria de Fluorescência/métodos
3.
Spectrochim Acta A Mol Biomol Spectrosc ; 224: 117394, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31351419

RESUMO

This study reports the utilization of engineered molecular networks between bacteriophage (or phage) and gold nanoparticles (AuNPs) prepared ablating a high purity gold target in water by nanosecond laser source. Gold colloids are assembled with P9b phage clone, displaying the specific peptide (QRKLAAKLT), able to bind P. aeruginosa. The single components and assembled systems were characterized by spectroscopic and electronic techniques, such as the conventional optical absorption and micro-Raman spectroscopies as well as the Dynamic Light Scattering (DLS) and Scanning Transmission Electron Microscopy (STEM) techniques. The performance of the AuNPs-phage assembly as substrate for Surface-Enhanced Raman Spectroscopy (SERS) was tested against the detection of the characteristics Raman vibrational features of the Pseudomonas aeruginosa bacteria.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Ouro/química , Nanopartículas Metálicas/química , Sondas Moleculares/química , Análise Espectral Raman/métodos , Bacteriófagos/química , Bacteriófagos/metabolismo , Sondas Moleculares/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo
4.
Biochim Biophys Acta Proteins Proteom ; 1867(11): 140263, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31421227

RESUMO

Pseudomonas species export the amyloid-forming protein FapC to strengthen bacterial biofilm. P. species also produce the biosurfactant rhamnolipid (Rhl) and its outer membrane contains lipopolysaccharide (LPS). Given the possible contacts between FapC, Rhl and LPS, we here investigate how Rhl and LPS affect FapC fibrillation compared with SDS, known to promote fibrillation of proteins at sub-micellar concentrations. Micelles of all three surfactants help FapC bypass the nucleation lag phase, leading to rapid fibrillation, which persists even at high concentrations of micelles and incorporates almost all available FapC monomers. Fibrils formed at high micellar concentrations of Rhl and SDS seed fibrillation at low surfactant concentrations while retaining the original fibril structure. FapC interacts strongly with SDS to form a dense network of narrow fibrils. Small angle X-ray scattering (SAXS) analyses reveal that surfactants reduce the population of intermediates in the fibrillation process and detect a fast aggregation step over the first 2-4 h which precedes the main fibrillation monitored by Thioflavin T. An additional SAXS-detected rearrangement of early aggregates occurs after 4-10 h. At high Rhl concentrations, the micelles are decorated with protein fibrils. SDS induces FapC fibrillation so efficiently that epigallocatechin-3-gallate (EGCG) is unable to inhibit this process. However, EGCG stimulates FapC oligomer formation and inhibits fibrillation both on its own and in the presence of Rhl and LPS. This oligomer could be modelled as a compact core with a flexible shell. This suggests that EGCG can override the natural amyloid-stimulatory properties of these biosurfactants and thus target biofilm.


Assuntos
Proteínas Amiloidogênicas/química , Proteínas de Bactérias/química , Glicolipídeos/química , Lipopolissacarídeos/química , Agregados Proteicos , Pseudomonas aeruginosa/química
5.
Curr Microbiol ; 76(11): 1270-1277, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31401778

RESUMO

In this study, iTRAQ analysis and bioinformatics analysis were used to reveal the changes in key proteins induced by different concentrations of petroleum hydrocarbons during the biodegradation of petroleum hydrocarbons in Pseudomonas aeruginosa P6. Sixty-three proteins were identified as differentially expressed proteins, and all of them were strongly associated with the cellular processes related to the biodegradation of petroleum hydrocarbons. The results further showed that among the differentially expressed proteins, 3 chemotaxis-related proteins, 10 terminal oxidation of short-chain alkane-related proteins, and 13 transmembrane transport-related proteins were down regulated, while 1 uptake of petroleum hydrocarbon-related protein, 3 terminal oxidation of long-chain alkane-related proteins, 4 dehydrogenation-related proteins, 12 ß-oxidation-related proteins, and 2 metabolisms of acyl-CoA-related proteins were up regulated. These results indicated that during the biodegradation of petroleum hydrocarbons in P. aeruginosa P6, the activity of chemotaxis, the terminal oxidation of short-chain alkanes, and transmembrane transport decreased, while the activity of the uptake of petroleum hydrocarbons, the terminal oxidation of long-chain alkanes, dehydrogenation, ß-oxidation, and the metabolism of acyl-CoA increased under the 20,000 mg/L petroleum hydrocarbon condition compared with the 500 mg/L petroleum hydrocarbon condition. The findings revealed changes in the key proteins and the corresponding cellular process of the biodegradation of petroleum hydrocarbons in P. aeruginosa P6 under high and low concentrations of petroleum hydrocarbons and provided references for future studies.


Assuntos
Proteínas de Bactérias/genética , Hidrocarbonetos/metabolismo , Petróleo/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Hidrocarbonetos/análise , Oxirredução , Petróleo/análise , Proteômica , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética
6.
Colloids Surf B Biointerfaces ; 181: 943-952, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31382344

RESUMO

Rhamnolipids produced by P. aeruginosa MR01 were fractionated into mono- and di-rhamnolipids, and their dominant congeners, Rha-C10-C10 and Rha-Rha-C10-C10, were shown by mass spectrometry. Minimum surface tensions and critical micelle concentrations (CMC) were determined as "≃34 mN/m; ≃26.17 mg/l;" and "≃29 mN/m; ≃29.63 mg/l" for mono- and di-rhamnolipids, respectively. Spectrophotometry measurements provided a close approximation of CMC. Contact angle and diameter of wet area were determined for rhamnolipid-containing drops on hydrophobic paper to display their capability for alteration of surface wettability. Wet area measurement is a simple, reliable method not requiring a Drop Shape Analyzer. Cell viabilities determined by MTT assay showed a decline in a dose-dependent manner and estimated IC50 values were 25.87 µg/ml and 31.00 µg/ml for mono- and di-rhamnolipids treating MCF-7 cells for 48 h. Morphological observations using the inverted phase-contrast microscopy and fluorescence microscopy via Hoechst staining revealed the apoptotic characteristics in treated MCF-7 cells. The semi-quantitative RT-PCR method demonstrated that expression of the p53 gene in mRNA levels significantly (P < 0.05) increased when treated with 30 µg/ml of each rhamnolipid compound for 12 h. It can be concluded that rhamnolipids derived from MR01 show significant anticancer potential against MCF-7 cell line and should be further investigated as natural, therapeutic anti-tumor agents.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Glicolipídeos/farmacologia , Pseudomonas aeruginosa/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Glicolipídeos/biossíntese , Glicolipídeos/química , Humanos , Células MCF-7 , Microscopia de Fluorescência , Tamanho da Partícula , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Relação Estrutura-Atividade , Propriedades de Superfície , Proteína Supressora de Tumor p53/genética
7.
ACS Chem Biol ; 14(7): 1515-1527, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31268665

RESUMO

Members of the AAA+ (ATPase associated with various cellular activities) family of ATPases couple chemical energy derived from ATP hydrolysis for generation of mechanical force, resulting in conformational changes. The hydrolysis is brought about by highly conserved domains and motifs. The sensor I motif is critical for sensing and hydrolysis of the nucleotide. Pseudomonas aeruginosa FleQ is an ATPase that is a positive regulator of flagellar gene expression. We have determined the crystal structures of the ATPase domain of wild-type FleQ and sensor I mutants H287N and H287A in complex with ATPγS and Mg2+ to 2.4, 1.95, and 2.25 Šresolution, respectively. The structural data highlight the role of sensor I in regulating the ATPase activity. The in vitro and in vivo data demonstrate that the moderate ATPase activity of FleQ due to the presence of histidine in sensor I is essential for maintaining the monotrichous phenotype and for the rapid motility to biofilm transition.


Assuntos
Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes , Pseudomonas aeruginosa/fisiologia , Transativadores/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutação Puntual , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Transativadores/química , Transativadores/genética
8.
J Agric Food Chem ; 67(29): 8191-8196, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31282662

RESUMO

Conversion of free fatty acids into monoacylglycerol gives rise to new structural properties, particularly amphipathic property. Therefore, monoacylglycerols are widely used in pharmaceutical and food industries and are also reported to facilitate better absorption into the human body. A functional fatty acid when transformed into a monoacylglycerol will possibly conserve both the original functionality and amphipathic property. The compound 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was generated from oleic acid by Pseudomonas aeruginosa PR3 and was known to contain antimicrobial activities against a broad range of food-borne and plant pathogenic bacteria. Here, we attempted to convert DOD into its monoacylglycerol form using lipase for producing an amphipathic antibacterial agent. Consequently, the monoacylglycerol of DOD (DOD-MAG) was successfully produced by coincubating DOD, glycerol, and lipase at 30 °C. The maximum conversion yield reached 70% after 12 h of incubation. Antibacterial activity of DOD-MAG was enhanced by 8 times from the original activity of DOD against food-borne bacteria.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Monoglicerídeos/química , Ácidos Oleicos/química , Ácidos Oleicos/farmacologia , Pseudomonas aeruginosa/química , Antibacterianos/metabolismo , Microbiologia de Alimentos , Ácidos Oleicos/metabolismo , Pseudomonas aeruginosa/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento
9.
Biomater Sci ; 7(9): 3594-3598, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31329200

RESUMO

Detection of the biofilm of bacteria would be a counter strategy to detect hidden bacteria in their camouflage. Through unbiased screening of bacteria biofilm, we discovered a long wavelength probe CDr15 with extracellular DNA as the molecular target. CDr15 revealed a real-time geometric distribution of eDNA in a 3D bacterial colony.


Assuntos
Biofilmes , DNA/química , Espaço Extracelular/química , Corantes Fluorescentes/química , Pseudomonas aeruginosa/química , Estrutura Molecular
10.
Environ Sci Pollut Res Int ; 26(24): 24695-24706, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31240645

RESUMO

The biotoxicity of heavy metals in sediments toward benthic organisms has evoked great concern for the health of freshwater ecosystems. This study applied a sediment toxicity testing protocol to investigate the single and joint toxicity of cadmium (Cd) and lead (Pb) on Bellamya aeruginosa. B. aeruginosa were exposed to different concentrations of Cd (5, 25, and 100 mg/kg), Pb (20, 100, and 400 mg/kg), and their different concentration combinations. A suite of biomarkers, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), metallothionein (MT), malondialdehyde (MDA), and acetylcholinesterase (AChE), were measured after 7, 14, 21, and 28 days of exposure to evaluate their oxidative stress status. Cell apoptosis of soft tissue was also determined after exposure. Results revealed that these endpoints represented sensitive biomarkers for the characterization of the oxidative stress response induced by these metals. Specifically, a decrease of SOD and GPx and an increase of MDA were indicative of the potential failure of the antioxidant defense system in neutralizing the reactive oxygen species (ROS) generated in the exposure of the Pb-treated group. The integrated biomarker response (IBR) index revealed the most significant sub-lethal toxicity for Pb-spiked sediments, leading to the highest rate of cell apoptosis (70.8%). Exposure to Cd resulted in a time- and dose-dependent effect on MT levels, which suggested active detoxification of this metal. Exposure to the mixture resulted in amelioration of Pb toxicity, likely due to the competitive binding of Cd to active enzyme, with the result of an observed antagonistic interaction. This study indicated that B. aeruginosa represents a good biomonitor for assessing Cd and Pb contamination of sediments, and laid the foundation for their potential risk assessments in freshwater ecosystems.


Assuntos
Cádmio/toxicidade , Chumbo/toxicidade , Metalotioneína/metabolismo , Metais Pesados/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Animais , Biomarcadores/metabolismo , Catalase/metabolismo , Ecossistema , Água Doce , Glutationa Peroxidase/metabolismo , Malondialdeído/metabolismo , Metalotioneína/química , Pseudomonas aeruginosa/química , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo
11.
Methods Mol Biol ; 1995: 383-393, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31148140

RESUMO

Biosurfactants, surface active molecules synthesized by microorganisms, represent a promising alternative to the synthetic surfactants in many different applications. Among them, rhamnolipids have attracted considerable attention in the last years due to their extraordinary surface-active properties and biological activities. Rhamnolipids are usually synthesized by the gram-negative bacterium Pseudomonas aeruginosa as complex mixtures of different congeners. In this chapter, we describe the most common techniques that can be used for the production, recovery and purification of rhamnolipids, using two sequential chromatographic techniques to recover and separate the monorhamnolipid and dirhamnolipid congeners.


Assuntos
Glicolipídeos/metabolismo , Pseudomonas aeruginosa/metabolismo , Tensoativos/metabolismo , Técnicas de Cultura de Células/métodos , Cromatografia em Camada Delgada/métodos , Glicolipídeos/análise , Glicolipídeos/isolamento & purificação , Microbiologia Industrial/métodos , Óleos Vegetais/química , Plantas/química , Pseudomonas aeruginosa/química , Tensoativos/análise , Tensoativos/isolamento & purificação
12.
Appl Microbiol Biotechnol ; 103(15): 6271-6285, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31161392

RESUMO

Proton magnetic resonance-based metabolomics analysis was performed to determine the global metabolite changes in pathogenic bacterium Pseudomonas aeruginosa PAO1 following exposure to quorum sensing (QS) inhibitor hordenine. Pyocyanin inhibition assay confirmed that hordenine exhibited potent QS inhibitory activity. A total of 40 metabolites were assigned by PMR spectra. Hordenine treatment resulted in the destruction of QS system in P. aeruginosa PAO1 by downregulating the expressions of genes involved in QS. The synthesis of antioxidant enzymes was repressed and the oxidative stress was enhanced due to the dysfunctional QS system of P. aeruginosa PAO1. The enhanced oxidative stress induced by the dysfunctional QS system of P. aeruginosa PAO1 altered the membrane components, enhanced membrane permeability, and disturbed energy metabolism, amino acid metabolism, and nucleotide metabolism, and would ultimately attenuate the pathogenicity of P. aeruginosa PAO1. Hordenine may have promising potential for controlling nosocomial pathogens.


Assuntos
Antibacterianos/metabolismo , Metaboloma , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Tiramina/análogos & derivados , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Metabolômica , Tiramina/metabolismo
13.
PLoS Pathog ; 15(6): e1007820, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31194839

RESUMO

Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. Pseudomonas aeruginosa, an opportunistic pathogen, employs multiple quorum-sensing systems to control behaviors including virulence factor production and biofilm formation. One P. aeruginosa quorum-sensing receptor, called RhlR, binds the cognate autoinducer N-butryl-homoserine lactone (C4HSL), and the RhlR:C4HSL complex activates transcription of target quorum-sensing genes. Here, we use a genetic screen to identify RhlR mutants that function independently of the autoinducer. The RhlR Y64F W68F V133F triple mutant, which we call RhlR*, exhibits ligand-independent activity in vitro and in vivo. RhlR* can drive wildtype biofilm formation and infection in a nematode animal model. The ability of RhlR* to properly regulate quorum-sensing-controlled genes in vivo depends on the quorum-sensing regulator RsaL keeping RhlR* activity in check. RhlR is known to function together with PqsE to control production of the virulence factor called pyocyanin. Likewise, RhlR* requires PqsE for pyocyanin production in planktonic cultures, however, PqsE is dispensable for RhlR*-driven pyocyanin production on surfaces. Finally, wildtype RhlR protein is not sufficiently stabilized by C4HSL to allow purification. However, wildtype RhlR can be stabilized by the synthetic ligand mBTL (meta-bromo-thiolactone) and RhlR* is stable without a ligand. These features enabled purification of the RhlR:mBTL complex and of RhlR* for in vitro examination of their biochemical activities. To our knowledge, this work reports the first RhlR protein purification.


Assuntos
Proteínas de Bactérias , Pseudomonas aeruginosa , Percepção de Quorum/fisiologia , Receptores de Superfície Celular , Substituição de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caenorhabditis elegans , Mutação de Sentido Incorreto , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Piocianina/química , Piocianina/genética , Piocianina/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo
14.
Curr Protein Pept Sci ; 20(12): 1189-1203, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31038064

RESUMO

Antimicrobial Resistance (AMR) represents a serious threat to health and the global economy. However, interest in antibacterial drug development has decreased substantially in recent decades. Meanwhile, anti-virulence drug development has emerged as an attractive alternative to fight AMR. Although several macromolecular targets have been explored for this goal, their druggability is a vital piece of information that has been overlooked. This review explores this subject by showing how structure- based freely available in silico tools, such as PockDrug and FTMap, might be useful for designing novel inhibitors of the pyocyanin biosynthesis pathway and improving the potency/selectivity of compounds that target the Pseudomonas aeruginosa quorum sensing mechanism. The information provided by hotspot analysis, along with binding site features, reveals novel druggable targets (PhzA and PhzS) that remain largely unexplored. However, it also highlights that in silico druggability prediction tools have several limitations that might be overcome in the near future. Meanwhile, anti-virulence drug targets should be assessed by complementary methods, such as the combined use of FTMap/PockDrug, once the consensus druggability classification reduces the risk of wasting resources on undruggable proteins.


Assuntos
Antibacterianos/química , Simulação por Computador , Inibidores Enzimáticos/química , Proteínas/química , Pseudomonas aeruginosa/química , Animais , Sítios de Ligação , Bases de Dados de Compostos Químicos , Resistência Microbiana a Medicamentos , Humanos , Conformação Proteica , Piocianina/biossíntese , Piocianina/metabolismo , Percepção de Quorum , Transdução de Sinais , Relação Estrutura-Atividade , Fatores de Virulência
15.
Eur J Med Chem ; 177: 212-220, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31146126

RESUMO

Pathogenic micro-organisms utilize protein receptors (lectins) in adhesion to host tissues, a process that in some cases relies on the interaction between lectins and human glycoconjugates. Oligosaccharide epitopes are recognized through their three-dimensional structure and their flexibility is a key issue in specificity. In this paper, we analysed by X-ray crystallography the structures of the LecB lectin from two strains of Pseudomonas aeruginosa in complex with Lewis x oligosaccharide present on cell surfaces of human tissues. An unusual conformation of the glycan was observed in all binding sites with a non-canonical syn orientation of the N-acetyl group of N-acetyl-glucosamine. A PDB-wide search revealed that such an orientation occurs only in 4% of protein/carbohydrate complexes. Theoretical chemistry calculations showed that the observed conformation is unstable in solution but stabilised by the lectin. A reliable description of LecB/Lewis x complex by force field-based methods had proven especially challenging due to the special feature of the binding site, two closely apposed Ca2+ ions which induce strong charge delocalisation. By comparing various force-field parametrisations, we propose a general strategy which will be useful in near future for designing carbohydrate-based ligands (glycodrugs) against other calcium-dependent protein receptors.


Assuntos
Lectinas/metabolismo , Oligossacarídeos/metabolismo , Sítios de Ligação , Cálcio/química , Cálcio/metabolismo , Cristalografia por Raios X , Glucosamina/química , Glucosamina/metabolismo , Lectinas/química , Lectinas/genética , Ligantes , Conformação Molecular , Simulação de Dinâmica Molecular , Mutação , Oligossacarídeos/química , Ligação Proteica , Pseudomonas aeruginosa/química
16.
Anal Chim Acta ; 1066: 146-153, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31027530

RESUMO

The analysis of bacterial volatile organic compounds has gained attraction as a non-invasive way to identify disease-causing organisms, given that bacteria have unique metabolisms and volatile metabolic byproducts. In the present research, different adsorbent materials (Carbopack Y, X, B, Carboxen 1000 and Tenax TA), packed singularly or in combination, were compared in terms of sampling performance (sensitivity, repeatability and selectivity) for the extraction of standards and bacterial volatile metabolites in vitro (from Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli). After extraction, bacterial volatile organic compounds were desorbed and analyzed in a comprehensive two-dimensional gas chromatography system coupled to a time-of-flight mass spectrometer (GC × GC-ToF MS). The results show that Tenax has the greater ability to extract the standard mix as well as volatile organic compounds with better repeatability (4-26 RSD%), higher sensitivity (on average ∼24 fold) compared to Carbopack Y, X and Carboxen 1000 tube, which followed in terms of performance. In addition, Tenax confirmed the best sensitivity and discriminatory power with no misclassification in the untargeted and unsupervised analysis for the differentiation of the bacterial species.


Assuntos
Adsorção , Escherichia coli/química , Pseudomonas aeruginosa/química , Staphylococcus aureus/química , Compostos Orgânicos Voláteis/análise , Cromatografia Gasosa , Espectrometria de Massas , Propriedades de Superfície
17.
Drug Des Devel Ther ; 13: 909-924, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30936684

RESUMO

Vaccines for Pseudomonas aeruginosa have been of longstanding interest to immunologists, bacteriologists, and clinicians, due to the widespread prevalence of hospital-acquired infection. As P. aeruginosa becomes increasingly antibiotic resistant, there is a dire need for novel treatments and preventive vaccines. Despite intense efforts, there currently remains no vaccine on the market to combat this dangerous pathogen. This article summarizes current and past vaccines under development that target various constituents of P. aeruginosa. Targeting lipopolysaccharides and O-antigens have shown some promise in preventing infection. Recombinant flagella and pili that target TLR5 have been utilized to combat P. aeruginosa by blocking its motility and adhesion. The type 3 secretion system components, such as needle-like structure PcrV or exotoxin PopB, are also potential vaccine targets. Outer membrane proteins including OprF and OprI are newer representatives of vaccine candidates. Live attenuated vaccines are a focal point in this review, and are also considered for novel vaccines. In addition, phage therapy is revived as an effective option for treating refractory infections after failure with antibiotic treatment. Many of the aforementioned vaccines act on a single target, thus lacking a broad range of protection. Recent studies have shown that mixtures of vaccines and combination approaches may significantly augment immunogenicity, thereby increasing their preventive and therapeutic potential.


Assuntos
Vacinas Bacterianas/imunologia , Terapia por Fagos , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/terapia , Pseudomonas aeruginosa/imunologia , Animais , Vacinas Bacterianas/química , Humanos , Lipopolissacarídeos/antagonistas & inibidores , Fenômenos Mecânicos , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/virologia , Pseudomonas aeruginosa/química
18.
Environ Pollut ; 250: 262-273, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30999203

RESUMO

Dibenzofuran (DBF) derivatives have caused serious environmental problems, especially those produced by paper pulp bleaching and incineration processes. Prominent for its resilient mutagenicity and toxicity, DBF poses a major challenge to human health. In the present study, a new strain of Pseudomonas aeruginosa, FA-HZ1, with high DBF-degrading activity was isolated and identified. The determined optimum conditions for cell growth of strain FA-HZ1 were a temperature of 30 °C, pH 5.0, rotation rate of 200 rpm and 0.1 mM DBF as a carbon source. The biochemical and physiological features as well as usage of different carbon sources by FA-HZ1 were studied. The new strain was positive for arginine double hydrolase, gelatinase and citric acid, while it was negative for urease and lysine decarboxylase. It could utilize citric acid as its sole carbon source, but was negative for indole and H2S production. Intermediates of DBF 1,2-dihydroxy-1,2-dihydrodibenzofuran, 1,2-dihydroxydibenzofuran, 2-hydroxy-4-(3'-oxo-3'H-benzofuran-2'-yliden)but-2-enoic acid, 2,3-dihydroxybenzofuran, 2-oxo-2-(2'-hydrophenyl)lactic acid, and 2-hydroxy-2-(2'-hydroxyphenyl)acetic acid were detected and identified through liquid chromatography-mass analyses. FA-HZ1 metabolizes DBF by both the angular and lateral dioxygenation pathways. The genomic study identified 158 genes that were involved in the catabolism of aromatic compounds. To identify the key genes responsible for DBF degradation, a proteomic study was performed. A total of 1459 proteins were identified in strain FA-HZ1, of which 100 were up-regulated and 104 were down-regulated. A novel enzyme "HZ6359 dioxygenase", was amplified and expressed in pET-28a in E. coli BL21(DE3). The recombinant plasmid was successfully constructed, and was used for further experiments to verify its function. In addition, the strain FA-HZ1 can also degrade halogenated analogues such as 2, 8-dibromo dibenzofuran and 4-(4-bromophenyl) dibenzofuran. Undoubtedly, the isolation and characterization of new strain and the designed pathways is significant, as it could lead to the development of cost-effective and alternative remediation strategies. The degradation pathway of DBF by P. aeruginosa FA-HZ1 is a promising tool of biotechnological and environmental significance.


Assuntos
Benzofuranos/metabolismo , Poluentes Ambientais/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Dioxigenases/genética , Dioxigenases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Resíduos Industriais , Redes e Vias Metabólicas/genética , Proteômica , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento
19.
Molecules ; 24(7)2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30979013

RESUMO

Rhamnolipids are a mixture of the homologs species due to variations in the rhamnose units and ß-hydroxy fatty acid moieties, mainly including Rha-C10-C10, Rha-Rha-C10-C10, and Rha-C10. In this study, strain P. aeruginosa YM4 was selected for its capacity to efficiently produce di-rhamnolipid (Rha-Rha-C10-C10) as the predominant component with soybean oil and glycerol as carbon source, accounting for 64.8% and 85.7% of total products, respectively. The critical micelle concentration (CMC) of rhamnolipid products varies with the content of di-rhamnolipid, whereby lower CMC values corresponding to higher di-rhamnolipid contents. The rhamnolipids containing 85.7% di-rhamnolipid had the lowest CMC value of 50 mg/L. Accordingly the viscosity-reducing efficiency and oil-washing efficiency of rhamnolipids increased with higher di-rhamnolipid component. At a concentration of 500 mg/L, the rhamnolipids containing 85.7% di-rhamnolipid worked best and showed 82.5% oil-washing efficiency, which offered great promise for applications in enhanced oil recovery. The results showed the variation of structure and composition of rhamnolipids had a significant effect on their application.


Assuntos
Glicolipídeos/biossíntese , Poluição por Petróleo/prevenção & controle , Pseudomonas aeruginosa/metabolismo , Ramnose/biossíntese , Carbono/química , Ácidos Graxos/química , Glicerol/química , Glicolipídeos/química , Humanos , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Ramnose/química , Óleo de Soja/química , Tensoativos/química
20.
Sensors (Basel) ; 19(7)2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30925832

RESUMO

In this paper we analyze an experiment for the use of low-cost gas sensors intended to detect bacteria in wounds using a non-intrusive technique. Seven different genera/species of microbes tend to be present in most wound infections. Detection of these bacteria usually requires sample and laboratory testing which is costly, inconvenient and time-consuming. The validation processes for these sensors with nineteen types of microbes (1 Candida, 2 Enterococcus, 6 Staphylococcus, 1 Aeromonas, 1 Micrococcus, 2 E. coli and 6 Pseudomonas) are presented here, in which four sensors were evaluated: TGS-826 used for ammonia and amines, MQ-3 used for alcohol detection, MQ-135 for CO2 and MQ-138 for acetone detection. Validation was undertaken by studying the behavior of the sensors at different distances and gas concentrations. Preliminary results with liquid cultures of 108 CFU/mL and solid cultures of 108 CFU/cm2 of the 6 Pseudomonas aeruginosa strains revealed that the four gas sensors showed a response at a height of 5 mm. The ammonia detection response of the TGS-826 to Pseudomonas showed the highest responses for the experimental samples over the background signals, with a difference between the values ​​of up to 60 units in the solid samples and the most consistent and constant values. This could suggest that this sensor is a good detector of Pseudomonas aeruginosa, and the recording made of its values ​​could be indicative of the detection of this species. All the species revealed similar CO2 emission and a high response rate with acetone for Micrococcus, Aeromonas and Staphylococcus.


Assuntos
Gases/análise , Compostos Orgânicos Voláteis/química , Infecção dos Ferimentos/diagnóstico , Álcoois/análise , Amônia/análise , Candida/química , Candida/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Humanos , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/metabolismo , Compostos Orgânicos Voláteis/análise , Infecção dos Ferimentos/microbiologia
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