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1.
Appl Opt ; 61(10): 2459-2472, 2022 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-35471319

RESUMO

A multispectral imaging system, based on a modified plenoptic camera, is presented. By adding a color filter in the aperture plane of the imaging system, it is possible to simultaneously image multiple discrete colors of light-seven in this design. To develop a measurement system that does not rely on in situ calibrations, each of the optical elements was characterized a priori. For the camera sensor, measurements of the exposure linearity, exposure duration, and quantum efficiency were measured. Additionally, the transmission of the optical filters, both spectral and neutral density, as well as the signal attenuation of the filter holder itself were measured. These measurements result in an instrument that can quantitatively image the emission of seven discrete spectral bands simultaneously. An example application of pyrometry is presented where the emission of a blackbody calibration source with known temperature was imaged. It was determined that by fitting the measured emission at seven wavelengths to Planck's law of radiation, the temperature could be determined to a mean difference of 0.65ºC across five temperatures from 600° to 1000ºC when compared to the set-point temperature.

2.
Mol Plant Microbe Interact ; 32(6): 729-739, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30589364

RESUMO

PdeR, a response regulator of the two-component system (TCS) with the cognate histidine kinase PdeK, has been shown to be an active phosphodiesterase (PDE) for intracellular cyclic dimeric guanosine monophosphate (c-di-GMP) turnover and positively regulates the virulence of Xanthomonas oryzae pv. oryzae, the causal pathogen of bacterial blight of rice. To further reveal the key components and pathways involved in the PdeR-mediated c-di-GMP regulation of virulence, 16 PdeR-interacting proteins were identified, using the yeast two-hybrid (Y2H) assay. Among them, PXO_04421 (named as TriP, a putative transcriptional regulator interacting with PdeR) was verified via Y2H and glutathione-S-transferase pull-down assays, and its regulatory functions in bacterial virulence and exopolysaccharide (EPS) production were assessed by biochemical and genetic analysis. The REC domain of TriP specifically interacted with the EAL domain of PdeR. TriP promoted the PDE activity of PdeR to degrade c-di-GMP in the presence of PdeK. In-frame deletion in triP abolished the polar localization of PdeR in the cell. Notably, the ∆triP mutant showed significantly reduced virulence on susceptible rice leaves and impaired EPS production compared with wild type, whereas the double mutant ∆triP∆pdeR, like ∆pdeR, caused shorter lesion lengths and produced less EPS than ∆triP. In addition, cross-complementation showed in trans expression of pdeR in ∆triP restored its EPS production to near wild-type levels but not vice versa. Taken together, our results suggest that TriP is a novel regulator that is epistatic to PdeR in positively regulating virulence expression in X. oryzae pv. oryzae.


Assuntos
Oryza , Virulência , Xanthomonas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Oryza/microbiologia , Diester Fosfórico Hidrolases/metabolismo , Doenças das Plantas/microbiologia , Virulência/genética , Xanthomonas/enzimologia , Xanthomonas/genética , Xanthomonas/patogenicidade
3.
Appl Environ Microbiol ; 81(13): 4358-67, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25911481

RESUMO

The PilZ domain proteins have been demonstrated to be one of the major types of receptors mediating cyclic di-GMP (c-di-GMP) signaling pathways in several pathogenic bacteria. However, little is known about the function of PilZ domain proteins in c-di-GMP regulation of virulence in the bacterial blight pathogen of rice Xanthomonas oryzae pv. oryzae. Here, the roles of PilZ domain proteins PXO_00049 and PXO_02374 in c-di-GMP binding, regulation of virulence and motility, and subcellular localization were characterized in comparison with PXO_02715, identified previously as an interactor with the c-di-GMP receptor Filp to regulate virulence. The c-di-GMP binding motifs in the PilZ domains were conserved in PXO_00049 and PXO_02374 but were less well conserved in PXO_02715. PXO_00049 and PXO_02374 but not PXO_02715 proteins bound to c-di-GMP with high affinity in vitro, and the R(141) and R(10) residues in the PilZ domains of PXO_00049 and PXO_02374, respectively, were crucial for c-di-GMP binding. Gene deletion of PXO_00049 and PXO_02374 resulted in significant increases in virulence and hrp gene transcription, indicating their negative regulation of virulence via type III secretion system expression. All mutants showed significant changes in sliding motility but not exopolysaccharide production and biofilm formation. In trans expression of the full-length open reading frame (ORF) of each gene in the relevant mutants led to restoration of the phenotype to wild-type levels. Moreover, PXO_00049 and PXO_02374 displayed mainly multisite subcellular localizations, whereas PXO_02715 showed nonpolar distributions in the X. oryzae pv. oryzae cells. Therefore, this study demonstrated the different functions of the PilZ domain proteins in mediation of c-di-GMP regulation of virulence and motility in X. oryzae pv. oryzae.


Assuntos
Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , Regulação Bacteriana da Expressão Gênica , Locomoção , Xanthomonas/fisiologia , Xanthomonas/patogenicidade , Motivos de Aminoácidos , Sítios de Ligação , GMP Cíclico/metabolismo , Deleção de Genes , Teste de Complementação Genética , Ligação Proteica , Virulência
4.
Mol Plant Microbe Interact ; 27(10): 1119-31, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25180688

RESUMO

The bacterial soft rot pathogen Dickeya dadantii utilizes the type III secretion system (T3SS) to suppress host defense responses, and secretes pectate lyase (Pel) to disintegrate the plant cell wall. A transposon mutagenesis fluorescence-activated cell sorting screen was used to identify mutants with altered promoter activities of the T3SS pilus gene hrpA. Several insertion mutations, resulting in changes in hrpA expression, were mapped to a new locus, opgGH, which encodes the gene cluster responsible for osmoregulated periplasmic glucan (OPG) synthesis proteins. Our data showed that OPG was involved in T3SS and Pel regulation by altering the expression of the regulatory small RNA RsmB. Through genome searching, the mechanism of two novel regulatory components, the RcsCD-RcsB phosphorelay and CsrD on OPG and the rsmB gene, was further investigated. The Rcs phosphorelay and OPG inversely regulated rsmB at transcriptional and post-transcriptional levels, respectively. CsrD exhibited dual functionality in T3SS and Pel regulation by manipulating levels of RsmB RNA and cyclic diguanylate monophosphate (c-di-GMP). CsrD positively regulated the promoter activity of the rsmB gene but negatively controlled RsmB RNA at the post-transcriptional level via OpgGH. In addition, CsrD contains both GGDEF and EAL domains but acted as a c-di-GMP phosphodiesterase. When the expression of the csrD gene was induced, CsrD regulated T3SS expression and Pel production through controlling intracellular c-di-GMP levels.


Assuntos
Proteínas de Bactérias/genética , Enterobacteriaceae/genética , Regulação Bacteriana da Expressão Gênica , Doenças das Plantas/microbiologia , Plantas/microbiologia , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Parede Celular/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/análise , GMP Cíclico/metabolismo , Enterobacteriaceae/enzimologia , Enterobacteriaceae/patogenicidade , Modelos Biológicos , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Fenótipo , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/metabolismo , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína , Análise de Sequência de DNA , Ativação Transcricional , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
5.
Biofouling ; 30(2): 213-22, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24404893

RESUMO

In this study, the impact of the exopolysaccharides Pel and Psl on the cell surface electron donor-electron acceptor (acid-base) properties and adhesion to quartz sand was investigated by using Pseudomonas aeruginosa PAO1 and its isogenic EPS-mutant strains Δpel, Δpsl and Δpel/Δpsl. The microbial adhesion to hydrocarbon (MATH) test and titration results showed that both Pel and Psl contribute to the surface hydrophobicity of the cell. The results of contact angle measurement, however, showed no correlation with the cell surface hydrophobicity measured by the MATH test and the titration method. Packed-bed column experiments indicated that the exopolysaccharides Pel and Psl are involved in the initial cell attachment to the sand surface and the extent of their impact is dependent on the ionic strength (IS) of the solution. Overall, the Δpel/Δpsl double mutant had the lowest adhesion coefficient to sand compared with the wild-type PAO1, the Δpel mutant and the Δpsl mutant. It is hypothesized that in addition to bacterial surface hydrophobicity and DLVO forces, other factors, eg steric repulsion caused by extracellular macromolecules, and cell surface appendages (flagella and pili) also contribute significantly to the interaction between the cell surface and a sand grain.


Assuntos
Aderência Bacteriana/genética , Incrustação Biológica , Polissacarídeos Bacterianos/fisiologia , Pseudomonas aeruginosa/fisiologia , Membrana Celular/química , Deleção de Genes , Interações Hidrofóbicas e Hidrofílicas , Polissacarídeos Bacterianos/genética , Pseudomonas aeruginosa/efeitos dos fármacos , Quartzo/química
6.
Appl Environ Microbiol ; 79(18): 5424-36, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23770912

RESUMO

Erwinia amylovora causes a devastating disease called fire blight in rosaceous plants. The type III secretion system (T3SS) is one of the important virulence factors utilized by E. amylovora in order to successfully infect its hosts. By using a green fluorescent protein (GFP) reporter construct combined with a high-throughput flow cytometry assay, a library of phenolic compounds and their derivatives was studied for their ability to alter the expression of the T3SS. Based on the effectiveness of the compounds on the expression of the T3SS pilus, the T3SS inhibitors 4-methoxy-cinnamic acid (TMCA) and benzoic acid (BA) and one T3SS inducer, trans-2-(4-hydroxyphenyl)-ethenylsulfonate (EHPES), were chosen for further study. Both the T3SS inhibitors (TMCA and BA) and the T3SS inducer (EHPES) were found to alter the expression of T3SS through the HrpS-HrpL pathway. Additionally, TMCA altered T3SS expression through the rsmBEa-RsmAEa system. Finally, we found that TMCA and BA weakened the hypersensitive response (HR) in tobacco by suppressing the T3SS of E. amylovora. In our study, we identified phenolic compounds that specifically targeted the T3SS. The T3SS inhibitor may offer an alternative approach to antimicrobial therapy by targeting virulence factors of bacterial pathogens.


Assuntos
Antibacterianos/metabolismo , Sistemas de Secreção Bacterianos/efeitos dos fármacos , Erwinia amylovora/efeitos dos fármacos , Erwinia amylovora/metabolismo , Expressão Gênica/efeitos dos fármacos , Extratos Vegetais/metabolismo , Plantas/química , Antibacterianos/isolamento & purificação , Citometria de Fluxo , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Extratos Vegetais/isolamento & purificação , Ativação Transcricional
7.
Am J Respir Cell Mol Biol ; 46(3): 389-96, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22033263

RESUMO

Experimental asthma increases eosinophil and collagen deposition in the lungs of sickle cell disease (SCD) mice to a greater extent than in control mice. However, the effects of asthma on inflammation and airway physiology remain unclear. To determine effects of asthma on pulmonary inflammation and airway mechanics in SCD mice, hematopoietic stem cell transplantation was used to generate chimeric SCD and hemoglobin A mice. Experimental asthma was induced by sensitizing mice with ovalbumin (OVA). Airway mechanics were assessed using forced oscillation techniques. Mouse lungs were examined histologically and physiologically. Cytokine, chemokine, and growth factors in bronchoalveolar lavage fluid were determined by multiplex. IgE was quantified by ELISA. LDH was quantified using a colorimetric enzymatic assay. At baseline (nonsensitized), chimeric SCD mice developed hemolytic anemia with sickled red blood cells, mild leukocytosis, and increased vascular endothelial growth factor and IL-13 compared with chimeric hemoglobin A mice. Experimental asthma increased perialveolar eosinophils, plasma IgE, and bronchoalveolar lavage fluid IL-1ß, IL-4, IL-6, and monocyte chemotactic protein 1 in chimeric hemoglobin A and SCD mice. IFN-γ levels were reduced in both groups. IL-5 was preferentially increased in chimeric SCD mice but not in hemoglobin A mice. Positive end-expiratory pressures and methacholine studies revealed that chimeric SCD mice had greater resistance in large and small airways compared with hemoglobin A mice at baseline and after OVA sensitization. SCD alone induces a baseline lung pathology that increases large and small airway resistance and primes the lungs to increased inflammation and airway hyperresponsiveness after OVA sensitization.


Assuntos
Resistência das Vias Respiratórias , Anemia Falciforme/complicações , Asma/complicações , Hiper-Reatividade Brônquica/etiologia , Pulmão/fisiopatologia , Pneumonia/etiologia , Anemia Falciforme/sangue , Anemia Falciforme/genética , Anemia Falciforme/fisiopatologia , Animais , Asma/imunologia , Asma/fisiopatologia , Hiper-Reatividade Brônquica/sangue , Hiper-Reatividade Brônquica/genética , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/fisiopatologia , Testes de Provocação Brônquica , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Broncoconstritores , Colorimetria , Citocinas/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Eosinófilos/imunologia , Hemoglobina A/genética , Hemoglobina A/metabolismo , Hemoglobina Falciforme/genética , Hemoglobina Falciforme/metabolismo , Humanos , Imunoglobulina E/sangue , Mediadores da Inflamação/metabolismo , L-Lactato Desidrogenase/metabolismo , Pulmão/imunologia , Pulmão/patologia , Cloreto de Metacolina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina , Pneumonia/sangue , Pneumonia/genética , Pneumonia/imunologia , Pneumonia/fisiopatologia , Respiração com Pressão Positiva , Fator A de Crescimento do Endotélio Vascular/metabolismo
8.
Antimicrob Agents Chemother ; 56(1): 36-43, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21968370

RESUMO

Antibiotic therapy is the most commonly used strategy to control pathogenic infections; however, it has contributed to the generation of antibiotic-resistant bacteria. To circumvent this emerging problem, we are searching for compounds that target bacterial virulence factors rather than their viability. Pseudomonas aeruginosa, an opportunistic human pathogen, possesses a type III secretion system (T3SS) as one of the major virulence factors by which it secretes and translocates T3 effector proteins into human host cells. The fact that this human pathogen also is able to infect several plant species led us to screen a library of phenolic compounds involved in plant defense signaling and their derivatives for novel T3 inhibitors. Promoter activity screening of exoS, which encodes a T3-secreted toxin, identified two T3 inhibitors and two T3 inducers of P. aeruginosa PAO1. These compounds alter exoS transcription by affecting the expression levels of the regulatory small RNAs RsmY and RsmZ. These two small RNAs are known to control the activity of carbon storage regulator RsmA, which is responsible for the regulation of the key T3SS regulator ExsA. As RsmY and RsmZ are the only targets directly regulated by GacA, our results suggest that these phenolic compounds affect the expression of exoS through the GacSA-RsmYZ-RsmA-ExsA regulatory pathway.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Fenóis/farmacologia , Pseudomonas aeruginosa/metabolismo , Fatores de Transcrição/metabolismo , Antibacterianos/química , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Genes Reguladores , Genes Reporter , Ensaios de Triagem em Larga Escala , Humanos , Fenóis/química , Extratos Vegetais/química , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
9.
Appl Environ Microbiol ; 77(1): 156-62, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21075884

RESUMO

The outbreaks caused by enterohemorrhagic Escherichia coli O157:H7 on leafy greens have raised serious and immediate food safety concerns. It has been suggested that several phytopathogens aid in the persistence and proliferation of the human enteropathogens in the phyllosphere. In this work, we examined the influence of virulence mechanisms of Dickeya dadantii 3937, a broad-host-range phytopathogen, on the proliferation of the human pathogen E. coli O157:H7 EDL933 (EDL933) on postharvest lettuce by coinoculation of EDL933 with D. dadantii 3937 derivatives that have mutations in virulence-related genes. A type II secretion system (T2SS)-deficient mutant of D. dadantii 3937, A1919 (ΔoutC), lost the capability to promote the multiplication of EDL933, whereas Ech159 (ΔrpoS), a stress-responsive σ factor RpoS-deficient mutant, increased EDL933 proliferation on lettuce leaves. A spectrophotometric enzyme activity assay revealed that A1919 (ΔoutC) was completely deficient in the secretion of pectate lyases (Pels), which play a major role in plant tissue maceration. In contrast to A1919 (ΔoutC), Ech159 (ΔrpoS) showed more than 2-fold-greater Pel activity than the wild-type D. dadantii 3937. Increased expression of pelD (encodes an endo-pectate lyase) was observed in Ech159 (ΔrpoS) in planta. These results suggest that the pectinolytic activity of D. dadantii 3937 is the dominant determinant of enhanced EDL933 proliferation on the lettuce leaves. In addition, RpoS, the general stress response σ factor involved in cell survival in suboptimal conditions, plays a role in EDL933 proliferation by controlling the production of pectate lyases in D. dadantii 3937.


Assuntos
Enterobacteriaceae/enzimologia , Enterobacteriaceae/crescimento & desenvolvimento , Lactuca/microbiologia , Interações Microbianas , Polissacarídeo-Liases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Enterobacteriaceae/genética , Escherichia coli O157/crescimento & desenvolvimento , Deleção de Genes , Humanos , Polissacarídeo-Liases/genética , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Virulência/genética
10.
Blood ; 112(6): 2529-38, 2008 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-18579795

RESUMO

Asthma is a comorbid condition associated with increased rates of pain, acute chest syndrome, and premature death in human sickle cell disease (SCD). We developed an experimental asthma model in SCD and control mice expressing either normal human or murine hemoglobin to determine its effect on mortality and lung pathology. To induce lung inflammation, experimental mice were sensitized to ovalbumin (OVA) by subcutaneous OVA implantation (Sen), allowed 2 weeks to recover, and then divided into 2 groups, each receiving over a subsequent 10-day period the same dosage of aerosolized OVA but 2 different levels of exposure: 15 minutes (LoSen) and 30 minutes (HiSen). During recovery, 10% of SCD mice died compared with no deaths in control mice. An additional 30% of HiSen SCD mice died during aerosolization compared with 10% in LoSen SCD. Histologic indices of lung inflammation (eg, eosinophil recruitment, airway and vessel wall thickening, and immunoreactive TGFbeta and fsp-1) and bronchial alveolar lavage fluid eosinophil peroxidase activity differentially increased in sensitized mice compared with unsensitized mice. Our findings indicate SCD mice with experimentally induced asthma are more susceptible to death and pulmonary inflammation compared with control mice, suggesting that asthma contributes significantly to morbidity and mortality in SCD.


Assuntos
Anemia Falciforme/complicações , Asma/patologia , Anemia Falciforme/mortalidade , Animais , Asma/induzido quimicamente , Asma/mortalidade , Modelos Animais de Doenças , Hemoglobinas , Humanos , Inflamação/etiologia , Pulmão/patologia , Camundongos , Ovalbumina/efeitos adversos , Ovalbumina/imunologia , Taxa de Sobrevida
11.
Front Microbiol ; 10: 1402, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31354637

RESUMO

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight of rice, one of the most devastating bacterial diseases of this staple crop worldwide. Xoo produces a range of virulence-related factors to facilitate its pathogenesis in rice, however, the regulatory mechanisms of Xoo virulence expression have been not fully elucidated. Recent studies have revealed that virulence factor production is regulated via cyclic dimeric guanosine monophosphate (c-di-GMP) signaling pathway that is well-conserved in Xoo and other Xanthomonas species. A set of GGDEF, EAL, HD-GYP, and PilZ domain proteins with diverse signal sensory domains for c-di-GMP synthesis, hydrolysis, and binding is encoded in the Xoo genome. Bioinformatic, genetic, and biochemical analysis has identified an array of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), as well as degenerate GGDEF/EAL, PilZ domain proteins along with a transcription regulator. These signaling components have been characterized to regulate various bacterial cellular processes, such as virulence, exopolysaccharide (EPS) production, biofilm formation, motility, and adaptation at the transcriptional, post-translational, and protein-protein interaction levels. This review summarized the recent progress in understanding the importance and complexity of c-di-GMP signaling in regulating bacterial virulence expression, highlighting the identified key signal elements and orthologs found in Xanthomonads, discussing the diverse functions of GGDEF/EAL/HD-GYP domains, existence of a complicated multifactorial network between DGCs, PDEs, and effectors, and further exploration of the new c-di-GMP receptor domains. These findings and knowledge lay the groundwork for future experimentation to further elucidate c-di-GMP regulatory circuits involved in regulation of bacterial pathogenesis.

12.
FEMS Microbiol Lett ; 366(20)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31697369

RESUMO

Bacteria, especially pathogenic bacteria, were detected in order to estimate the safety of drinking water distribution systems (DWDSs). Sixteen biofilms and 12 water samples (six retained and six flowing) were collected from a city DWDS in eastern China. Biofilms were observed using scanning electron microscopy. Cultivable bacteria of biofilms were counted by heterotrophic plate counts, ranging from 3.61 × 101 to 1.67 × 106 CFU·cm-2. Coliforms, Salmonella, Shigella, Vibrio and Legionella were separated by Eosin-Methylene Blue (EMB) agar, Salmonella chromogenic medium, Shigella chromogenic medium, Thiosulfate Citrate Bile Salts Sucrose (TCBS) agar and Buffered Charcoal Yeast Extract (BCYE) agar and 13/16, 8/16, 7/16, 6/16, 0/16 biofilm samples were found to be positive, respectively. Retained and flowing water samples were collected to estimate the influence of hydrodynamic conditions on biofilm detachment. All six retained water samples were positive for bacteria, the count ranged from 1.2 × 103 to 2.8 × 104 CFU·mL-1 and 2/6, 3/6, 2/6, 0/6, 0/6 samples were positive for coliforms, Salmonella, Shigella, Legionella and Vibrio, respectively. While only three of six flowing water samples were bacteria positive, the counts ranged from 102 to 103 CFU·mL-1, 2/6 were coliform positive and no pathogens were detected under testing. The results show that there are pathogens in DWDS biofilms, which can cause health-related problems if detached from their surfaces.


Assuntos
Bactérias/isolamento & purificação , Biofilmes , Água Potável/microbiologia , Microbiologia da Água , Bactérias/classificação , Bactérias/patogenicidade , Bactérias/ultraestrutura , China
13.
Cardiovasc Res ; 72(1): 143-51, 2006 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-16930572

RESUMO

OBJECTIVE: The relative contributions of the fraction of inspired oxygen (FIO2) and atmospheric pressure (ATM) to cardioprotection are unknown. We determined whether the product of FIO2 x ATM (oxygen partial pressure) controls the extent of hyperoxic+hyperbaric-induced cardioprotection and involves activation of nitric oxide synthase (NOS). METHODS: Adult Sprague Dawley rats (n = 10/gp) were treated for 1 h with (1) normoxia+normobaria (21% O2 at 1 ATM), (2) hyperoxia+normobaria (100% O2 at 1 ATM), (3) normoxia+hyperbaria (21% O2 at 2 ATM) and (4) hyperoxia+hyperbaria (100% O2 at 2 ATM). RESULTS: Infarct size following 25 min ischemia and 180 min reperfusion was decreased following hyperoxia+normobaria and normoxia+hyperbaria compared with normoxia+normobaria and further decreased following hyperoxia+hyperbaria treatment. l-NAME (200 microM) reversed the cardioprotective effects of hyperoxia+hyperbaria. Nitrite plus nitrate content was increased 2.2-fold in rats treated with normoxia+hyperbaria and hyperoxia+hyperbaria. NOS3 protein increased 1.2-fold and association of hsp90 with NOS3 four-fold in hyperoxic+hyperbaric rats. CONCLUSIONS: Cardioprotection conferred by hyperoxia+hyperbaria is directly dependent on oxygen availability and mediated by NOS.


Assuntos
Oxigenoterapia Hiperbárica , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/química , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Ativação Enzimática , Proteínas de Choque Térmico HSP90/metabolismo , Heme Oxigenase-1/metabolismo , Masculino , Isquemia Miocárdica/metabolismo , Reperfusão Miocárdica , Traumatismo por Reperfusão Miocárdica/metabolismo , Nitratos/análise , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Perfusão , Ratos , Ratos Sprague-Dawley
14.
Mol Plant Pathol ; 18(4): 555-568, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27084974

RESUMO

The targeting of bacterial type III secretion systems (T3SSs), which are critical virulence factors in most Gram-negative pathogens, is regarded as an alternative strategy for the development of novel anti-microbial drugs. Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) are two of the most important bacterial pathogens on rice, which cause leaf blight and leaf streak diseases, respectively. To identify potential anti-virulence drugs against these two pathogens, we screened a library of plant phenolic compounds and derivatives for their effects on the Xoo T3SS. Ten of 56 compounds significantly inhibited the promoter activity of a harpin gene, hpa1. These inhibitors were further tested for their impact on the hypersensitive response (HR) caused by Xoo on non-host tobacco plants. The results showed that pretreatment of Xoo with TS006 (o-coumaric acid, OCA), TS010, TS015 and TS018 resulted in significantly attenuated HR without affecting bacterial growth or survival. In addition, Cya translocation assays demonstrated that the translocation of two T3 effectors was suppressed by the four inhibitors. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that mRNA levels of representative genes in the hrp (hypersensitive response and pathogenicity) cluster, as well as the regulatory genes hrpG and hrpX, were reduced by treatment with the four inhibitors, suggesting that expression of the Xoo T3SS was suppressed. The expression of other virulence factors was not suppressed, which indicated possible T3SS-specific inhibition. Finally, we demonstrated that these inhibitors reduced the disease symptoms of Xoo and Xoc on the rice cultivar (Oryza sativa) IR24 to varying extents.


Assuntos
Oryza/microbiologia , Fenóis/farmacologia , Sistemas de Secreção Tipo III/metabolismo , Xanthomonas/metabolismo , Xanthomonas/patogenicidade , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citometria de Fluxo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Oryza/efeitos dos fármacos , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Nicotiana/efeitos dos fármacos , Nicotiana/imunologia , Nicotiana/microbiologia , Virulência/efeitos dos fármacos , Virulência/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Água , Xanthomonas/genética , Xanthomonas/crescimento & desenvolvimento
15.
Environ Sci Pollut Res Int ; 22(21): 16914-26, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26109219

RESUMO

Commonly used flame retardants, such as polybrominated diphenyl ethers, are extremely persistent in the environment, causing serious environmental risks. Certain strains of bacteria are able to degrade several low brominated congeners of PBDEs aerobically. However, the aerobic degradation pathway is not yet well understood, particularly at the genetic level. In this study, we isolated Cupriavidus sp. WS from the environment that could degrade diphenyl ether (DE), 4-bromodiphenyl ether, and 4,4'-bromodiphenyl ether. DE was completely degraded in 6 days without any detectable end-product. Using transposon mutagenesis, several DE degradation-deficient mutants were obtained. Knocking out bphA1, bphA2, and bphA3 eliminated the ability of the Cupriavidus sp. WS bacterium to degrade DE, indicating that the bph genes play a crucial role in DE degradation by this strain. The specific roles of bphA, bphB, and bphC were identified by systematically expressing these genes in Escherichia coli. The dihydrodiol product of BphA was dehydrogenated into 2,3-dihydroxydiphenyl ether by BphB. 2,3-Dihydroxydiphenyl ether was then decomposed into phenol and 2-pyrone-6-carboxylic acid by BphC. Thus, BphA, BphB, and BphC act sequentially in the aerobic degradation of DE, 4-bromodiphenyl ether, and 4,4'-dibromodiphenyl ether by the Cupriavidus sp. WS bacterium.


Assuntos
Cupriavidus/crescimento & desenvolvimento , Poluentes Ambientais/análise , Retardadores de Chama/análise , Éteres Difenil Halogenados/análise , Biodegradação Ambiental , Cromatografia Líquida de Alta Pressão , Cupriavidus/genética , Cupriavidus/metabolismo , Poluentes Ambientais/metabolismo , Escherichia coli/genética , Retardadores de Chama/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Éteres Difenil Halogenados/metabolismo , Pironas/análise , Especificidade da Espécie
16.
Mol Plant Pathol ; 16(2): 150-63, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24986378

RESUMO

The type III secretion system (T3SS) is a major virulence factor in many Gram-negative bacterial pathogens and represents a particularly appealing target for antimicrobial agents. Previous studies have shown that the plant phenolic compound p-coumaric acid (PCA) plays a role in the inhibition of T3SS expression of the phytopathogen Dickeya dadantii 3937. This study screened a series of derivatives of plant phenolic compounds and identified that trans-4-hydroxycinnamohydroxamic acid (TS103) has an eight-fold higher inhibitory potency than PCA on the T3SS of D. dadantii. The effect of TS103 on regulatory components of the T3SS was further elucidated. Our results suggest that TS103 inhibits HrpY phosphorylation and leads to reduced levels of hrpS and hrpL transcripts. In addition, through a reduction in the RNA levels of the regulatory small RNA RsmB, TS103 also inhibits hrpL at the post-transcriptional level via the rsmB-RsmA regulatory pathway. Finally, TS103 inhibits hrpL transcription and mRNA stability, which leads to reduced expression of HrpL regulon genes, such as hrpA and hrpN. To our knowledge, this is the first inhibitor to affect the T3SS through both the transcriptional and post-transcriptional pathways in the soft-rot phytopathogen D. dadantii 3937.


Assuntos
Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/metabolismo , Proteínas de Escherichia coli/metabolismo , Fenóis/farmacologia , Transdução de Sinais/efeitos dos fármacos
17.
PLoS One ; 7(2): e31855, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22384086

RESUMO

Fructose-bisphophate aldolase (FbaB), is an enzyme in glycolysis and gluconeogenesis in living organisms. The mutagenesis in a unique fbaB gene of Xanthomonas oryzae pv. oryzicola, the causal agent of rice bacterial leaf streak, led the pathogen not only unable to use pyruvate and malate for growth and delayed its growth when fructose was used as the sole carbon source, but also reduced extracellular polysaccharide (EPS) production and impaired bacterial virulence and growth in rice. Intriguingly, the fbaB promoter contains an imperfect PIP-box (plant-inducible promoter) (TTCGT-N(9)-TTCGT). The expression of fbaB was negatively regulated by a key hrp regulatory HrpG and HrpX cascade. Base substitution in the PIP-box altered the regulation of fbaB with the cascade. Furthermore, the expression of fbaB in X. oryzae pv. oryzicola RS105 strain was inducible in planta rather than in a nutrient-rich medium. Except other hrp-hrc-hpa genes, the expression of hrpG and hrpX was repressed and the transcripts of hrcC, hrpE and hpa3 were enhanced when fbaB was deleted. The mutation in hrcC, hrpE or hpa3 reduced the ability of the pathogen to acquire pyruvate and malate. In addition, bacterial virulence and growth in planta and EPS production in RΔfbaB mutant were completely restored to the wild-type level by the presence of fbaB in trans. This is the first report to demonstrate that carbohydrates, assimilated by X. oryzae pv. oryzicola, play critical roles in coordinating hrp gene expression through a yet unknown regulator.


Assuntos
Carbono/metabolismo , Frutose-Bifosfato Aldolase/fisiologia , Oryza/microbiologia , Xanthomonas/metabolismo , Proteínas de Bactérias/genética , Códon , Meios de Cultura/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Reporter , Teste de Complementação Genética , Variação Genética , Genoma Bacteriano , Mutagênese Sítio-Dirigida , Mutação , Fases de Leitura Aberta , Doenças das Plantas/microbiologia , Plasmídeos/metabolismo , Polissacarídeos/química , Fatores de Transcrição/genética
18.
J Cardiovasc Pharmacol ; 50(5): 497-502, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18030058

RESUMO

Chronic hypoxia increases resistance to myocardial ischemia in infants. Activation of the mitochondrial big conductance Ca(2+) -sensitive K channel (mitoBKCa) has been shown to be protective in adult hearts; however, its role in infant hearts is unknown. Hearts from normoxic or hypoxic infant rabbits were perfused with a mitoKCa opener, NS1619, or blocker Paxilline before ischemia and reperfusion. Hypoxic hearts were more resistant to ischemia than normoxic hearts as manifested by a reduction in infarct size (9 +/- 5% versus 14 +/- 5%) and an increase in recovery of left ventricular developed pressure (LVDP) (69 +/- 7% versus 51 +/- 2%). NS1619 decreased infarct size in normoxic hearts from 14 +/- 5% to 10 +/- 5% and increased recovery of LVDP from 51 +/- 2% to 65 +/- 4%, but it had no effect on hypoxic hearts. Paxilline did not affect normoxic or hypoxic hearts. Activation of mitoBKCa protects normoxic infant rabbit hearts; however, cardioprotection by chronic hypoxia in infant rabbits does not appear involve mitoBKCa.


Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Mitocôndrias/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Animais , Animais Recém-Nascidos , Benzimidazóis/farmacologia , Circulação Coronária/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/fisiopatologia , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/fisiologia , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Indóis/farmacologia , Precondicionamento Isquêmico Miocárdico , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/agonistas , Canais de Potássio Ativados por Cálcio de Condutância Alta/antagonistas & inibidores , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/patologia , Perfusão , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/agonistas , Coelhos , Função Ventricular/efeitos dos fármacos
19.
Am J Physiol Heart Circ Physiol ; 288(1): H62-8, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15319200

RESUMO

Hypoxia from birth increases resistance to myocardial ischemia in infant rabbits. We hypothesized that increased cardioprotection in hearts chronically hypoxic from birth persists following development in a normoxic environment and involves increased activation of nitric oxide synthase (NOS) and ATP-dependent K (K(ATP)) channels. Resistance to myocardial ischemia was determined in rabbits raised from birth to 10 days of age in a normoxic (Fi(O(2)) = 0.21) or hypoxic (Fi(O(2)) = 0.12) environment and subsequently exposed to normoxia for up to 60 days of age. Isolated hearts (n = 8/group) were subjected to 30 min of global ischemia followed by 35 min of reperfusion. At 10 days of age, resistance to myocardial ischemia (percent recovery postischemic recovery left ventricular developed pressure) was higher in chronically hypoxic hearts (68 +/- 4%) than normoxic controls (43 +/- 4%). At 10 days of age, N(G)-nitro-L-arginine methyl ester (200 microM) and glibenclamide (3 microM) abolished the cardioprotective effects of chronic hypoxia (45 +/- 4% and 46 +/- 5%, respectively) but had no effect on normoxic hearts. At 30 days of age resistance to ischemia in normoxic hearts declined (36 +/- 5%). However, in hearts subjected to chronic hypoxia from birth to 10 days and then exposed to normoxia until 30 days of age, resistance to ischemia persisted (63 +/- 4%). L-NAME or glibenclamide abolished cardioprotection in previously hypoxic hearts (37 +/- 4% and 39 +/- 5%, respectively) but had no effect on normoxic hearts. Increased cardioprotection was lost by 60 days. We conclude that cardioprotection conferred by adaptation to hypoxia from birth persists on subsequent exposure to normoxia and is associated with enhanced NOS activity and activation of K(ATP) channels.


Assuntos
Trifosfato de Adenosina/metabolismo , Citoproteção , Hipóxia/fisiopatologia , Isquemia Miocárdica/prevenção & controle , Óxido Nítrico Sintase/metabolismo , Canais de Potássio/metabolismo , Adaptação Fisiológica , Animais , Animais Recém-Nascidos , Peso Corporal , Doença Crônica , Coração/fisiopatologia , Hipóxia/metabolismo , Hipóxia/patologia , Miocárdio/patologia , Tamanho do Órgão , Coelhos
20.
Am J Physiol Heart Circ Physiol ; 288(1): H175-84, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15388508

RESUMO

We determined whether isoflurane can confer delayed cardioprotection in the adult rat by triggering increased production of reactive oxygen (ROS) and nitrogen species (RNS). Our objectives were to determine 1) the concentration of isoflurane that confers delayed cardioprotection in the adult rat, 2) the role of ROS and RNS in the induction of delayed cardioprotection, and 3) the cellular sources of ROS and RNS responsible for induction of delayed cardioprotection by isoflurane. Male Sprague-Dawley rats at 8 wk of age (n = 8 rats/group) were exposed to 0.5%, 0.8%, 1%, and 2% (vol/vol) isoflurane-100% oxygen for 2 h. Isoflurane conferred delayed cardioprotection 24 h later at a concentration of 0.8% (vol/vol). Administration of manganese (III) tetrakis (4-benzoic acid)porphyrin chloride (MnTBAP), a superoxide scavenger (15 mg/kg ip), or N(G)-nitro-L-arginine methyl ester (L-NAME), a general nitric oxide synthase inhibitor (15 mg/kg ip), 15 min before isoflurane treatment abolished the delayed cardioprotective effects of isoflurane. MnTBAP and L-NAME had no effect on delayed cardioprotection in untreated hearts. Perfusion of isolated hearts with hydroethidine, a fluorescent probe for superoxide, after isoflurane treatment resulted in a twofold increase in ethidine staining of isoflurane-treated hearts compared with untreated controls, which was attenuated by myxothiazol, an inhibitor of the mitochondrial electron transport chain (0.2 mg/kg ip) and L-NAME (15 mg/kg ip). Nitrite and nitrate content in isoflurane-treated hearts was 1.5-fold higher than in untreated hearts, whereas myocardial reduced glutathione levels were decreased by 13% in 0.8% but not in 1.0% isoflurane-treated hearts. We conclude that isoflurane confers delayed cardioprotection in the adult rat, triggered by ROS and RNS.


Assuntos
Anestésicos Inalatórios/farmacologia , Citoproteção , Coração/efeitos dos fármacos , Isoflurano/farmacologia , Miocárdio/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Gases/sangue , Glutationa/metabolismo , Masculino , Miocárdio/citologia , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/metabolismo , Oxirredução/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/metabolismo , Fatores de Tempo
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