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1.
Sci Rep ; 11(1): 22465, 2021 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-34789815

RESUMO

After a century of investigations, the function of the obligate betaproteobacterial endosymbionts accommodated in leaf nodules of tropical Rubiaceae remained enigmatic. We report that the α-D-glucose analogue (+)-streptol, systemically supplied by mature Ca. Burkholderia kirkii nodules to their Psychotria hosts, exhibits potent and selective root growth inhibiting activity. We provide compelling evidence that (+)-streptol specifically affects meristematic root cells transitioning to anisotropic elongation by disrupting cell wall organization in a mechanism of action that is distinct from canonical cellulose biosynthesis inhibitors. We observed no inhibitory or cytotoxic effects on organisms other than seed plants, further suggesting (+)-streptol as a bona fide allelochemical. We propose that the suppression of growth of plant competitors is a major driver of the formation and maintenance of the Psychotria-Burkholderia association. In addition to potential agricultural applications as a herbicidal agent, (+)-streptol might also prove useful to dissect plant cell and organ growth processes.


Assuntos
Alelopatia/fisiologia , Burkholderia/metabolismo , Cicloexanóis/farmacologia , Feromônios/farmacologia , Extratos Vegetais/farmacologia , Folhas de Planta/química , Folhas de Planta/microbiologia , Psychotria/química , Psychotria/microbiologia , Simbiose/fisiologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Germinação/efeitos dos fármacos , Lactuca/efeitos dos fármacos , Lactuca/crescimento & desenvolvimento , Meristema/efeitos dos fármacos , Meristema/crescimento & desenvolvimento , Mostardeira/efeitos dos fármacos , Mostardeira/crescimento & desenvolvimento , Filogenia , Folhas de Planta/metabolismo , Psychotria/metabolismo , Plântula/efeitos dos fármacos , Plântula/crescimento & desenvolvimento , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimento
2.
Antimicrob Agents Chemother ; 56(5): 2314-25, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22314537

RESUMO

In relation to emerging multiresistant bacteria, development of antimicrobials and new treatment strategies of infections should be expected to become a high-priority research area. Quorum sensing (QS), a communication system used by pathogenic bacteria like Pseudomonas aeruginosa to synchronize the expression of specific genes involved in pathogenicity, is a possible drug target. Previous in vitro and in vivo studies revealed a significant inhibition of P. aeruginosa QS by crude garlic extract. By bioassay-guided fractionation of garlic extracts, we determined the primary QS inhibitor present in garlic to be ajoene, a sulfur-containing compound with potential as an antipathogenic drug. By comprehensive in vitro and in vivo studies, the effect of synthetic ajoene toward P. aeruginosa was elucidated. DNA microarray studies of ajoene-treated P. aeruginosa cultures revealed a concentration-dependent attenuation of a few but central QS-controlled virulence factors, including rhamnolipid. Furthermore, ajoene treatment of in vitro biofilms demonstrated a clear synergistic, antimicrobial effect with tobramycin on biofilm killing and a cease in lytic necrosis of polymorphonuclear leukocytes. Furthermore, in a mouse model of pulmonary infection, a significant clearing of infecting P. aeruginosa was detected in ajoene-treated mice compared to a nontreated control group. This study adds to the list of examples demonstrating the potential of QS-interfering compounds in the treatment of bacterial infections.


Assuntos
Antibacterianos/farmacologia , Dissulfetos/farmacologia , Alho/química , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Animais , Antibacterianos/isolamento & purificação , Antibacterianos/uso terapêutico , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Fracionamento Químico , Dissulfetos/isolamento & purificação , Dissulfetos/uso terapêutico , Sinergismo Farmacológico , Genes Reporter , Glicolipídeos/antagonistas & inibidores , Camundongos , Neutrófilos/efeitos dos fármacos , Extratos Vegetais/química , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Percepção de Quorum/genética , Infecções Respiratórias/tratamento farmacológico , Infecções Respiratórias/microbiologia , Sulfóxidos , Tobramicina/administração & dosagem , Tobramicina/uso terapêutico , Fatores de Virulência/antagonistas & inibidores
3.
Proteomics ; 9(13): 3564-79, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19609963

RESUMO

Members of the genus Cronobacter are opportunistic pathogens for neonates and are often associated with contaminated milk powder formulas. At present little is known about the virulence mechanisms or the natural reservoir of these organisms. The proteome of Cronobacter turicensis 3032, which has recently caused two deaths, was mapped aiming at a better understanding of physiology and putative pathogenic traits of this clinical isolate. Our analyses of extracellular, surface-associated and whole-cell proteins by two complementary proteomics approaches, 1D-SDS-PAGE combined with LC-ESI-MS/MS and 2D-LC-MALDI-TOF/TOF MS, lead to the identification of 832 proteins corresponding to a remarkable 19% of the theoretically expressed protein complement of C. turicensis. The majority of the identified proteins are involved in central metabolic pathways, translation, protein folding and stability. Several putative virulence factors, whose expressions were confirmed by phenotypic assays, could be identified: a macrophage infectivity potentiator involved in C. turicensis persistence in host cells, a superoxide dismutase protecting the pathogen against reactive oxygen species and an enterobactin-receptor protein for the uptake of siderophore-bound iron. Most interestingly, a chitinase and a metalloprotease that might act against insects and fungi but no casein hydrolysing enzymes were found, suggesting that there is an environmental natural habitat of C. turicensis 3032.


Assuntos
Proteínas de Bactérias/metabolismo , Enterobacteriaceae/metabolismo , Doenças Transmitidas por Alimentos/microbiologia , Proteínas de Bactérias/química , Quimiotaxia , Enterobacteriaceae/química , Enterobacteriaceae/crescimento & desenvolvimento , Enterobacteriaceae/patogenicidade , Dobramento de Proteína , Transporte Proteico , Proteômica , Estresse Fisiológico , Fatores de Virulência/química , Fatores de Virulência/metabolismo
4.
Microb Pathog ; 45(5-6): 331-6, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18771721

RESUMO

This work describes the isolation and characterization of an acyl carrier protein (ACP) mutant from Burkholderia cenocepacia J2315, a strain of the Burkholderia cepacia complex (Bcc). Bcc comprises at least 9 species that emerged as opportunistic pathogens able to cause life-threatening infections, particularly severe among cystic fibrosis patients. Bacterial ACPs are the donors of the acyl moiety involved in the biosynthesis of fatty acids, which play a central role in metabolism. The mutant was found to exhibit an increased ability to form biofilms in vitro, a more hydrophobic cell surface and reduced ability to colonize and kill the nematode Caenorhabditis elegans, used as a model of infection. The B. cenocepacia J2315 ACP protein is composed of 79 amino acid residues, with a predicted molecular mass and pI of 8.71kDa and 4.08, respectively. The ACP amino acid sequence was found to be 100% conserved within the genomes of the 52 Burkholderia strains sequenced so far. These data, together with results showing that the predicted structure of B. cenocepacia J2315 ACP is remarkably similar to the Escherichia coli AcpP, highlight its potential as a target to develop antibacterial agents to combat infections caused not only by Bcc species, but also by other Burkholderia species, especially B. pseudomallei and B. mallei.


Assuntos
Proteína de Transporte de Acila/metabolismo , Proteínas de Bactérias/metabolismo , Complexo Burkholderia cepacia/fisiologia , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biofilmes , Infecções por Burkholderia/tratamento farmacológico , Infecções por Burkholderia/microbiologia , Complexo Burkholderia cepacia/química , Complexo Burkholderia cepacia/genética , Caenorhabditis elegans , Avaliação Pré-Clínica de Medicamentos , Humanos , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Alinhamento de Sequência
5.
Microbiology (Reading) ; 151(Pt 12): 3873-3880, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16339933

RESUMO

The opportunistic human pathogen Pseudomonas aeruginosa is the predominant micro-organism of chronic lung infections in cystic fibrosis patients. P. aeruginosa colonizes the lungs by forming biofilm microcolonies throughout the lung. Quorum sensing (QS) renders the biofilm bacteria highly tolerant to otherwise lethal doses of antibiotics, and protects against the bactericidal activity of polymorphonuclear leukocytes (PMNs). It has been previously demonstrated that QS is inhibited by garlic extract. In this study, the synergistic effects of garlic and tobramycin, and PMNs activities have been evaluated. P. aeruginosa was grown in vitro in continuous-culture once-through flow chambers with and without garlic extract. The garlic-treated biofilms were susceptible to both tobramycin and PMN grazing. Furthermore, the PMNs showed an increase in respiratory burst activation, when incubated with the garlic-treated biofilm. Garlic extract was administered as treatment for a mouse pulmonary infection model. Mice were treated with garlic extract or placebo for 7 days, with the initial 2 days being prophylactic before P. aeruginosa was instilled in the left lung of the mice. Bacteriology, mortality, histopathology and cytokine production were used as indicators. The garlic treatment initially provoked a higher degree of inflammation, and significantly improved clearing of the infecting bacteria. The results indicate that a QS-inhibitory extract of garlic renders P. aeruginosa sensitive to tobramycin, respiratory burst and phagocytosis by PMNs, as well as leading to an improved outcome of pulmonary infections.


Assuntos
Antibacterianos/uso terapêutico , Alho/química , Extratos Vegetais/uso terapêutico , Infecções por Pseudomonas/prevenção & controle , Pseudomonas aeruginosa/efeitos dos fármacos , Animais , Antibacterianos/farmacocinética , Fibrose Cística/microbiologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Extratos Vegetais/farmacocinética , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Transdução de Sinais/efeitos dos fármacos
6.
J Bacteriol ; 187(5): 1799-814, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15716452

RESUMO

With the widespread appearance of antibiotic-resistant bacteria, there is an increasing demand for novel strategies to control infectious diseases. Furthermore, it has become apparent that the bacterial life style also contributes significantly to this problem. Bacteria living in the biofilm mode of growth tolerate conventional antimicrobial treatments. The discovery that many bacteria use quorum-sensing (QS) systems to coordinate virulence and biofilm development has pointed out a new, promising target for antimicrobial drugs. We constructed a collection of screening systems, QS inhibitor (QSI) selectors, which enabled us to identify a number of novel QSIs among natural and synthetic compound libraries. The two most active were garlic extract and 4-nitro-pyridine-N-oxide (4-NPO). GeneChip-based transcriptome analysis revealed that garlic extract and 4-NPO had specificity for QS-controlled virulence genes in Pseudomonas aeruginosa. These two QSIs also significantly reduced P. aeruginosa biofilm tolerance to tobramycin treatment as well as virulence in a Caenorhabditis elegans pathogenesis model.


Assuntos
4-Butirolactona/análogos & derivados , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana/métodos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , 4-Butirolactona/metabolismo , Animais , Biofilmes/efeitos dos fármacos , Caenorhabditis elegans , Simulação por Computador , Regulação para Baixo , Farmacorresistência Bacteriana , Alho , Extratos Vegetais/farmacologia , Plantas Medicinais , Pseudomonas aeruginosa/genética
7.
Environ Microbiol ; 5(12): 1350-69, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14641579

RESUMO

The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen which is responsible for severe nosocomial infections in immunocompromised patients and is the major pathogen in cystic fibrosis. The bacterium utilizes two interrelated quorum-sensing (QS) systems, which rely on N-acyl-homoserine lactone (AHL) signal molecules, to control the expression of virulence factors and biofilm development. In this study, we compared the protein patterns of the intracellular, extracellular and surface protein fractions of the PAO1 parent strain with those of an isogenic lasI rhlI double mutant by means of two-dimensional gel electrophoresis (2-DE). This analysis showed that the intensities of 23.7% of all detected protein spots differed more than 2.5-fold between the two strains. We only considered those protein spots truly QS regulated that were changed in the mutant in the absence of signal molecules but were rescued to the wild-type situation when the medium was supplemented with AHLs. These protein spots were characterized by MALDI-TOF peptide mapping. Twenty-seven proteins were identified that were previously reported to be AHL controlled, among them several well-characterized virulence factors. For one of the identified proteins, the serine protease PrpL, a biochemical assay was established to verify that expression of this factor is indeed QS regulated. Furthermore, it is shown that the quorum-sensing blocker C-30 specifically interferes with the expression of 67% of the AHL-controlled protein spots of the surface fraction, confirming the high specificity of the compound. Importantly, 20 novel QS-regulated proteins were identified, many of which are involved in iron utilization, suggesting a link between quorum sensing and the iron regulatory system. Two of these proteins, PhuR and HasAp, are components of the two distinct haem-uptake systems present in P. aeruginosa. In agreement with the finding that both proteins are positively regulated by the QS cascade, we show that the lasI rhlI double mutant grows poorly with haemoglobin as the only iron source when compared with the wild type. These results add haemoglobin utilization to the list of phenotypes controlled through QS in P. aeruginosa. The surprisingly high number of AHL-regulated proteins relative to the number of regulated genes suggests that quorum-sensing control also operates via post-transcriptional mechanisms. To strengthen this hypothesis we investigated the role of quorum sensing in the post-translational modification of HasAp, an extracellular protein required for the uptake of free and haemoglobin-bound haem.


Assuntos
Proteínas de Bactérias/análise , Proteínas de Bactérias/isolamento & purificação , Proteômica , Pseudomonas aeruginosa/química , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Eletroforese em Gel Bidimensional , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Heme/metabolismo , Ferro/metabolismo , Ligases , Mutação , Processamento de Proteína Pós-Traducional , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Transcrição/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
8.
Appl Environ Microbiol ; 68(7): 3328-38, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12089011

RESUMO

To study the effect of plant species on the abundance and diversity of bacterial antagonists, the abundance, the phenotypic diversity, and the genotypic diversity of rhizobacteria isolated from potato, oilseed rape, and strawberry and from bulk soil which showed antagonistic activity towards the soilborne pathogen Verticillium dahliae Kleb. were analyzed. Rhizosphere and soil samples were taken five times over two growing seasons in 1998 and 1999 from a randomized field trial. Bacterial isolates were obtained after plating on R2A (Difco, Detroit, Mich.) or enrichment in microtiter plates containing high-molecular-weight substrates followed by plating on R2A. A total of 5,854 bacteria isolated from the rhizosphere of strawberry, potato, or oilseed rape or bulk soil from fallow were screened by dual testing for in vitro antagonism towards VERTICILLIUM: The proportion of isolates with antagonistic activity was highest for strawberry rhizosphere (9.5%), followed by oilseed rape (6.3%), potato (3.7%), and soil (3.3%). The 331 Verticillium antagonists were identified by their fatty acid methyl ester profiles. They were characterized by testing their in vitro antagonism against other pathogenic fungi; their glucanolytic, chitinolytic, and proteolytic activities; and their BOX-PCR fingerprints. The abundance and composition of Verticillium antagonists was plant species dependent. A rather high proportion of antagonists from the strawberry rhizosphere was identified as Pseudomonas putida B (69%), while antagonists belonging to the Enterobacteriaceae (Serratia spp., Pantoea agglomerans) were mainly isolated from the rhizosphere of oilseed rape. For P. putida A and B plant-specific genotypes were observed, suggesting that these bacteria were specifically enriched in each rhizosphere.


Assuntos
Pseudomonas putida/isolamento & purificação , Verticillium/fisiologia , Ecossistema , Variação Genética , Genótipo , Fenótipo , Doenças das Plantas/microbiologia , Pseudomonas putida/química , Pseudomonas putida/genética , Pseudomonas putida/fisiologia , Solanum tuberosum/microbiologia , Verticillium/efeitos dos fármacos
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