RESUMO
The in situ stimulation of Fe(III) oxide reduction in the subsurface stimulates the growth of Geobacter spp. and the precipitation of U(VI) from groundwater. As with Fe(III) oxide reduction, the reduction of uranium by Geobacter spp. requires the expression of their conductive pili. The pili bind the soluble uranium and catalyse its extracellular reductive precipitation along the pili filaments as a mononuclear U(IV) complexed by carbon-containing ligands. Although most of the uranium is immobilized by the pili, some uranium deposits are also observed in discreet regions of the outer membrane, consistent with the participation of redox-active foci, presumably c-type cytochromes, in the extracellular reduction of uranium. It is unlikely that cytochromes released from the outer membrane could associate with the pili and contribute to the catalysis, because scanning tunnelling microscopy spectroscopy did not reveal any haem-specific electronic features in the pili, but, rather, showed topographic and electronic features intrinsic to the pilus shaft. Pili not only enhance the rate and extent of uranium reduction per cell, but also prevent the uranium from traversing the outer membrane and mineralizing the cell envelope. As a result, pili expression preserves the essential respiratory activities of the cell envelope and the cell's viability. Hence the results support a model in which the conductive pili function as the primary mechanism for the reduction of uranium and cellular protection in Geobacter spp.
Assuntos
Fímbrias Bacterianas/metabolismo , Geobacter/metabolismo , Urânio/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/fisiologia , Precipitação Química , Grupo dos Citocromos c/metabolismo , Grupo dos Citocromos c/fisiologia , Transporte de Elétrons , Compostos Férricos/metabolismo , Fímbrias Bacterianas/ultraestrutura , Geobacter/ultraestrutura , Heme/metabolismo , Viabilidade Microbiana , Oxirredução , Periplasma/metabolismo , Periplasma/ultraestrutura , Urânio/químicaRESUMO
The Gram-negative rod Actinobacillus pleuropneumoniae is a facultative anaerobic pathogen of the porcine respiratory tract, and HlyX, the A. pleuropneumoniae homologue of fumarate and nitrate reduction regulator (FNR), has been shown to be important for persistence. An A. pleuropneumoniae hlyX deletion mutant has a decreased generation time but highly prolonged survival in comparison to its wild type parent strain when grown anaerobically in glucose-supplemented medium. Applying a combination of proteomic and transcriptomic approaches as well as in silico analyses, we identified 23 different proteins and 418 genes to be modulated by HlyX (> or = twofold up- or down-regulated). A putative HlyX-box was identified upstream of 54 of these genes implying direct control by HlyX. Consistent with its role as a strong positive regulator, HlyX induced the expression of genes for anaerobic metabolism encoding alternative terminal reductases and hydrogenases. In addition, expression of virulence-associated genes encoding iron uptake systems, a putative DNA adenine modification system, and an autotransporter serine protease were induced by HlyX under anaerobic growth conditions. With respect to virulence-associated genes, we focused on the iron-regulated protein B (FrpB) as it is the outer membrane protein most strongly up-regulated by HlyX. An frpB deletion mutant of A. pleuropneumoniae had the same growth characteristics as wild type grown aerobically and anaerobically. In contrast, A. pleuropneumoniae DeltafrpB did not cause any disease and could not be re-isolated from experimentally infected pigs, thereby identifying FrpB as a previously unknown virulence factor.
Assuntos
Actinobacillus pleuropneumoniae/fisiologia , Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Regulon , Fatores de Transcrição/genética , Actinobacillus pleuropneumoniae/genética , Actinobacillus pleuropneumoniae/crescimento & desenvolvimento , Actinobacillus pleuropneumoniae/patogenicidade , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Biologia Computacional , Simulação por Computador , Proteínas de Ligação a DNA/fisiologia , Eletroforese em Gel Bidimensional , Regulação Bacteriana da Expressão Gênica , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de Sequência , Suínos , Fatores de Transcrição/fisiologia , Fatores de Virulência/genética , Fatores de Virulência/fisiologiaRESUMO
Using a series of efflux mutants of Pseudomonas aeruginosa, the MexAB-OprM pump was identified as contributing to this organism's tolerance to the antimicrobial agent tea tree (Melaleuca alternifolia) oil and its monoterpene components terpinen-4-ol, 1,8-cineole, and alpha-terpineol. These data show that a multidrug efflux system of P. aeruginosa can extrude monoterpenes and related alcohols.
Assuntos
Proteínas da Membrana Bacteriana Externa/fisiologia , Melaleuca/química , Monoterpenos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Óleo de Melaleuca/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Monoterpenos Cicloexânicos , Cicloexanóis/química , Cicloexanóis/farmacologia , Cicloexenos/química , Cicloexenos/farmacologia , Eucaliptol , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/fisiologia , Monoterpenos/química , Mutação , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Óleo de Melaleuca/químicaRESUMO
Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, utilize a plasmid encoded type III secretion system (T3SS) to promote infection by delivering Yersinia outer proteins (Yops) into the cytosol of mammalian cells. This T3SS is absolutely required for Yersinia virulence, which makes T3SS an attractive target in the development of novel therapeutics for treatment of plague and other Yersinia infections. In this study, a new method for high throughput screening (HTS) of small molecules for the ability to inhibit type III secretion (T3S) in Y. pestis has been developed. In comparison with screening assays employed by others, this method is very simple and rapid, and thus well suited for examining very large compound sets. Using this method, we screened a diverse collection of libraries at the US National Screening Laboratory. The initial examination of 70,966 compounds and mixtures from 13 libraries resulted in 431 primary hits. Strong positive indications of inhibition were observed at a rate of 0.01%, while moderate and weak but potentially meaningful signals were observed at rates of 0.056% and 0.54% respectively. Further characterizations were conducted on selected primary hits in Y. pestis. Of the eight compounds examined in secondary assays, four show good promise as leads for structure activity relationship studies. They are a diverse group, each having chemical scaffolds not only distinct from one another, but also distinct from previously described candidate T3S inhibitors.
Assuntos
Antibacterianos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Yersinia pestis/efeitos dos fármacos , Animais , Antibacterianos/química , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas da Membrana Bacteriana Externa/fisiologia , Células Cultivadas , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/estatística & dados numéricos , Humanos , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/fisiologia , Sensibilidade e Especificidade , Virulência/efeitos dos fármacos , Virulência/fisiologia , Yersinia pestis/patogenicidade , Yersinia pestis/fisiologiaRESUMO
A ferrichrome receptor, FhuA, was identified in Actinobacillus pleuropneumoniae serotype 7. An isogenic mutant with a deletion in the ferrichrome uptake receptor gene (fhuA) was constructed and examined in an aerosol infection model. The disease caused by the mutant was indistinguishable from disease induced by A. pleuropneumoniae serotype 7 wild-type; an isogenic mutant lacking expression of the exbB gene that is required for the uptake of transferrin-bound iron retained the ability to utilize ferrichrome, thereby indicating that an energy-coupling mechanism involved in ferrichrome transport remains to be identified.
Assuntos
Infecções por Actinobacillus/veterinária , Actinobacillus pleuropneumoniae/patogenicidade , Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Escherichia coli/fisiologia , Pneumonia Bacteriana/veterinária , Receptores Virais/fisiologia , Doenças dos Suínos/microbiologia , Infecções por Actinobacillus/microbiologia , Actinobacillus pleuropneumoniae/classificação , Actinobacillus pleuropneumoniae/genética , Actinobacillus pleuropneumoniae/metabolismo , Aerossóis , Animais , Proteínas da Membrana Bacteriana Externa/genética , Líquido da Lavagem Broncoalveolar/microbiologia , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , DNA Complementar/genética , Proteínas de Escherichia coli/genética , Ferricromo/metabolismo , Deleção de Genes , Transporte de Íons , Ferro/metabolismo , Pneumonia Bacteriana/microbiologia , Fatores R , Receptores Virais/genética , Sorotipagem , Suínos , Transferrina/metabolismo , Virulência/genéticaRESUMO
We previously showed that heat-killed Borrelia burgdorferi spirochetes and lipidated outer surface protein A (L-OspA) stimulated the in vitro production of interleukin-10 (IL-10) in peripheral blood mononuclear cells (PBMC) from uninfected humans and rhesus monkeys (G. Giambartolomei et al., Infect. Immun. 66:2691-2697, 1998). Here we demonstrate that uninfected human peripheral blood monocytes, but not B or T cells, are the cells that transcribe the IL-10 cytokine gene in response to heat-killed B. burgdorferi. B. burgdorferi similarly induced an upregulation of the IL-1beta and IL-6 cytokine genes in monocytes and the production of IL-10 and IL-6 in culture supernatants of the human monocytic cell line THP-1. Purified L-OspA (but not unlipidated OspA [U-OspA] or U-OspC) also stimulated the production of both cytokines in THP-1 cells in a dose-dependent fashion, suggesting that acylation of the OspA protein molecule is required for the production of both anti- and pro-inflammatory cytokines in naive monocytes. A lipohexapeptide that contained the tripalmitoyl-modified cysteine motif (Pam3Cys-Hex) of B. burgdorferi lipoproteins but with an arbitrary peptide sequence had the same effect. Monoclonal antibodies (MAbs) MY4 and 60bca, both of which bind to CD14 and are known to block lipopolysaccharide (LPS)-mediated cytokine production, were able to block L-OspA-mediated IL-10 and IL-6 cytokine production. In contrast, MAb 26ic, which also binds to CD14 but does not block LPS function, failed to inhibit L-OspA-mediated cytokine production. These data suggest that activation of monocytes and production of both anti- and pro-inflammatory cytokines induced by lipoproteins proceeds via the CD14 receptor. LPS binding protein was not required for OspA-induced cytokine production. Our results demonstrate that pro- and anti-inflammatory cytokines induced by B. burgdorferi lipoproteins in PBMC are produced by monocytes and that lipoprotein and LPS signaling pathways share at least the initial signaling event that involves the CD14 receptor.
Assuntos
Proteínas de Fase Aguda , Antígenos de Superfície/fisiologia , Proteínas da Membrana Bacteriana Externa/fisiologia , Grupo Borrelia Burgdorferi/fisiologia , Citocinas/biossíntese , Receptores de Lipopolissacarídeos/fisiologia , Lipoproteínas/fisiologia , Glicoproteínas de Membrana , Monócitos/metabolismo , Monócitos/microbiologia , Adjuvantes Imunológicos/metabolismo , Adjuvantes Imunológicos/farmacologia , Vacinas Bacterianas , Proteínas de Transporte/fisiologia , Linhagem Celular , Cisteína/análogos & derivados , Cisteína/metabolismo , Cisteína/farmacologia , Citocinas/fisiologia , Humanos , Inflamação/etiologia , Inflamação/microbiologia , Inflamação/prevenção & controle , Interleucina-1/genética , Interleucina-10/genética , Interleucina-6/genética , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Lipoproteínas/metabolismo , Lipoproteínas/farmacologia , Monócitos/imunologia , Transcrição Gênica/imunologiaRESUMO
A confocal microscopy study was undertaken to characterize the bactericidal effects of the Fab fragments of CB2, an immunoglobulin G1kappa murine monoclonal antibody, to an epitope in the carboxy region of the outer surface protein B (OspB) of Borrelia burgdorferi. Simultaneous direct labeling of both fixed and live spirochetes with fluorochrome-labeled Fab-CB2 and 11G1, and an immunoglobulin Mkappa monoclonal antibody to OspA, showed that OspA and OspB seem to colocalize in dead spirochetes but do not appear to be physically associated when the organisms are alive. A polar bleb composed of a Fab-CB2-OspB complex, followed by incorporation of 11G1-OspA, precedes the formation of a spheroplast. The spheroplasts contain both OspA and OspB and are a terminal stage in the bactericidal process induced by Fab-CB2. Outer membrane destabilization by Fab-CB2, but not cell wall or cytoplasmic membrane alterations, was demonstrated experimentally by the sequential treatment of spirochetes with Fab-CB2 and monoclonal antibodies to flagellin and DnaK. The action of Fab-CB2 is epitope specific, as another monoclonal antibody to an epitope in the amino terminus of OspB was not bactericidal. The bactericidal effect of Fab-CB2 is not dependent on the induction of spirochetal proteases but is dependent on the presence of Ca2+ and Mg2+. Supplementation of Ca2(+)- and Mg2(+)-free medium with these cations restored the bactericidal effects of Fab-CB2. The mechanism by which a Fab fragment of an antibody destroys a bacterium directly may represent a novel form of antibody-organism interaction.