RESUMO
Electron storage rings used for the production of synchrotron radiation (SR) have an output photon brightness that is limited by the equilibrium beam emittance. By using interleaved injection and ejection of bunches from a source with repetition rate greater than 1 kHz, we show that it is practicable to overcome this limit in rings of energy â¼1 GeV. Sufficiently short kicker pulse lengths enable effective currents of many milliamperes, which can deliver a significant flux of diffraction-limited soft x-ray photons. Thus, either existing SR facilities may be adapted for nonequilibrium operation, or the technique applied to construct SR rings smaller than their storage ring equivalent.
RESUMO
Our previous studies showed that both Sae and Fur are required for the induction of eap and emp expression in low iron. In this study, we show that expression of sae is also iron-regulated, as sae expression is activated by Fur in low iron. We also demonstrate that both Fur and Sae are required for full induction of the oxidative stress response and expression of non-covalently bound surface proteins in low-iron growth conditions. In addition, Sae is required for the induced expression of the important virulence factors isdA and isdB in low iron. Our studies also indicate that Fur is required for the induced expression of the global regulators Agr and Rot in low iron and a number of extracellular virulence factors such as the haemolysins which are also Sae- and Agr-regulated. Hence, we show that Fur is central to a complex regulatory network that is required for the induced expression of a number of important S. aureus virulence determinants in low iron.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Quinases/metabolismo , Proteínas Repressoras/metabolismo , Staphylococcus aureus/patogenicidade , Fatores de Virulência/biossíntese , Perfilação da Expressão Gênica , Ferro/metabolismo , Fatores de Transcrição , VirulênciaRESUMO
Enteropathogenic Escherichia coli cause protracted diarrhoea and malnutrition in infants by cytoskeletal depolymerisation and effacement of enterocyte microvilli. In this study, outer membrane proteins of wild-type enteropathogenic E. coli and an intimin-deficient mutant are shown to induce apoptosis by up-regulation of tumour necrosis factor alpha and activation of c-jun N-terminal kinase. Fluorescence-activated cell sorter analysis revealed apoptosis of cells treated with outer membrane proteins of wild-type and intimin-deficient strains. Proteinase K treatment of outer membrane proteins reduced apoptosis significantly, as did neutralising tumour necrosis factor alpha with specific antibodies. Elevated tumour necrosis factor receptor 1-associated death domain and caspase-3 expression were also observed on treatment with both types of outer membrane proteins. Furthermore, apoptosis was associated with suppression of Bcl-2 protein expression, up-regulation of Bax mRNA levels and increased cytochrome c release from mitochondria. Elevated phospho-c-jun N-terminal kinase, c-jun mRNA and activator protein-1 expression were observed, and phosphorylation of activator protein-1 was also observed by DNA-binding assays. Inhibition of c-jun N-terminal kinase, but not inhibition of p38 mitogen-activated protein kinase, resulted in reduction of tumour necrosis factor alpha mRNA levels and caspase-3 protein levels, and a reduction in apoptosis as observed by fluorescence-activated cell sorter analysis. From the host point of view, this study suggests a possible interplay between the death receptor and mitochondrial pathways when cell-free bacterial outer membrane preparations are used to trigger apoptosis.
Assuntos
Apoptose , Proteínas da Membrana Bacteriana Externa/imunologia , Escherichia coli Enteropatogênica/imunologia , Células Epiteliais/microbiologia , Caspase 3/biossíntese , Linhagem Celular Tumoral , Citocromos c/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossíntese , Regulação para Cima , Proteína X Associada a bcl-2/biossínteseRESUMO
During routine quality control testing of diagnostic methods for Shiga toxin-producing Escherichia coli (STEC) using stool samples spiked with STEC, it was observed that the Shiga toxin could not be detected in 32 out of 82 samples tested. Strains of E. coli isolated from such stool samples were shown to be responsible for this inhibition. One particular isolate, named E. coli 1307, was intensively studied because of its highly effective inhibitory effect; this strain significantly reduced growth and Shiga toxin levels in coculture of several STEC strains regardless of serovar or Shiga toxin type. The probiotic E. coli Nissle 1917 inhibited growth and reduced Shiga toxin levels in STEC cultures to an extent similar to E. coli 1307, but commensal E. coli strains and several other known probiotic bacteria (enterococci, Bacillus sp., Lactobacillus acidophilus) showed no, or only small, inhibitory effects. Escherichia coli 1307 lacks obvious fitness factors, such as aerobactin, yersiniabactin, microcins and a polysaccharide capsule, that are considered to promote the growth of pathogenic bacteria. We therefore propose strain E. coli 1307 as a candidate probiotic for use in the prevention and treatment of infections caused by STEC.
Assuntos
Antibiose , Escherichia coli/crescimento & desenvolvimento , Probióticos , Toxina Shiga/metabolismo , Escherichia coli Shiga Toxigênica/crescimento & desenvolvimento , Técnicas de Cocultura , Meios de Cultura , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Escherichia coli O157/crescimento & desenvolvimento , Escherichia coli O157/metabolismo , Fezes/microbiologia , Humanos , Sorotipagem , Escherichia coli Shiga Toxigênica/metabolismoRESUMO
Campylobacter jejuni NCTC 11168 was capable of growth to levels comparable with FeSO4 in defined iron-limited medium (minimal essential medium alpha [MEMalpha]) containing ferrilactoferrin, ferritransferrin, or ferri-ovotransferrin. Iron was internalized in a contact-dependent manner, with 94% of cell-associated radioactivity from either 55Fe-loaded transferrin or lactoferrin associated with the soluble cell fraction. Partitioning the iron source away from bacteria significantly decreased cellular growth. Excess cold transferrin or lactoferrin in cultures containing 55Fe-loaded transferrin or lactoferrin resulted in reduced levels of 55Fe uptake. Growth of C. jejuni in the presence of ferri- and an excess of apoprotein reduced overall levels of growth. Following incubation of cells in the presence of ferrilactoferrin, lactoferrin became associated with the cell surface; binding levels were higher after growth under iron limitation. A strain carrying a mutation in the cj0178 gene from the iron uptake system Cj0173c-Cj0178 demonstrated significantly reduced growth promotion in the presence of ferrilactoferrin in MEMalpha compared to wild type but was not affected in the presence of heme. Moreover, this mutant acquired less 55Fe than wild type when incubated with 55Fe-loaded protein and bound less lactoferrin. Complementation restored the wild-type phenotype when cells were grown with ferrilactoferrin. A mutant in the ABC transporter system permease gene (cj0174c) showed a small but significant growth reduction. The cj0176c-cj0177 intergenic region contains two separate Fur-regulated iron-repressible promoters. This is the first demonstration that C. jejuni is capable of acquiring iron from members of the transferrin protein family, and our data indicate a role for Cj0178 in this process.
Assuntos
Campylobacter jejuni/metabolismo , Ferro/metabolismo , Lactoferrina/metabolismo , Transferrina/metabolismo , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/genética , Campylobacter jejuni/crescimento & desenvolvimento , Divisão Celular/efeitos dos fármacos , Conalbumina/metabolismo , Conalbumina/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Humanos , Radioisótopos de Ferro , Lactoferrina/farmacologia , Mutação , Ligação Proteica , Transferrina/farmacologiaRESUMO
Norepinephrine promotes the growth of Salmonella enterica in vitro in iron-restricted conditions imposed by the iron-binding proteins serum transferrin and egg-white ovotransferrin by facilitating the release of bound iron and subsequent uptake by the bacteria. Moreover, significantly increased colonisation and systemic spread were observed in mouse and chicken models of S. enterica infection following pre-treatment of animals with norepinephrine. Both ent and tonB mutants showed no growth promotion by norepinephrine either in liquid medium containing serum or on plates containing hens' egg-white, indicating that the process is dependent both on the ability to synthesise enterobactin and on TonB-dependent uptake of iron. An entS mutant (formerly designated ybdA) and an iroB mutant behaved as wild type in both assays, showing that neither secretion of enterobactin nor conversion of enterobactin to salmochelin S4 is necessary for the effect. On the other hand, the presence of mutations in fes or iroD resulted in loss of growth promotion by norepinephrine in both assays. Since the fes and iroD genes encode enzymes that hydrolyse enterobactin and salmochelin S4 respectively to monomers, these data suggest that excretion of monomeric forms of these siderophores may be important for the uptake of iron released by norepinephrine from transferrin or ovotransferrin. A similar pattern of behaviour was observed with S. enterica serovar Typhimurium in a mouse model of infection; treatment of animals with norepinephrine before intragastric challenge resulted in increased intestinal colonisation and systemic spread of both wild-type and entS mutant strains, while the fes mutant was significantly attenuated in vivo. This suggests that excretion of 2,3-dihydroxybenzoylserine may be essential for norepinephrine-dependent growth promotion in the iron-restricted environment of the infected host. Unlike the situation in vitro, however, tonB mutants of S. enterica serovars Typhimurium and Enteritidis behaved the same as wild type in mouse and chick infection models, respectively, suggesting that norepinephrine-dependent growth stimulation may also occur by TonB-independent uptake of the enterobactin precursor 2,3-dihydroxybenzoic acid.
Assuntos
Ferro/metabolismo , Norepinefrina/metabolismo , Salmonella enteritidis/efeitos dos fármacos , Salmonella enteritidis/fisiologia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ceco/microbiologia , Galinhas , Contagem de Colônia Microbiana , Enterobactina/análogos & derivados , Enterobactina/genética , Enterobactina/metabolismo , Feminino , Glucosídeos/genética , Glucosídeos/metabolismo , Fígado/microbiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Salmonelose Animal , Salmonella enteritidis/crescimento & desenvolvimento , Salmonella typhimurium/crescimento & desenvolvimento , VirulênciaRESUMO
Enteropathogenic Escherichia coli (EPEC) is a Gram-negative bacterial pathogen that adheres to human intestinal epithelial cells, resulting in watery, persistent diarrhoea. Despite the advances made in understanding EPEC-host cell interactions, the molecular mechanisms underlying watery diarrhoea have not been understood fully. Loss of transepithelial resistance and increased monolayer permeability by disruption of tight junctions has been implicated in this process. Apart from disruption of tight junctions, an important factor known to regulate monolayer permeability is E-cadherin and its interaction with beta-catenin, both of which constitute the adherens junctions. Our previous studies using HEp-2 cells demonstrated the morphological and cytoskeletal changes caused by cell-free outer membrane preparations (OMPs) of EPEC. In this study, we have shown that EPEC and its OMP induce significant changes in the adherens junctions of Caco-2 monolayers. We also observed significant phosphorylation of protein kinase Calpha (PKCalpha) in cells treated with either whole EPEC or its OMP. Immunoprecipitation of cell lysates with anti-E-cadherin and probing with phospho-PKCalpha monoclonal antibodies and anti-beta-catenins revealed that in these cells, phosphorylated PKCalpha is associated with cadherins, leading to the dissociation of the cadherin/beta-catenin complex. Immunofluorescence showed beta-catenins dissociated from the membrane-bound cadherins and redistributed into the cytoplasm. Expression of dominant negative PKCalpha reversed these effects caused by either whole EPEC or its OMP and also reduced the associated increase in monolayer permeability. It is possible that this mechanism may complement the earlier known pathways for loss of barrier function involving myosin light chain kinase activation and also may play a role in causing host cell death by apoptosis.
Assuntos
Junções Aderentes/metabolismo , Caderinas/metabolismo , Escherichia coli O157/patogenicidade , Proteína Quinase C/metabolismo , Proteínas da Membrana Bacteriana Externa/farmacologia , Transporte Biológico , Células CACO-2 , Proteínas do Citoesqueleto/metabolismo , Citosol/metabolismo , Relação Dose-Resposta a Droga , Disenteria/microbiologia , Ativação Enzimática , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/química , Proteínas de Escherichia coli/farmacologia , Humanos , Isoenzimas/metabolismo , Permeabilidade/efeitos dos fármacos , Proteína Quinase C-alfa , Transativadores/metabolismo , beta CateninaRESUMO
Trauma is well recognized to result in the immediate and sustained release of stress-related neurochemicals such as the catecholamine norepinephrine. Past work has shown that in addition to their ability to function as neurotransmitters, catecholamines can also directly stimulate the growth of a number of pathogenic bacteria. The development of trauma-associated sepsis has often been linked to the ability of otherwise normal commensal bacteria to invade and penetrate the gut mucosal barrier. Therefore, the aim of our study was to examine whether catecholamines could also stimulate the growth of commensal Escherichia coli strains of the type present in the intestinal tract at the time of a traumatic event. Herein we report that the growth of a range of non-pathogenic isolates of E. coli of human and environmental origin was significantly increased in the presence of catecholamines. A primary mechanism by which catecholamines increase bacterial growth was shown to be iron removal from lactoferrin and transferrin and subsequent acquisition by bacteria. The 3,4-dihydroxybenzoyl (catechol) structure of the catecholamines was further demonstrated to be critical to iron acquisition. The synthetic catecholamine inotropes dobutamine and isoprenaline, as well as norepinephrine metabolites that retained the catechol structure were also active, whereas norepinephrine metabolites in which the catechol moiety had been modified were not. A role for catecholamine-mediated bacterial iron supply in the pathophysiology of gut-derived sepsis due to trauma is proposed.
Assuntos
Catecolaminas/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Intestinos/microbiologia , Sepse/etiologia , Ferimentos e Lesões/complicações , Catecolaminas/metabolismo , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Humanos , Técnicas In Vitro , Ferro/metabolismo , Modelos Biológicos , Norepinefrina/farmacologia , Sepse/metabolismo , Sepse/microbiologia , Ferimentos e Lesões/metabolismoRESUMO
Exposure of bacteria to members of the stress-associated family of catecholamine hormones, principally norepinephrine, has been demonstrated to increase both growth and production of virulence-related factors. Mutation of genes for enterobactin synthesis and uptake revealed an absolute requirement for enterobactin in norepinephrine-stimulated growth of Escherichia coli O157:H7. The autoinducer produced by norepinephrine-stimulated E. coli could not substitute for enterobactin. We also demonstrate that norepinephrine promotes iron shuttling between transferrin molecules, thereby enabling the bacterial siderophore enterobactin to more readily acquire iron for growth. These results suggest one of the possible mechanisms by which the hormonal output of stress may affect enterohaemorrhagic E. coli pathogenicity.
Assuntos
Enterobactina/metabolismo , Escherichia coli O157/metabolismo , Ferro/farmacocinética , Norepinefrina/farmacologia , Simpatomiméticos/farmacologia , Transferrina/metabolismo , Escherichia coli O157/efeitos dos fármacos , Escherichia coli O157/crescimento & desenvolvimento , Radioisótopos de FerroRESUMO
Enteropathogenic Escherichia coli (EPEC) is an important aetiological agent of persistent infantile diarrhoea. EPEC pathogenicity is not mediated through known toxins and the role played by outer-membrane proteins (OMPs) in the initial adherence of the bacterium, intimate attachment to epithelial cells and ultimately in the effacement of the intestinal epithelium is being pursued vigorously. In this study of the different cellular fractions of the bacterium investigated, only the outer-membrane fraction was able to disrupt HEp-2 cells. The outer-membrane fraction was also found to be cytotoxic and caused actin accumulation around the periphery of the host cells. To understand the role of OMPs in pathogenesis, protein profiles of outer-membrane preparations of wild-type and attenuated mutants lacking either the EPEC adherence factor (EAF) mega-plasmid or EPEC attaching and effacing gene A (eaeA) coding for a 94-kDa OMP, intimin or EPEC secretory protein gene B (espB) coding for a 34-kDa translocated signal transducing protein were compared and correlated with their cytopathic effects. A 43-kDa protein seen along with intimin in the outer membrane of EPEC was identified as maltoporin, an E. coli outer-membrane porin normally expressed only in response to maltose in the growth medium. In the case of EPEC, not only was this regulation lost, but also the expression of maltoporin was found to be tightly coupled to the expression of the secretory virulence factor EspB. Maltoporin per se is not toxic, as evidenced by the treatment of HEp-2 cells with the outer-membrane preparation of E. coli DH5a grown in the presence of maltose and the significance of this pathogenic adaptation is not clear. However, when maltoporin and possibly other unidentified proteins were not present as a component of the outer-membrane preparation, as in the outer-membrane preparation of an espB-negative strain, cellular disruption as well as actin accumulation proceeded at a very slow rate even though the cytotoxic effects were comparable to those of the wild-type EPEC strains.
Assuntos
Adesinas Bacterianas , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas de Transporte , Proteínas de Escherichia coli , Escherichia coli/patogenicidade , Receptores Virais/biossíntese , Linhagem Celular , Densitometria , Eletroforese em Gel de Poliacrilamida , Humanos , Fenótipo , Porinas , Transdução de SinaisRESUMO
Enteropathogenic Escherichia coli (EPEC) infects the human intestinal epithelium and is a major cause of infantile diarrhea in developing countries. Nitric oxide (NO) is an important modulator of intestinal inflammatory response. The aim of the present study was to investigate whether EPEC outer membrane proteins (OMPs) up regulate epithelial cell expression of inducible nitric oxide synthase (iNOS) and to examine the role of NF-kappaB and MAP kinases (MAPK) on nitrite production. iNOS mRNA expression was assessed by RT-PCR. Nitrite levels were measured by Griess reaction. NF-kappaB activation by OMPs was evaluated by EMSA and immunoblotting was done to detect MAPK activation. EPEC OMP up regulated iNOS, induced nitrite production and NF-kappaB and MAPK were activated in caco-2 cells. The nitrite levels decreased when NF-kappaB and MAPK inhibitors were used. Thus, EPEC OMPs induce iNOS expression and NO production through activation of NF-kappaB and MAPK.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/patogenicidade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico Sintase/biossíntese , Células CACO-2 , Ensaio de Desvio de Mobilidade Eletroforética , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Flavonoides/farmacologia , Humanos , Imidazóis/farmacologia , Medições Luminescentes , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Nitrilas/farmacologia , Nitritos/análise , Nitritos/antagonistas & inibidores , Piridinas/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfonas/farmacologiaRESUMO
Campylobacter requires iron for successful colonization of the host. In the last 7 years, a wealth of data has been generated allowing detailed molecular characterization of Campylobacter iron-uptake systems. Several exogenous siderophores have been identified as sources of ferric iron for Campylobacter. Ferri-enterochelin uptake requires both the outer-membrane receptor protein CfrA and the inner-membrane ABC transporter system CeuBCDE. Ferrichrome has been shown to support growth of some Campylobacter jejuni strains and the presence of homologues of Escherichia coli fhuABD genes was proposed; the Cj1658-Cj1663 system appears to be involved in the uptake of ferri-rhodotorulic acid. In addition to siderophores, the importance of host iron sources was highlighted by recent studies demonstrating that C. jejuni can exploit haem compounds and the transferrins using ChuABCDZ and Cj0173c-Cj0178, respectively. An additional putative receptor, Cj0444, present in some, but not all, strains has not yet been characterized. Following diffusion through the outer membrane, inner-membrane transport of ferrous iron can occur via the FeoB protein. While it may be assumed that all systems are not essential, there is growing evidence supporting the need for multiple iron-uptake systems for successful host colonization by Campylobacter. In light of this, comparative molecular characterization of iron systems in all Campylobacter strains is necessary to gain further insight into the pathogenesis of members of this genus.
Assuntos
Campylobacter/metabolismo , Ferro/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transporte BiológicoRESUMO
For lack of simple inexpensive early detection methods for Shigella spp. and enteroinvasive Escherichia coli (EIEC), bacillary dysentery remains a major cause of childhood mortality and morbidity in India and other developing countries. Rapid stool testing for apyrase, a specific periplasmic enzyme essential for the pathogen's intracellular spread, may provide a solution. We have developed a whole-cell colorimetric pyrophosphate hydrolysis assay based on cheap, stable, and locally available reagents. An innovative filtration-cum-inoculation step eliminates interfering stool solids and ensures sufficient bacterial growth and apyrase expression in 6 to 7 h at 37 degrees C. In a limited double-blind study of 57 clinical isolates of common enterobacteria, the test showed 100% sensitivity and 80% specificity for Shigella spp. and EIEC. Requiring only widely available equipment and inexpensive consumables, this affordable test is readily adaptable for determining antibiograms and for surveillance of food and water samples for the presence of Shigella and EIEC.
Assuntos
Apirase/análise , Proteínas de Bactérias/análise , Técnicas Bacteriológicas/métodos , Colorimetria/métodos , Disenteria Bacilar/diagnóstico , Infecções por Escherichia coli/diagnóstico , Fezes/microbiologia , Difosfatos/metabolismo , Disenteria Bacilar/microbiologia , Escherichia coli/enzimologia , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Microbiologia de Alimentos , Índia , Testes de Sensibilidade Microbiana , Sensibilidade e Especificidade , Shigella/enzimologia , Shigella/isolamento & purificaçãoRESUMO
Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea. In this work we investigated the effect of outer membrane proteins (OMP) of EPEC on barrier integrity and the role of actin, junctional adhesion molecule (JAM) and signaling pathways contributing to these changes. Barrier function was assessed by transepithelial electrical resistance (TER). OMP of wild type EPEC, eaeA and maltoporin mutants decreased TER levels of Caco-2 cells. The OMP of espB mutant was deficient in decreasing TER of Caco-2 cells. The proteinase K-digested wild type OMP and EAF mutant OMP did not cause any change in barrier function. Our previous studies have demonstrated that EPEC OMP induced changes in cadherin junctions of Caco-2 cells. Immunofluorescence revealed disruption in actin cytoskeleton by EPEC OMP. However, no change in expression of junctional adhesion molecule-1 was observed. NF-kappaB inhibitor slightly blocked the decrease in TER and protected against actin disruption while ERK1/2 inhibitor had no effect in blocking these changes. In conclusion, our data suggest that the OMP of EPEC alter intestinal barrier function by disrupting actin cytoskeleton and signaling pathways like NF-kappaB may have a role in regulating barrier changes.
Assuntos
Actinas/fisiologia , Proteínas da Membrana Bacteriana Externa/fisiologia , Moléculas de Adesão Celular/fisiologia , Escherichia coli/patogenicidade , Junções Íntimas/fisiologia , Adesinas Bacterianas/fisiologia , Células CACO-2 , Infecções por Escherichia coli/fisiopatologia , Proteínas de Escherichia coli/fisiologia , Flavonoides/farmacologia , Humanos , Moléculas de Adesão Juncional , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Nitrilas/farmacologia , Porinas , Receptores Virais/fisiologia , Sulfonas/farmacologia , Junções Íntimas/efeitos dos fármacosRESUMO
Clinical isolates of enterohaemorrhagic Escherichia coli, both O157 and non-O157 serotypes, were investigated for siderophore production, for growth promotion by haem and esculetin in iron-restricted conditions, for production of enterohaemolysin and esculin hydrolase, and for the presence of the chuA and ehx genes by PCR. As expected, all the strains produced enterobactin, but the prevalence of other factors varied among the serovars tested. None of the O157 and O26 strains produced aerobactin or "colibactin", whereas among other enterohaemorrhagic E. coli non-O157 serovars the frequencies of aerobactin and "colibactin" production were similar to those of commensal E. coli strains. The ability to use ferric esculetin for growth in iron-limited media was markedly more prevalent among non-O157 serovars and less prevalent among O157 strains compared with commensal E. coli strains. Almost all O157, O26 and O103 strains expressed enterohaemolysin, compared with only 50% of other non-O157 strains. Similarly, almost all O157 and O26 strains utilised haem as a host iron source; the frequency of haem use by other non-O157 strains was generally lower and variable among serovars, such that none of the O103:H2 isolates tested used haem as an iron source. The gene chuA, which encodes the haem transport protein ChuA and which is prevalent in O157:H7 strains, was only rarely noted among non-O157 serovars of enterohaemorrhagic E. coli, even among isolates that could use haem as an iron source. Overall our data demonstrate that O157:H7 and non-O157 serovars, in particular O26:H(-)/H11 and O103:H2, use distinctly different strategies for obtaining iron, and suggest two evolutionary distinct lines of enterhaemorrhagic E. coli.
Assuntos
Infecções por Escherichia coli/microbiologia , Escherichia coli O157/metabolismo , Escherichia coli/metabolismo , Ferro/metabolismo , Antígenos de Bactérias/análise , Proteínas da Membrana Bacteriana Externa/genética , Toxinas Bacterianas/genética , DNA Bacteriano/genética , Enterobactina/biossíntese , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Heme/metabolismo , Proteínas Hemolisinas/biossíntese , Humanos , Ácidos Hidroxâmicos/análise , Antígenos O/análise , Peptídeos/metabolismo , Policetídeos/metabolismo , Reação em Cadeia da Polimerase , Receptores de Superfície Celular/genética , Sideróforos/biossíntese , Umbeliferonas/metabolismoRESUMO
We have shown that Staphylococcus aureus biofilm production is induced in iron-restricted conditions and is repressed by iron via a Fur-independent mechanism, while Fur has both positive and negative regulatory roles in low iron. Furthermore, there is no significant increase in polymeric N-acetylglucosamine polysaccharide expression to account for induction of biofilms in low iron.
Assuntos
Biofilmes/crescimento & desenvolvimento , Ferro , Staphylococcus aureus/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Meios de Cultura , Regulação Bacteriana da Expressão Gênica , Mutação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Staphylococcus aureus/genéticaRESUMO
For survival, pathogenic organisms such as Candida albicans must possess an efficient mechanism for acquiring iron in the iron-restricted environment of the human body. C. albicans can use iron from a variety of sources found within the host. However, it is not clear how biologically active ferrous iron is obtained from these sources. One strategy adopted by some organisms is to reduce iron extracellularly and then specifically transport the ferrous iron into the cell. We have shown that clinical isolates of C. albicans do have a cell-associated ferric-reductase activity. The determination of ferric-reductase activity of cells growing exponentially in either low- or high-iron media over a period of time indicated that C. albicans reductase activity is induced when in low-iron conditions. Moreover, we have demonstrated that C. albicans reductase activity is also regulated in response to the growth phase of the culture, with induction occurring upon exit from stationary phase and maximal levels being reached in early exponential stage irrespective of the iron content of the medium. These results suggest that C. albicans reductase activity is regulated in a very similar manner to the Saccharomyces cerevisiae ferric-reductase. Iron reduction and uptake in S. cerevisiae are closely connected to copper reduction, and possibly copper uptake. In this report we show that iron and copper reduction also appear to be linked in C. albicans. The ferric-reductase activity is negatively regulated by copper. Moreover, quantitative cupric-reductase assays indicated that C. albicans is capable of reducing copper and that this cupric-reductase activity is negatively regulated by both iron and copper. This is the first report that C. albicans has an iron- and copper-mediated ferri-reductase activity.
Assuntos
Candida albicans/enzimologia , FMN Redutase , NADH NADPH Oxirredutases/metabolismo , Candida albicans/crescimento & desenvolvimento , Cobre , Meios de Cultura , FerroRESUMO
Copper and iron uptake in Saccharomyces cerevisiae are linked through a high-affinity ferric/cupric-reductive uptake system. Evidence suggests that a similar system operates in Candida albicans. The authors have identified a C. albicans gene that is able to rescue a S. cerevisiae ctr1/ctr3-null mutant defective in high-affinity copper uptake. The 756 bp ORF, designated CaCTR1, encodes a 251 amino acid protein with a molecular mass of 27.8 kDa. Comparisons between the deduced amino acid sequence of the C. albicans Ctr1p and S. cerevisiae Ctr1p indicated that they share 39.6 % similarity and 33.0 % identity over their entire length. Within the predicted protein product of CaCTR1 there are putative transmembrane regions and sequences that resemble copper-binding motifs. The promoter region of CaCTR1 contains four sequences with significant identity to S. cerevisiae copper response elements. CaCTR1 is transcriptionally regulated in S. cerevisiae in response to copper availability by the copper-sensing transactivator Mac1p. Transcription of CaCTR1 in C. albicans is also regulated in a copper-responsive manner. This raises the possibility that CaCTR1 may be regulated in C. albicans by a Mac1p-like transactivator. A C. albicans ctr1-null mutant displays phenotypes consistent with the lack of copper uptake including growth defects in low-copper and low-iron conditions, a respiratory deficiency and sensitivity to oxidative stress. Furthermore, changes in morphology were observed in the C. albicans ctr1-null mutant. It is proposed that CaCTR1 facilitates transport of copper into the cell.
Assuntos
Candida albicans/genética , Candida albicans/metabolismo , Proteínas de Transporte de Cátions , Cobre/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Sequência de Bases , Candida albicans/crescimento & desenvolvimento , Transportador de Cobre 1 , Meios de Cultura , DNA Fúngico/genética , Proteínas Fúngicas/química , Ferro/metabolismo , Proteínas de Membrana/química , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estresse Oxidativo , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Candida albicans, like other pathogens, has to compete with the host for a limited supply of available iron. Consequently, iron acquisition is likely to be an important factor for the growth, survival and virulence of this organism. It was previously demonstrated that C. albicans has a surface-associated ferric reductase similar to that of Saccharomyces cerevisiae. Therefore, functional rescue of a S. cerevisiae fre1 mutant was used to isolate a C. albicans ferric reductase gene (CFL1). This gene has been previously identified. However, the workers had not observed any functional reductase activity associated with the gene. The discrepancy with the findings in this report appears to be due to the clone previously reported carrying a non-contiguous piece of C. albicans DNA. Results shown in this paper demonstrate that CFL1 transcription is regulated in response to levels of iron and copper. This is the first demonstration of a functional ferric reductase gene from C. albicans.
Assuntos
Candida albicans/genética , FMN Redutase , Proteínas Fúngicas/genética , Genes Fúngicos , NADH NADPH Oxirredutases/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , Oxirredutases/genéticaRESUMO
BipA is a novel member of the ribosome binding GTPase superfamily and is widely distributed in bacteria and plants. We report here that it regulates -multiple cell surface- and virulence-associated -components in the enteropathogenic Escherichia coli (EPEC) strain E2348/69. The regulated components include bacterial flagella, the espC pathogenicity island and a type III secretion system specified by the locus of enterocyte effacement (LEE). BipA positively regulated the espC and LEE gene clusters through transcriptional control of the LEE-encoded regulator, Ler. Additionally, it affected the pattern of proteolysis of intimin, a key LEE-encoded adhesin specified by the LEE. BipA control of the LEE operated independently of the previously characterized regulators Per, integration host factor and H-NS. In contrast, it negatively regulated the flagella-mediated motility of EPEC and in a Ler-independent manner. Our results indicate that the BipA GTPase functions high up in diverse regulatory cascades to co-ordinate the expression of key pathogenicity islands and other virulence-associated factors in E. coli.