RESUMO
Metal(loid) salts were used to treat infectious diseases in the past due to their exceptional biocidal properties at low concentrations. However, the mechanism of their toxicity has yet to be fully elucidated. The production of reactive oxygen species (ROS) has been linked to the toxicity of soft metal(loid)s such as Ag(I), Au(III), As(III), Cd(II), Hg(II), and Te(IV). Nevertheless, few reports have described the direct, or ROS-independent, effects of some of these soft-metal(loid)s on bacteria, including the dismantling of iron-sulfur clusters [4Fe-4S] and the accumulation of porphyrin IX. Here, we used genome-wide genetic, proteomic, and biochemical approaches under anaerobic conditions to evaluate the direct mechanisms of toxicity of these metal(loid)s in Escherichia coli. We found that certain soft-metal(loid)s promote protein aggregation in a ROS-independent manner. This aggregation occurs during translation in the presence of Ag(I), Au(III), Hg(II), or Te(IV) and post-translationally in cells exposed to Cd(II) or As(III). We determined that aggregated proteins were involved in several essential biological processes that could lead to cell death. For instance, several enzymes involved in amino acid biosynthesis were aggregated after soft-metal(loid) exposure, disrupting intracellular amino acid concentration. We also propose a possible mechanism to explain how soft-metal(loid)s act as proteotoxic agents.
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The tellurium oxyanion tellurite (TeO3 (2-)) is extremely harmful for most organisms. It has been suggested that a potential bacterial tellurite resistance mechanism would consist of an enzymatic, NAD(P)H-dependent, reduction to the less toxic form elemental tellurium (Te(0)). To date, a number of enzymes such as catalase, type II NADH dehydrogenase and terminal oxidases from the electron transport chain, nitrate reductases, and dihydrolipoamide dehydrogenase (E3), among others, have been shown to display tellurite-reducing activity. This activity is generically referred to as tellurite reductase (TR). Bioinformatic data resting on some of the abovementioned enzymes enabled the identification of common structures involved in tellurite reduction including vicinal catalytic cysteine residues and the FAD/NAD(P)(+)-binding domain, which is characteristic of some flavoproteins. Along this line, thioredoxin reductase (TrxB), alkyl hydroperoxide reductase (AhpF), glutathione reductase (GorA), mercuric reductase (MerA), NADH: flavorubredoxin reductase (NorW), dihydrolipoamide dehydrogenase, and the putative oxidoreductase YkgC from Escherichia coli or environmental bacteria were purified and assessed for TR activity. All of them displayed in vitro TR activity at the expense of NADH or NADPH oxidation. In general, optimal reducing conditions occurred around pH 9-10 and 37°C. Enzymes exhibiting strong TR activity produced Te-containing nanostructures (TeNS). While GorA and AhpF generated TeNS of 75 nm average diameter, E3 and YkgC produced larger structures (>100 nm). Electron-dense structures were observed in cells over-expressing genes encoding TrxB, GorA, and YkgC.
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Introducción: la osteogénesis imperfecta es un trastorno congénito caracterizado por la fragilidad del hueso, causado por mutaciones en los genes del procolágeno tipo I. La incidencia es variable, encontrando un caso por cada 5.000 a 70.000 nacidos vivos. Debido a las limitaciones en el tratamiento se ha planteado el uso de bifosfonatoscomo el ácido zoledrónico. Objetivo: describir el efecto del tratamiento con ácido zoledrónico endovenosoen pacientes pediátricos con osteogénesis imperfecta atendidos en el Hospital Universitario San José (Popayán,Colombia) durante 2013 y 2014. Materiales y métodos: se realizó un estudio observacional tipo cohorte de carácter cerrado y sin grupo de comparación. El tratamiento con ácido zoledrónico se realizó trimestralmente durante un año, tiempo en el que se determinaron los efectos positivos y adversos del tratamiento. Resultados: seis pacientes presentaron diagnóstico de osteogénesis imperfecta; uno fue excluido porque inició tratamiento quirúrgico, los demás iniciaron tratamiento con ácido zoledrónico. Un paciente se retiró voluntariamente y cuatro completaron el estudio, dos sin fracturas y dos con una fractura durante este período. La densidad mineral ósea de la columna lumbar y femoral presentó mejoría, documentándose en dos pacientes valores normales a los tres meses de finalizado el tratamiento. La intensidad del dolor óseo, según la escala visual análoga al inicio del estudio, fue entre 5 y 8, y al final entre 0 y 2. No se encontraron cambios en la limitación funcional ni efectos adversos...
Introduction: osteogenesis imperfecta is a congenital disorder characterized by bone fragility, caused by mutationsin genes encoding the type I procollagen. The incidence is variable with ranges reported from one case per 5,000 to 70,000 live births. Due to limitations in the treatment, it has proposed the use of bisphosphonate as zoledronic acid. Objective: to describe the effect of treatment with intravenous zoledronic acid in of pediatric patients diagnosed with osteogenesis imperfecta attended at the Hospital Universitario San José (Popayán Colombia) between 2013 and 2014. Material and methods: an observational cohort study, closed and without comparison group was conducted. The zoledronic acid treatment was conducted quarterly for one year and during this time the positive and adverse effects of treatment were determined. Results: six patients had a diagnosis of osteogenesis imperfecta, of which one was excluded because it began surgical treatment, and five of them started treatment with zoledronic acid; subsequently, one patient voluntarily withdrew from the study found. From the four patients who completed one year of treatment with zoledronic acid, two not showed fractures during it and the other two each presented one fracture. Bone mineral density of the lumbar spine and femoral showed improvement; having two patients in the normal range at three months of treatment ends. According to the visual analogue scale, bone pain intensity at baseline ranged between 5 and 8, and at the end between 0 and 2. Regarding functional limitation were not found significant changes nor adverse effects were found throughout follow-up...
Assuntos
Humanos , Fraturas Ósseas , Osteogênese Imperfeita , PediatriaRESUMO
The tellurium oxyanion tellurite induces oxidative stress in most microorganisms. In Escherichia coli, tellurite exposure results in high levels of oxidized proteins and membrane lipid peroxides, inactivation of oxidation-sensitive enzymes and reduced glutathione content. In this work, we show that tellurite-exposed E. coli exhibits transcriptional activation of the zwf gene, encoding glucose 6-phosphate dehydrogenase (G6PDH), which in turn results in augmented synthesis of reduced nicotinamide adenine dinucleotide phosphate (NADPH). Increased zwf transcription under tellurite stress results mainly from reactive oxygen species (ROS) generation and not from a depletion of cellular glutathione. In addition, the observed increase of G6PDH activity was paralleled by accumulation of glucose-6-phosphate (G6P), suggesting a metabolic flux shift toward the pentose phosphate shunt. Upon zwf overexpression, bacterial cells also show increased levels of antioxidant molecules (NADPH, GSH), better-protected oxidation-sensitive enzymes and decreased amounts of oxidized proteins and membrane lipids. These results suggest that by increasing NADPH content, G6PDH plays an important role in E. coli survival under tellurite stress.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Glucosefosfato Desidrogenase/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Telúrio/farmacologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Glucose-6-Fosfato/metabolismo , Glucosefosfato Desidrogenase/genética , NADP/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
This work shows that the recently described Escherichia coli BtuE peroxidase protects the bacterium against oxidative stress that is generated by tellurite and by other reactive oxygen species elicitors (ROS). Cells lacking btuE (ΔbtuE) displayed higher sensitivity to K(2)TeO(3) and other oxidative stress-generating agents than did the isogenic, parental, wild-type strain. They also exhibited increased levels of cytoplasmic reactive oxygen species, oxidized proteins, thiobarbituric acid reactive substances, and lipoperoxides. E. coli ΔbtuE that was exposed to tellurite or H(2)O(2) did not show growth changes relative to wild type cells either in aerobic or anaerobic conditions. Nevertheless, the elimination of btuE from cells deficient in catalases/peroxidases (Hpx(-)) resulted in impaired growth and resistance to these toxicants only in aerobic conditions, suggesting that BtuE is involved in the defense against oxidative damage. Genetic complementation of E. coli ΔbtuE restored toxicant resistance to levels exhibited by the wild type strain. As expected, btuE overexpression resulted in decreased amounts of oxidative damage products as well as in lower transcriptional levels of the oxidative stress-induced genes ibpA, soxS and katG.
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Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Estresse Oxidativo , Proteínas Periplásmicas de Ligação/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas Periplásmicas de Ligação/genética , Peroxidases/metabolismo , Transcrição GênicaRESUMO
Data regarding tellurium (Te) toxicity are scarce. Studies on its metabolism, performed mainly in bacteria, underline a major role of reactive oxygen species (ROS). We investigated whether tellurite undergoes redox cycling leading to ROS formation and cancer cell death. The murine hepatocarcinoma Transplantable Liver Tumor (TLT) cells were challenged with tellurite either in the presence or in the absence of different compounds as N-acetylcysteine (NAC), 3-methyladenine, BAPTA-AM, and catalase. NAC inhibition of tellurite-mediated toxicity suggested a major role of oxidative stress. Tellurite also decreased both glutathione (GSH) and ATP content by 57 and 80%, respectively. In the presence of NAC however, the levels of such markers were almost fully restored. Tellurite-mediated ROS generation was assessed both by using the fluorescent, oxidation-sensitive probe dichlorodihydrofluorescein diacetate (DCHF-DA) and electron spin resonance (ESR) spectroscopy to detect hydroxyl radical formation. Cell death occurs by a caspase-independent mechanism, as shown by the lack of caspase-3 activity and no cleavage of poly(ADP-ribose)polymerase (PARP). The presence of gamma-H2AX suggests tellurite-induced DNA strand breaking, NAC being unable to counteract it. Although the calcium chelator BAPTA-AM did show no effect, the rapid phosphorylation of eIF2alpha suggests that, in addition to oxidative stress, an endoplasmic reticulum (ER) stress may be involved in the mechanisms leading to cell death by tellurite.
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Carcinoma Hepatocelular/metabolismo , Morte Celular/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Telúrio/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Caspase 3/metabolismo , Linhagem Celular Tumoral , Glutationa/metabolismo , Humanos , Camundongos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Evidence that Escherichia coli YqhD is involved in bacterial response to compounds that generate membrane lipid peroxidation is presented. Overexpression of yqhD results in increased resistance to the reactive oxygen species-generating compounds hydrogen peroxide, paraquat, chromate, and potassium tellurite. Increased tolerance was also observed for the lipid peroxidation-derived aldehydes butanaldehyde, propanaldehyde, acrolein, and malondialdehyde and the membrane-peroxidizing compound tert-butylhydroperoxide. Expression of yqhD was also associated with changes in the concentration of intracellular peroxides and cytoplasmic protein carbonyl content and with a reduction in intracellular acrolein levels. When compared with the wild type strain, an yqhD mutant exhibited a sensitive phenotype to all these compounds and also augmented levels of thiobarbituric acid-reactive substances, which may indicate an increased level of lipid peroxidation. Purified YqhD catalyzes the in vitro reduction of acetaldehyde, malondialdehyde, propanaldehyde, butanaldehyde, and acrolein in a NADPH-dependent reaction. Finally, yqhD transcription was induced in cells that had been exposed to conditions favoring lipid peroxidation. Taken together these results indicate that this enzyme may have a physiological function by protecting the cell against the toxic effect of aldehydes derived from lipid oxidation. We speculate that in Escherichia coli YqhD is part of a glutathione-independent, NADPH-dependent response mechanism to lipid peroxidation.
Assuntos
Aldeído Desidrogenase/biossíntese , Aldeído Redutase/biossíntese , Aldeídos/metabolismo , Proteínas de Escherichia coli/biossíntese , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Peroxidação de Lipídeos/fisiologia , Aldeído Desidrogenase/química , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/isolamento & purificação , Aldeído Redutase/genética , Aldeídos/química , Catálise , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Oxidantes/farmacologia , Peróxidos/química , Peróxidos/metabolismoRESUMO
Biochemical, genetic, enzymatic and molecular approaches were used to demonstrate, for the first time, that tellurite (TeO(3) (2-)) toxicity in E. coli involves superoxide formation. This radical is derived, at least in part, from enzymatic TeO(3) (2-) reduction. This conclusion is supported by the following observations made in K(2)TeO(3)-treated E. coli BW25113: i) induction of the ibpA gene encoding for the small heat shock protein IbpA, which has been associated with resistance to superoxide, ii) increase of cytoplasmic reactive oxygen species (ROS) as determined with ROS-specific probe 2'7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA), iii) increase of carbonyl content in cellular proteins, iv) increase in the generation of thiobarbituric acid-reactive substances (TBARs), v) inactivation of oxidative stress-sensitive [Fe-S] enzymes such as aconitase, vi) increase of superoxide dismutase (SOD) activity, vii) increase of sodA, sodB and soxS mRNA transcription, and viii) generation of superoxide radical during in vitro enzymatic reduction of potassium tellurite.