RESUMEN
Mycobacterium tuberculosis (Mtb) senses and responds to host-derived gasotransmitters NO and CO via heme-containing sensor kinases DosS and DosT and the response regulator DosR. Hydrogen sulfide (H2S) is an important signaling molecule in mammals, but its role in Mtb physiology is unclear. We have previously shown that exogenous H2S can modulate expression of genes in the Dos dormancy regulon via an unknown mechanism(s). Here, we test the hypothesis that Mtb senses and responds to H2S via the DosS/T/R system. Using UV-Vis and EPR spectroscopy, we show that H2S binds directly to the ferric (Fe3+) heme of DosS (KDapp = 5.30 µM) but not the ferrous (Fe2+) form. No interaction with DosT(Fe2+-O2) was detected. We found that the binding of sulfide can slowly reduce the DosS heme iron to the ferrous form. Steered Molecular Dynamics simulations show that H2S, and not the charged HS- species, can enter the DosS heme pocket. We also show that H2S increases DosS autokinase activity and subsequent phosphorylation of DosR, and H2S-mediated increases in Dos regulon gene expression is lost in Mtb lacking DosS. Finally, we demonstrate that physiological levels of H2S in macrophages can induce DosR regulon genes via DosS. Overall, these data reveal a novel mechanism whereby Mtb senses and responds to a third host gasotransmitter, H2S, via DosS(Fe3+). These findings highlight the remarkable plasticity of DosS and establish a new paradigm for how bacteria can sense multiple gasotransmitters through a single heme sensor kinase.
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Gasotransmisores , Mycobacterium tuberculosis , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ácido Dioctil Sulfosuccínico/metabolismo , Gasotransmisores/metabolismo , Regulación Bacteriana de la Expresión Génica , Hemo/metabolismo , Hierro/metabolismo , Mamíferos/genética , Mamíferos/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Protamina Quinasa/química , Protamina Quinasa/genética , Protamina Quinasa/metabolismo , RegulónRESUMEN
Nitric oxide (NO) chemiluminescence detectors (CLDs) are specialized and sensitive spectroscopic instruments capable of directly measuring NO flux rates. NO CLDs have been instrumental in the characterization of mammalian nitrite-dependent NO synthases. However, no detailed description of NO flux analysis using NO CLD is available. Herein, a detailed review of the NO CL methodology is provided with guidelines for measuring NO-production rates from aqueous samples, such as isolated enzymes or protein homogenates. Detailed description of the types of signals one can encounter, data processing, and potential pitfalls related to NO flux measurements will also be covered.
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Mediciones Luminiscentes/métodos , Óxido Nítrico Sintasa , Óxido Nítrico , Ozono/química , Animales , Diseño de Equipo , Cinética , Óxido Nítrico/análisis , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa/metabolismoRESUMEN
H2S is a potent gasotransmitter in eukaryotes and bacteria. Host-derived H2S has been shown to profoundly alter M. tuberculosis (Mtb) energy metabolism and growth. However, compelling evidence for endogenous production of H2S and its role in Mtb physiology is lacking. We show that multidrug-resistant and drug-susceptible clinical Mtb strains produce H2S, whereas H2S production in non-pathogenic M. smegmatis is barely detectable. We identified Rv3684 (Cds1) as an H2S-producing enzyme in Mtb and show that cds1 disruption reduces, but does not eliminate, H2S production, suggesting the involvement of multiple genes in H2S production. We identified endogenous H2S to be an effector molecule that maintains bioenergetic homeostasis by stimulating respiration primarily via cytochrome bd. Importantly, H2S plays a key role in central metabolism by modulating the balance between oxidative phosphorylation and glycolysis, and it functions as a sink to recycle sulfur atoms back to cysteine to maintain sulfur homeostasis. Lastly, Mtb-generated H2S regulates redox homeostasis and susceptibility to anti-TB drugs clofazimine and rifampicin. These findings reveal previously unknown facets of Mtb physiology and have implications for routine laboratory culturing, understanding drug susceptibility, and improved diagnostics.
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Organic nitrate esters, long-recognized therapies for cardiovascular disorders, have not been detected biologically. We characterize in rat stomach unsaturated fatty acid nitration reactions that proceed by generation of nitro-nitrate intermediates (NO2-ONO2-FA) via oxygen and nitrite dependent reactions. NO2-ONO2-lipids represent â¼70% of all nitrated lipids in the stomach and they decay in vitro at neutral or basic pH by the loss of the nitrate ester group (-ONO2) from the carbon backbone upon deprotonation of the α-carbon (pKa â¼7), yielding nitrate, nitrite, nitrosative species, and an electrophilic fatty acid nitroalkene product (NO2-FA). Of note, NO2-FA are anti-inflammatory and tissue-protective signaling mediators, which are undergoing Phase II trials for the treatment of kidney and pulmonary diseases. The decay of NO2-ONO2-FA occurs during intestinal transit and absorption, leading to the formation of NO2-FA that were subsequently detected in circulating plasma triglycerides. These observations provide new insight into unsaturated fatty acid nitration mechanisms, identify nitro-nitrate ester-containing lipids as intermediates in the formation of both secondary nitrogen oxides and electrophilic fatty acid nitroalkenes, and expand the scope of endogenous products stemming from metabolic reactions of nitrogen oxides.
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Ácidos Grasos , Nitratos , Animales , Ésteres , Nitrocompuestos , Óxidos de Nitrógeno , RatasRESUMEN
For centuries, hydrogen sulfide (H2S) was considered primarily as a poisonous gas and environmental hazard. However, with the discovery of prokaryotic and eukaryotic enzymes for H2S production, breakdown, and utilization, H2S has emerged as an important signaling molecule in a wide range of physiological and pathological processes. Hence, H2S is considered a gasotransmitter along with nitric oxide (â¢NO) and carbon monoxide (CO). Surprisingly, despite having overlapping functions with â¢NO and CO, the role of host H2S in microbial pathogenesis is understudied and represents a gap in our knowledge. Given the numerous reports that followed the discovery of â¢NO and CO and their respective roles in microbial pathogenesis, we anticipate a rapid increase in studies that further define the importance of H2S in microbial pathogenesis, which may lead to new virulence paradigms. Therefore, this review provides an overview of sulfide chemistry, enzymatic production of H2S, and the importance of H2S in metabolism and immunity in response to microbial pathogens. We then describe our current understanding of the role of host-derived H2S in tuberculosis (TB) disease, including its influences on host immunity and bioenergetics, and on Mycobacterium tuberculosis (Mtb) growth and survival. Finally, this review discusses the utility of H2S-donor compounds, inhibitors of H2S-producing enzymes, and their potential clinical significance.
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Sulfuro de Hidrógeno , Mycobacterium tuberculosis , Tuberculosis , Monóxido de Carbono , Humanos , Óxido NítricoRESUMEN
Electron Paramagnetic Resonance (EPR) spectroscopy coupled with spin traps/probes enables quantitative determination of reactive nitrogen and oxygen species (RNOS). Even with numerous studies using spin probes, the methodology has not been rigorously investigated. The autoxidation of spin probes has been commonly overlooked. Using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH), the present study has tested the effects of metal chelators, temperature, and oxygen content on the autoxidation of spin probes, where an optimized condition is refined for cell studies. The apparent rate of CMH autoxidation under this condition is 7.01 ± 1.60 nM/min, indicating low sensitivity and great variation of the CMH method and that CMH autoxidation rate should be subtracted from the generation rate of CMH-detectable oxidants (simplified as oxidants below) in samples. Oxidants in RAW264.7 cells are detected at an initial rate of 4.0 ± 0.7 pmol/min/106 cells, which is not considered as the rate of basal oxidants generation because the same method has failed to detect oxidant generation from the stimulation of phorbol-12-mysirate-13-acetate (PMA, 0.1 nmol/106 cells) in cells (2.5 ± 0.9 for PMA vs. 2.1 ± 1.5 pmol/min/106 cells for dimethyl sulfoxide (DMSO)-treated cells). In contrast, the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), which exhibits minimal autoxidation, reveals differences between PMA and DMSO treatment (0.26 ± 0.09 vs. -0.06 ± 0.12 pmol/min/106 cells), which challenges previous claims that spin probes are more sensitive than spin traps. We have also found that low temperature EPR measurements of frozen samples of CMH autoxidation provide lower signal intensity and greater variation compared to RT measurements of fresh samples. The current study establishes an example for method development of RNOS detection, where experimental details are rigorously considered and tested, and raises questions on the applications of spin probes and spin traps.
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Oxidantes , Oxígeno , Frío , Óxidos N-Cíclicos , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Especies Reactivas de Oxígeno , Marcadores de SpinRESUMEN
The award of the 1998 Nobel Prize in Physiology or Medicine to Robert F. Furchgott, Louis J. Ignarro, and Ferid Murad "for their discoveries concerning nitric oxide as a signaling molecule in the cardiovascular system" highlighted the discovery of NO in mammals. This breakthrough also coincided with the discoveries of the role of NO as a cytotoxic effector in the immune system and as an intercellular neurotransmitter in the nervous system. This brief overview describes the chronological development of this trilinear convergence in 1986-1988, including background chemistry and history of human/nitrogen oxide interactions in general.
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Óxido Nítrico/historia , Premio Nobel , Transducción de Señal , Animales , Sistema Cardiovascular/metabolismo , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Mamíferos/metabolismo , Sistema Nervioso/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico/fisiologíaRESUMEN
Hydrogen sulfide (H2S) is involved in numerous pathophysiological processes and shares overlapping functions with CO and â¢NO. However, the importance of host-derived H2S in microbial pathogenesis is unknown. Here we show that Mtb-infected mice deficient in the H2S-producing enzyme cystathionine ß-synthase (CBS) survive longer with reduced organ burden, and that pharmacological inhibition of CBS reduces Mtb bacillary load in mice. High-resolution respirometry, transcriptomics and mass spectrometry establish that H2S stimulates Mtb respiration and bioenergetics predominantly via cytochrome bd oxidase, and that H2S reverses â¢NO-mediated inhibition of Mtb respiration. Further, exposure of Mtb to H2S regulates genes involved in sulfur and copper metabolism and the Dos regulon. Our results indicate that Mtb exploits host-derived H2S to promote growth and disease, and suggest that host-directed therapies targeting H2S production may be potentially useful for the management of tuberculosis and other microbial infections.
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Sulfuro de Hidrógeno/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , Animales , Cobre/metabolismo , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Citocinas/sangre , Modelos Animales de Enfermedad , Complejo IV de Transporte de Electrones/metabolismo , Metabolismo Energético , Femenino , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Homeostasis , Pulmón/patología , Macrófagos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mycobacterium tuberculosis/genética , Células RAW 264.7 , Regulón , Azufre/metabolismo , Transcriptoma , TuberculosisRESUMEN
Cells evolved robust homeostatic mechanisms to protect against oxidation or alkylation by electrophilic species. Glutathione (GSH) is the most abundant intracellular thiol, protects cellular components from oxidation and is maintained in a reduced state by glutathione reductase (GR). Nitro oleic acid (NO2-OA) is an electrophilic fatty acid formed under digestive and inflammatory conditions that both reacts with GSH and induces its synthesis upon activation of Nrf2 signaling. The effects of NO2-OA on intracellular GSH homeostasis were evaluated. In addition to upregulation of GSH biosynthesis, we observed that NO2-OA increased intracellular GSSG in an oxidative stress-independent manner. NO2-OA directly inhibited GR in vitro by covalent modification of the catalytic Cys61, with kon of (3.45 ± 0.04)â¯×â¯103 M-1 s-1, koff of (4.4 ± 0.4)â¯×â¯10-4 s-1, and Keq of (1.3 ± 0.1)â¯×â¯10-7 M. Akin to NO2-OA, the electrophilic Nrf2 activators bardoxolone-imidazole (CDDO-Im), bardoxolone-methyl (CDDO-Me) and dimethyl fumarate (DMF) also upregulated GSH biosynthesis while promoting GSSG accumulation, but without directly inhibiting GR activity. In vitro assays in which GR was treated with increasing GSH concentrations and GSH depletion experiments in cells revealed that GR activity is finely regulated via product inhibition, an observation further supported by theoretical (kinetic modeling of cellular GSSG:GSH levels) approaches. Together, these results describe two independent mechanisms by which electrophiles modulate the GSH/GSSG couple, and provide a novel conceptual framework to interpret experimentally determined values of GSH and GSSG.
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Glutatión Reductasa/química , Glutatión Reductasa/metabolismo , Glutatión/biosíntesis , Algoritmos , Alquilación , Secuencia de Aminoácidos , Animales , Catálisis , Dominio Catalítico , Disulfuro de Glutatión/metabolismo , Espacio Intracelular , Cinética , Ratones , Modelos Teóricos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Óxido Nítrico/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Células RAW 264.7 , Especies Reactivas de Oxígeno , Compuestos de SulfhidriloRESUMEN
Liver failure induced by systemic inflammatory response (SIRS) is often associated with mitochondrial dysfunction but the mechanism linking SIRS and mitochondria-mediated liver failure is still a matter of discussion. Current hypotheses suggest that causative events could be a drop in ATP synthesis, opening of mitochondrial permeability transition pore, specific changes in mitochondrial morphology, impaired Ca2+ uptake, generation of mitochondrial reactive oxygen species (mtROS), turnover of mitochondria and imbalance in electron supply to the respiratory chain. The aim of this review is to critically analyze existing hypotheses, in order to highlight the most promising research lines helping to prevent liver failure induced by SIRS. Evaluation of the literature shows that there is no consistent support that impaired Ca++ metabolism, electron transport chain function and ultrastructure of mitochondria substantially contribute to liver failure. Moreover, our analysis suggests that the drop in ATP levels has protective rather than a deleterious character. Recent data suggest that the most critical mitochondrial event occurring upon SIRS is the release of mtROS in cytoplasm, which can activate two specific intracellular signaling cascades. The first is the mtROS-mediated activation of NADPH-oxidase in liver macrophages and endothelial cells; the second is the acceleration of the expression of inflammatory genes in hepatocytes. The signaling action of mtROS is strictly controlled in mitochondria at three points, (i) at the site of ROS generation at complex I, (ii) the site of mtROS release in cytoplasm via permeability transition pore, and (iii) interaction with specific kinases in cytoplasm. The systems controlling mtROS-signaling include pro- and anti-inflammatory mediators, nitric oxide, Ca2+ and NADPH-oxidase. Analysis of the literature suggests that further research should be focused on the impact of mtROS on organ failure induced by inflammation and simultaneously providing a new theoretical basis for a targeted therapy of overwhelmed inflammatory response.
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Mitocondrias/metabolismo , Insuficiencia Multiorgánica/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Animales , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , HumanosRESUMEN
The biological mechanisms of de novo formation of cellular nitrosothiols (as opposed to transnitrosation) are reviewed. The approach is to introduce chemical foundations for each mechanism, followed by evidence in biological systems. The general categories include mechanisms involving nitrous acid, NO autoxidation and oxidant stress, redox active and inactive metal ions, and sulfide/persulfide. Important conclusions/speculations are that de novo cellular thiol nitrosation (1) is an oxidative process, and so should be considered within the family of other thiol oxidative modifications, (2) may not involve a single dominant process but depends on the specific conditions, (3) does not involve O2 under at least some conditions, and (4) may serve to provide a "substrate pool" of protein cysteine nitrosothiol which could, through subsequent enzymatic transnitrosation/denitrosation, be "rearranged" to accomplish the specificity and regulatory control required for effective post-translational signaling.
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Óxido Nítrico/química , Nitrógeno/química , Oxígeno/química , S-Nitrosotioles/química , Animales , Cisteína/química , Hemo/química , Humanos , Iones , Hierro/química , Ligandos , Metales/química , Nitrosación , Oxidación-Reducción , Estrés Oxidativo , Procesamiento Proteico-Postraduccional , Transducción de SeñalRESUMEN
The transport of oxygen and other nonelectrolytes across lipid membranes is known to depend on both diffusion and solubility in the bilayer, and to be affected by changes in the physical state and by the lipid composition, especially the content of cholesterol and unsaturated fatty acids. However, it is not known how these factors affect diffusion and solubility separately. Herein we measured the partition coefficient of oxygen in liposome membranes of dilauroyl-, dimiristoyl- and dipalmitoylphosphatidylcholine in buffer at different temperatures using the equilibrium-shift method with electrochemical detection. The apparent diffusion coefficient was measured following the fluorescence quenching of 1-pyrenedodecanoate inserted in the liposome bilayers under the same conditions. The partition coefficient varied with the temperature and the physical state of the membrane, from below 1 in the gel state to above 2.8 in the liquid-crystalline state in DMPC and DPPC membranes. The partition coefficient was directly proportional to the partial molar volume and was then associated to the increase in free-volume in the membrane as a function of temperature. The apparent diffusion coefficients were corrected by the partition coefficients and found to be nearly the same, with a null dependence on viscosity and physical state of the membrane, probably because the pyrene is disturbing the surrounding lipids and thus becoming insensitive to changes in membrane viscosity. Combining our results with those of others, it is apparent that both solubility and diffusion increase when increasing the temperature or when comparing a membrane in the gel to one in the fluid state.
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1,2-Dipalmitoilfosfatidilcolina/química , Colesterol/química , Dimiristoilfosfatidilcolina/química , Liposomas/química , Oxígeno/química , Fosfatidilcolinas/química , Tampones (Química) , Difusión , Técnicas Electroquímicas , Colorantes Fluorescentes/química , Lauratos/química , Membrana Dobles de Lípidos/química , Transición de Fase , Pirenos/química , Temperatura , Termodinámica , ViscosidadRESUMEN
Mycobacterium tuberculosis (Mtb) is a facultative intracellular pathogen and the second largest contributor to global mortality caused by an infectious agent after HIV. In infected host cells, Mtb is faced with a harsh intracellular environment including hypoxia and the release of nitric oxide (NO) and carbon monoxide (CO) by immune cells. Hypoxia, NO and CO induce a state of in vitro dormancy where Mtb senses these gases via the DosS and DosT heme sensor kinase proteins, which in turn induce a set of â¼47 genes, known as the Mtb Dos dormancy regulon. On the contrary, both iNOS and HO-1, which produce NO and CO, respectively, have been shown to be important against mycobacterial disease progression. In this review, we discuss the impact of O2, NO and CO on Mtb physiology and in host responses to Mtb infection as well as the potential role of another major endogenous gas, hydrogen sulfide (H2S), in Mtb pathogenesis.
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Gasotransmisores/fisiología , Mycobacterium tuberculosis/fisiología , Tuberculosis Pulmonar/metabolismo , Monóxido de Carbono/fisiología , Humanos , Sulfuro de Hidrógeno/metabolismo , Mycobacterium tuberculosis/genética , Óxido Nítrico/fisiología , Oxígeno/fisiología , Especies Reactivas de Oxígeno/metabolismo , Tuberculosis Pulmonar/microbiologíaRESUMEN
The current perspective holds that the generation of secondary signaling mediators from nitrite (NO2(-)) requires acidification to nitrous acid (HNO2) or metal catalysis. Herein, the use of stable isotope-labeled NO2(-) and LC-MS/MS analysis of products reveals that NO2(-) also participates in fatty acid nitration and thiol S-nitrosation at neutral pH. These reactions occur in the absence of metal centers and are stimulated by autoxidation of nitric oxide ((â¢)NO) via the formation of symmetrical dinitrogen trioxide (nitrous anhydride, symN2O3). Although theoretical models have predicted physiological symN2O3 formation, its generation is now demonstrated in aqueous reaction systems, cell models and in vivo, with the concerted reactions of (â¢)NO and NO2(-) shown to be critical for symN2O3 formation. These results reveal new mechanisms underlying the NO2(-) propagation of (â¢)NO signaling and the regulation of both biomolecule function and signaling network activity via NO2(-)-dependent nitrosation and nitration reactions.
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Macrófagos/química , Nitratos/química , Óxido Nítrico/química , Nitritos/química , Óxidos de Nitrógeno/química , Ácido Nitroso/química , Animales , Línea Celular , Glutatión/química , Glutatión/metabolismo , Concentración de Iones de Hidrógeno , Inflamación/inducido químicamente , Inflamación/metabolismo , Cinética , Ácidos Linoleicos Conjugados/química , Ácidos Linoleicos Conjugados/metabolismo , Ácidos Linoleicos Conjugados/farmacología , Lipopolisacáridos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Nitritos/farmacología , Isótopos de Nitrógeno , Óxidos de Nitrógeno/metabolismo , Nitrosación , Ácido Nitroso/metabolismo , Isótopos de OxígenoRESUMEN
1-Hydroxyphenazine (1-HP) is a virulence factor produced by Pseudomonas aeruginosa. In this study,supercoiled plasmid DNA was employed as an analytical tool for the detection of ROS generation mediated by 1-HP. These assays provided evidence that 1-HP, in conjunction with NADPH alone or NADPH and the enzyme NADPH:cytochrome P450 reductase, mediated the production of superoxide radical under physiological conditions. Experiments with murine macrophage RAW264.7 cells and profluorescent ROS probes dichlorodihydrofluorescein or dihydroethidine provided preliminary evidence that 1-HP mediates the generation of intracellular oxidants. Generation of reactive oxygen species may contribute to the virulence properties of 1-HP in P. aeruginosa infections.
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Fenazinas/química , Fenazinas/metabolismo , Pseudomonas aeruginosa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Virulencia/metabolismo , Animales , Células Cultivadas , Ratones , Estructura Molecular , NADP/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Pseudomonas aeruginosa/química , Especies Reactivas de Oxígeno/química , Factores de Virulencia/químicaRESUMEN
Reactive oxygen species (ROS) produced by an oxidative burst are an important component of the wound response in algae, vascular plants, and animals. In all taxa, ROS production is usually attributed solely to a defense-related enzyme like NADPH-oxidase (Nox). However, here we show that the initial, wound-induced oxidative burst of the kelp Saccharina latissima depends on light and photosynthetic electron transport. We measured oxygen evolution and ROS production at different light levels and in the presence of a photosynthetic inhibitor, and we used spin trapping and electron paramagnetic resonance as an orthogonal method. Using an in vivo chemical probe, we provide data suggesting that wound-induced ROS production in two distantly related and geographically isolated species of Antarctic macroalgae may be light dependent as well. We propose that electron transport chains are an important and as yet unaddressed component of the wound response, not just for photosynthetic organisms, but for animals via mitochondria as well. This component may have been obscured by the historic use of diphenylene iodonium, which inhibits not only Noxes but also photosynthetic and respiratory electron transport as well. Finally, we anticipate physiological and/or ecological consequences of the light dependence of macroalgal wound-induced ROS since pathogens and grazers do not disappear in the dark.
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Nitric oxide (nitrogen monoxide, â¢NO) has been intensively studied by chemists and physicists for over 200 years and thus there is an extensive database of information that determines its biological actions. This is a very brief overview of the chemical and physical properties of â¢NO that are most relevant to Biology in general and to the development of â¢NO releasing materials in particular.
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Dinitrosyliron complexes (DNIC) have been found in a variety of pathological settings associated with (â¢)NO. However, the iron source of cellular DNIC is unknown. Previous studies on this question using prolonged (â¢)NO exposure could be misleading due to the movement of intracellular iron among different sources. We here report that brief (â¢)NO exposure results in only barely detectable DNIC, but levels increase dramatically after 1-2 h of anoxia. This increase is similar quantitatively and temporally with increases in the chelatable iron, and brief (â¢)NO treatment prevents detection of this anoxia-induced increased chelatable iron by deferoxamine. DNIC formation is so rapid that it is limited by the availability of (â¢)NO and chelatable iron. We utilize this ability to selectively manipulate cellular chelatable iron levels and provide evidence for two cellular functions of endogenous DNIC formation, protection against anoxia-induced reactive oxygen chemistry from the Fenton reaction and formation by transnitrosation of protein nitrosothiols (RSNO). The levels of RSNO under these high chelatable iron levels are comparable with DNIC levels and suggest that under these conditions, both DNIC and RSNO are the most abundant cellular adducts of (â¢)NO.
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Hipoxia de la Célula/fisiología , Hierro/metabolismo , Óxido Nítrico/metabolismo , Óxidos de Nitrógeno/metabolismo , S-Nitrosotioles/metabolismo , Animales , Línea Celular , Deferoxamina/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Peróxido de Hidrógeno/metabolismo , Quelantes del Hierro/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Pseudomonas aeruginosa (PA) is a common bacterial pathogen, responsible for a high incidence of nosocomial and respiratory infections. KatA is the major catalase of PA that detoxifies hydrogen peroxide (H2O2), a reactive oxygen intermediate generated during aerobic respiration. Paradoxically, PA displays elevated KatA activity under anaerobic growth conditions where the substrate of KatA, H2O2, is not produced. The aim of the present study is to elucidate the mechanism underlying this phenomenon and define the role of KatA in PA during anaerobiosis using genetic, biochemical and biophysical approaches. We demonstrated that anaerobic wild-type PAO1 cells yielded higher levels of katA transcription and expression than aerobic cells, whereas a nitrite reductase mutant ΔnirS produced â¼50% the KatA activity of PAO1, suggesting that a basal NO level was required for the increased KatA activity. We also found that transcription of the katA gene was controlled, in part, by the master anaerobic regulator, ANR. A ΔkatA mutant and a mucoid mucA22 ΔkatA bacteria demonstrated increased sensitivity to acidified nitrite (an NO generator) in anaerobic planktonic and biofilm cultures. EPR spectra of anaerobic bacteria showed that levels of dinitrosyl iron complexes (DNIC), indicators of NO stress, were increased significantly in the ΔkatA mutant, and dramatically in a ΔnorCB mutant compared to basal levels of DNIC in PAO1 and ΔnirS mutant. Expression of KatA dramatically reduced the DNIC levels in ΔnorCB mutant. We further revealed direct NO-KatA interactions in vitro using EPR, optical spectroscopy and X-ray crystallography. KatA has a 5-coordinate high spin ferric heme that binds NO without prior reduction of the heme iron (Kd â¼6 µM). Collectively, we conclude that KatA is expressed to protect PA against NO generated during anaerobic respiration. We proposed that such protective effects of KatA may involve buffering of free NO when potentially toxic concentrations of NO are approached.
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Catalasa/metabolismo , Óxido Nítrico/metabolismo , Pseudomonas aeruginosa/metabolismo , Anaerobiosis/efectos de los fármacos , Antibacterianos/farmacología , Catalasa/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Hierro/metabolismo , Nitritos/metabolismo , Óxidos de Nitrógeno/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , Transcripción Genética/efectos de los fármacosRESUMEN
Following nitric oxide (nitrogen monoxide) and carbon monoxide, hydrogen sulfide (or its newer systematic name sulfane, H2S) became the third small molecule that can be both toxic and beneficial depending on the concentration. In spite of its impressive therapeutic potential, the underlying mechanisms for its beneficial effects remain unclear. Any novel mechanism has to obey fundamental chemical principles. H2S chemistry was studied long before its biological relevance was discovered, however, with a few exceptions, these past works have received relatively little attention in the path of exploring the mechanistic conundrum of H2S biological functions. This review calls attention to the basic physical and chemical properties of H2S, focuses on the chemistry between H2S and its three potential biological targets: oxidants, metals and thiol derivatives, discusses the applications of these basics into H2S biology and methodology, and introduces the standard terminology to this youthful field.